WO2010140694A1 - Method for screening of inhibitor using factor capable of enhancing production of amyloid-beta peptide, and inhibitor obtained by same - Google Patents

Method for screening of inhibitor using factor capable of enhancing production of amyloid-beta peptide, and inhibitor obtained by same Download PDF

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WO2010140694A1
WO2010140694A1 PCT/JP2010/059568 JP2010059568W WO2010140694A1 WO 2010140694 A1 WO2010140694 A1 WO 2010140694A1 JP 2010059568 W JP2010059568 W JP 2010059568W WO 2010140694 A1 WO2010140694 A1 WO 2010140694A1
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protein
refseq
substance
gene
secretase
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PCT/JP2010/059568
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French (fr)
Japanese (ja)
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オー． ラース シャンバリィ
スザンヌ フリークマン
ジエン フー
康博 寺西
博之 中川
順 左海
孝洋 木原
泰三 石川
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大日本住友製薬株式会社
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Publication of WO2010140694A1 publication Critical patent/WO2010140694A1/en

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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
    • 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
    • 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
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention relates to a ⁇ -amyloid production inhibitor that is the cause of Alzheimer's disease, a prophylactic and therapeutic agent for Alzheimer's disease, and a screening method for such a drug.
  • ⁇ -amyloid (A ⁇ ) peptide which accumulates in the brain of Alzheimer's disease (AD), is produced by being excised from the precursor protein amyloid precursor protein (APP) by ⁇ -secretase and ⁇ -secretase, which is polymerized to form senile plaques Is formed. Accumulation of A ⁇ is a lesion that occurs early in AD with high specificity in AD brain. Furthermore, in familial Alzheimer's disease (FAD), A ⁇ is considered to be a causative agent of AD because the mutation of the etiological genes APP and presenilin (PS) increases the production of highly aggregated A ⁇ 42 (amyloid hypothesis). It is regarded as a promising therapeutic target for radical treatment of AD (Non-patent Document 1).
  • a ⁇ peptide production can be suppressed by inhibiting ⁇ -secretase, the responsible enzyme responsible for the final cleavage.
  • ⁇ -secretase inhibitors that have been reported so far also inhibit cleavage of Notch, which is another physiological substrate of the enzyme, and inhibit signals involved in development and differentiation.
  • Peripheral side effects such as immune abnormalities are a problem (Non-patent Document 2).
  • an abnormality occurs in the signal pathway via Notch, not only a serious abnormality occurs in the generation and formation of tissues and organs, but it may cause cancer.
  • the Notch gene has been clarified as an oncogenic gene, and it is also known that regulating the activity of ⁇ -secretase that cleaves Notch has an anticancer effect (Non-patent Document 3).
  • NSAIDs non-steroidal anti-inflammatory drugs
  • ⁇ -secretase modulator activity ⁇ -secretase modulator activity
  • ⁇ -secretase is a transmembrane aspartic protease composed of presenilin (PS), nicastrin (NCT), Aph-1 and Pen-2 (Non-patent Document 5).
  • PS presenilin
  • NCT nicastrin
  • Aph-1 and Pen-2 Non-patent Document 5
  • TMP21 was identified as a molecule that binds to ⁇ -secretase and controls its activity (Non-patent Document 6).
  • the existence of other ⁇ -secretase activity regulators is not known at all.
  • a gene encoding a membrane-bound protein (ATP2A2, FLOT2) is knocked down by the RNAi method, production of A ⁇ is reported to decrease (Non-patent Documents 7 and 8).
  • An object of the present invention is to identify a novel factor that binds to ⁇ -secretase and promotes / enhances its activity, and to suppress the expression and function of the factor, a ⁇ -secretase inhibitor or modulator, A ⁇ It is to provide a production inhibitor and an AD preventive / therapeutic agent, and to provide a method for screening such a drug using the factor.
  • the present inventors have conceived that there are molecules other than TMP21 that bind to ⁇ -secretase and control A ⁇ production in brain tissue, and that bind to ⁇ -secretase using an inhibitor pull-down method.
  • HEK-APP human embryonic kidney cell line
  • APP human embryonic kidney cell line
  • the present invention is as follows.
  • the following proteins (P1) Probable phospholipid-transporting ATPase IIA; (P2) BDNF / NT-3 growth factors receptor precursor; (P3) ELAV-like protein 4; (P4) Coiled-coil domain-containing protein 136; (P5) Potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 2; (P6) DnaJ homolog subfamily A member 2; (P7) Vesicle-associated membrane protein-associated protein A; (P8) Proton myo-inositol cotransporter; (P9) Leukocyte surface antigen CD47; (P10) Flotillin-1; (P11) Band 4.1 like 1; (P12) Regulator of G-protein signaling 7; (P13) Phospholipase D3; (P14) Ectoderm-neural cortex protein 1; (P15) synaptophysin; (P16) solute carrier family 2 (
  • [6] In mammals, the following proteins: (P1) Probable phospholipid-transporting ATPase IIA; (P2) BDNF / NT-3 growth factors receptor precursor; (P3) ELAV-like protein 4; (P4) Coiled-coil domain-containing protein 136; (P5) Potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 2; (P6) DnaJ homolog subfamily A member 2; (P7) Vesicle-associated membrane protein-associated protein A; (P8) Proton myo-inositol cotransporter; (P9) Leukocyte surface antigen CD47; (P10) Flotillin-1; (P11) Band 4.1 like 1; (P12) Regulator of G-protein signaling 7; (P13) Phospholipase D3; (P14) Ectoderm-ne
  • the method comprises selecting a substance that decreases the expression level of at least one gene selected from (G1) to (G25) or the activity of at least one protein selected from (P1) to (P25).
  • a method for screening an A ⁇ production inhibitor comprises selecting a substance that decreases the expression level of at least one gene selected from (G1) to (G25) or the activity of at least one protein selected from (P1) to (P25).
  • a method for screening an A ⁇ production inhibitor comprising the following steps (a) to (c): A reporter gene under the control of the transcriptional regulatory region of (a) (a1) gene Gn (n is any integer from 1 to 25) or (a2) gene Gn (n is any integer from 1 to 25) A step of contacting a test substance with a cell to be expressed (b) a step of measuring the expression level of (b1) gene Gn or (b2) reporter gene in the cell (c) a measurement in the absence of the test substance [14] A method for screening an A ⁇ production inhibitory substance, comprising the following steps (a) to (c): selecting a substance that reduces the expression level as a candidate for an A ⁇ production inhibitory substance compared to (A) contacting a cell expressing amyloid precursor protein (APP) with at least one protein selected from the above (P1) to (P25) and a test substance (b) measuring the activity of the protein ( c) The step of selecting a substance that reduces the activity of the protein compared to the
  • a method for screening an A ⁇ production inhibitor comprising the following steps (a) to (c): (A) contacting at least one protein selected from (P1) to (P25) and a test substance with a cell expressing ⁇ -secretase or a cell membrane fraction thereof; and (b) measuring ⁇ -secretase activity. Step (c) selecting a substance that reduces or modulates ⁇ -secretase activity compared to that measured in the absence of the test substance [18]
  • the protein is provided by the cell itself, The method according to [17].
  • a screening method for an A ⁇ production inhibitor which comprises selecting a substance that inhibits the binding activity between protein Pn (n is an integer of 1 to 25) and ⁇ -secretase.
  • a screening method for an A ⁇ production inhibitor comprising the following steps (a) to (c): (A) contacting protein Pn (n is an integer of 1 to 25) and ⁇ -secretase with a test substance (b) measuring the binding activity between protein Pn and ⁇ -secretase (c ) A step of selecting a substance that reduces the binding activity as compared with the case where it is measured in the absence of the test substance [21] non-introducing at least one gene selected from (G1) to (G25) A method for screening or evaluating an efficacy of a therapeutic or prophylactic agent for Alzheimer's disease, comprising selecting a substance that improves the phenotype reflecting the pathological condition of Alzheimer's disease in a human animal.
  • a screening or drug efficacy evaluation method for a therapeutic or prophylactic agent for Alzheimer's disease comprising the following steps (a) to (c): (A) a step of administering a test substance to a non-human animal into which at least one gene selected from the above (G1) to (G25) has been introduced; (b) at least one expression reflecting the pathological condition of Alzheimer's disease in the animal A step of evaluating the type (c) a step of selecting a substance that improves the phenotype as compared with the case where the test substance is not administered, [23] the phenotype is brain tissue, cerebrospinal fluid, Selected from the group consisting of A ⁇ amount in tissues such as blood, neuronal cell death, central nervous system inflammatory response, cognitive ability, amyloid plaque accumulation, cerebral blood flow and cerebral glucose metabolism 22].
  • a method for determining the onset or risk of developing Alzheimer's disease or cancer comprising the following steps (a) to (c): (A) Providing a sample derived from a test animal (b) Expression level of at least one gene selected from (G1) to (G25) in the sample or selected from (P1) to (P25) A step of measuring the activity of at least one protein (c) is the subject animal having an increased expression level or activity compared to the case of measuring in a sample derived from a normal animal developing Alzheimer's disease or cancer? The process of determining that the risk of developing in the future is high
  • the present invention by using a novel protein that regulates the activity of ⁇ -secretase, it becomes possible to screen for compounds that inhibit the production of A ⁇ and to develop medical treatments using antibodies and nucleic acids. Thus, it is useful for the prevention and treatment of diseases such as AD and cancer.
  • FIG. 2 is a schematic diagram (A) of the principle of an inhibitor pull-down method and a diagram showing that a known component of ⁇ -secretase is pulled down by the method. It is a figure which shows the production suppression of endogenous A (beta) 42 by the gene knockdown by siRNA with respect to a HEK-APP cell (transduction siRNA amount: 0.1pmol / well).
  • the present invention includes the following proteins: (P1) Probable phospholipid-transporting ATPase IIA; (P2) BDNF / NT-3 growth factors receptor precursor; (P3) ELAV-like protein 4; (P4) Coiled-coil domain-containing protein 136; (P5) Potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 2; (P6) DnaJ homolog subfamily A member 2; (P7) Vesicle-associated membrane protein-associated protein A; (P8) Proton myo-inositol cotransporter; (P9) Leukocyte surface antigen CD47; (P10) Flotillin-1; (P11) Band 4.1 like 1; (P12) Regulator of G-protein signaling 7; (P13) Phospholipase D3; (P14) Ectoderm-neural cortex protein 1; (P15) synaptophysin; (P16) solute carrier family 2 (facilitated glucose transporter), member 3;
  • Protein Pn [(P1) Probable phospholipid-transporting ATPase IIA; (P2) BDNF / NT-3 growth factors receptor precursor; (P3) ELAV-like protein 4; (P4) Coiled-coil domain-containing protein 136; (P5) Potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 2; (P6) DnaJ homolog subfamily A member 2; (P7) Vesicle-associated membrane protein-associated protein A; (P8) Proton myo-inositol cotransporter; (P9) Leukocyte surface antigen CD47; (P10) Flotillin-1; (P11) Band 4.1 like 1; (P12) Regulator of G-protein signaling 7; (P13) Phospholipase D3; (P14) Ectoderm-neural cortex protein 1; (P15) ) synaptophysin; (P16) solute carrier family 2 (facilitated glucose transporter), member 3; (P
  • Protein Pn is a cell of humans and other warm-blooded animals (eg, guinea pigs, rats, mice, chickens, rabbits, dogs, pigs, sheep, cows, monkeys, etc.) [eg, hepatocytes, spleen cells, neurons, glia.
  • Cells pancreatic ⁇ cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, lung cells, endothelial cells, smooth muscle cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg, macrophages) , T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts , Mammary cells or stromal cells, or precursor cells of these cells, stem cells, cancer cells, etc.] or any tissue in which these cells are present [eg, brain, brain regions (eg, olfactory bulb, amygdala, cerebrum) Basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach
  • amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2n (a) an amino acid sequence having about 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2n; (b) in the amino acid sequence represented by SEQ ID NO: 2n, an amino acid sequence in which 1 to 50 amino acids are substituted and / or deleted and / or inserted and / or added; (c) the amino acid sequence of an ortholog in another mammal of the human protein consisting of the amino acid sequence represented by SEQ ID NO: 2n; or (d) The amino acid sequence of the human protein consisting of the amino acid sequence represented by SEQ ID NO: 2n or the splice variant, allelic variant or polymorphism of the ortholog of (c) above.
  • homology refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm uses a sequence of sequences for optimal alignment). The ratio of the same amino acid residue and similar amino acid residues to all overlapping amino acid residues in the case of introducing a gap into one or both).
  • similar amino acids means amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn).
  • NCBI BLAST National Center for Biotechnology Information Basic Local Alignment Search Tool
  • Other algorithms for determining amino acid sequence homology include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [the algorithms include NBLAST and XBLAST] Embedded in the program (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 1997 (1997))], Needleman et al., J. Mol.
  • the “amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2n” is about 80% or more of the amino acid sequence represented by SEQ ID NO: 2n,
  • the amino acid sequence preferably has about 90% or more, more preferably about 95% or more, still more preferably about 97% or more, particularly preferably about 98% or more, and most preferably about 99% or more.
  • a protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2n includes the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2n, and the sequence No .: A protein having substantially the same activity as the protein consisting of the amino acid sequence represented by 2n.
  • activity refers to an activity that promotes the production of A ⁇ .
  • substantially the same quality means that the properties are qualitatively the same, for example, physiologically or pharmacologically. Therefore, it is preferable that the A ⁇ production promoting activities are equivalent, but quantitative factors such as the degree of these activities (eg, about 0.1 to about 10 times) and the molecular weight of the protein may be different.
  • a ⁇ production promoting activity can be measured by measuring and comparing the amount of A ⁇ produced (preferably the amount of A ⁇ 40 or A ⁇ 42 produced) in the presence and absence of a protein according to a method known per se. .
  • the protein Pn in the present invention as shown in (b) above, for example, (i) 1 to 50, preferably 1 to 30, more preferably 1 in the amino acid sequence represented by SEQ ID NO: 2n ⁇ 10, more preferably 1 to several (5, 4, 3 or 2) amino acid sequences deleted, (ii) 1 to 50, preferably 1 to the amino acid sequence represented by SEQ ID NO: 2n An amino acid sequence to which 1 to 30, more preferably 1 to 10, more preferably 1 to several (5, 4, 3, or 2) amino acids are added; (iii) an amino acid sequence represented by SEQ ID NO: 2n An amino acid sequence into which 1 to 50, preferably 1 to 30, more preferably 1 to 10, more preferably 1 to several (5, 4, 3 or 2) amino acids are inserted, (iv) a sequence 1 to 50, preferably 1 to 30, more preferably 1 to 10, more preferably in the amino acid sequence represented by the number: 2n It is also included so-called muteins such as proteins containing 1 to several (5, 4, 3 or 2) amino acid sequence number
  • Preferred examples of the protein P1 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (RefSeq No. NP_006036), or their orthologs in other mammals (for example, , Mice (RefSeq No. NP_056546), chimpanzees (RefSeq No. XP_001168714), dogs (RefSeq No. XP_534457), and their splice variants (eg UniProtKB Nos. O75110-1, O75110-2 etc.), alleles Examples include mutants and polymorphisms.
  • Preferred examples of the protein P2 include, for example, a human protein (RefSeq No. NP_006171) consisting of the amino acid sequence represented by SEQ ID NO: 4, or those in other mammals.
  • Orthologs eg, mice (RefSeq No. NP_001020245), rats (RefSeq No. NP_036863), cows (RefSeq No. NP_001068693), chimpanzees (RefSeq No. XP_001135401), dogs (RefSeq No. XP_856422) etc.
  • those Examples include splice variants (for example, RefSeq No.
  • NP_001007098, NP_001018074, NP_001018075, NP_001018076, etc. allelic variants, polymorphisms, and the like.
  • Preferred examples of the protein P3 include, for example, a human protein (RefSeq No. NP_068771) consisting of the amino acid sequence represented by SEQ ID NO: 6, or an ortholog thereof (for example, Mice (RefSeq No. NP_034618), rats (RefSeq No.NP_001071119), cows (RefSeq No. NP_001075075), chimpanzees (RefSeq No.
  • protein P4 oiled-coil domain-containing protein 136) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 8 (RefSeq No. NP_073579), or an ortholog thereof in other mammals (For example, mouse (RefSeq No.
  • NP_663549 rat (RefSeq No. XP_342653), cattle (RefSeq No. XP_872956), chimpanzee (RefSeq No. XP_519361), etc.) and their splice variants (for example, UniProtKB Nos. Q96JN2 -1, Q96JN2-2, Q96JN2-3, Q96JN2-4, etc.), allelic variants, polymorphisms (eg refSNP Nos. Rs3816887 (Asp / His), rs4728137 (Glu / Gln), rs8180870 (Thr / Ala), etc.) ) Etc.
  • Preferred examples of the protein P5 include, for example, a human protein (RefSeq No. NP_001185) consisting of the amino acid sequence represented by SEQ ID NO: 10, or other mammals Orthologs (eg mice (RefSeq No. NP_032252), rats (RefSeq No. NP_446136), dogs (RefSeq No. XP_855233), etc.), and their splice variants, allelic variants, polymorphisms, etc. .
  • Preferred examples of the protein P6 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 12 (RefSeq No. NP_005871), or an ortholog thereof in other mammals (for example, , Mice (RefSeq No. NP_062768), rats (RefSeq No. NP_114468), cattle (RefSeq No. NP_001035581), chimpanzees (RefSeq No. XP_528644), dogs (RefSeq No.
  • protein P7 Vehicle-associated membrane protein-associated protein A
  • a human protein consisting of the amino acid sequence represented by SEQ ID NO: 14, or those in other mammals.
  • Orthologs for example, mice (RefSeq No. NP_038961), rats (RefSeq No. NP_113819), cattle (RefSeq No. NP_001069201), chimpanzees (RefSeq No.
  • XP_512022) dogs
  • dogs dogs
  • RefSeq No. XP_537332 dogs
  • Examples include splice variants (for example, RefSeq No. NP_919415), allelic variants, and polymorphisms.
  • Preferred examples of the protein P8 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 16 (RefSeq No. NP_443117), or an ortholog thereof in other mammals (for example, Mice (RefSeq No. NP_001028805), rats (RefSeq No. NP_598295), cattle (RefSeq No.
  • Preferred examples of the protein P9 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 18 (RefSeq No. NP_001768), or an ortholog thereof (for example, mouse) in other mammals (RefSeq No. NP_034711), rat (RefSeq No. NP_062068), cattle (RefSeq No.
  • NP_777133 chimpanzee
  • chimpanzee RefSeq No. XP_516636
  • dog RefSeq No. NP_001074190
  • splice variants for example, UniProtKB Nos. Q08722-1, Q08722-2, Q08722-3, Q08722-4, etc.
  • allelic variants for example, refSNP No. rs11546646 (Pro / Ala), etc.
  • Preferred examples of the protein P10 include, for example, a human protein (RefSeq No.
  • NP_005794 consisting of the amino acid sequence represented by SEQ ID NO: 20, or an ortholog thereof (for example, mouse ( RefSeq No. NP_032053), rat (RefSeq No. NP_073192), cattle (RefSeq No. NP_001070355), chimpanzee (RefSeq No. NP_001035840), dog (RefSeq No. XP_848778) etc.), and their splice variants and allelic variants And polymorphism (for example, refSNP No. rs1802470 (Asp / Tyr) etc.) and the like.
  • Preferred examples of the protein P11 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 22 (RefSeq No. NP_818932), or an ortholog thereof (eg, rat) in other mammals.
  • a human protein consisting of the amino acid sequence represented by SEQ ID NO: 22 (RefSeq No. NP_818932), or an ortholog thereof (eg, rat) in other mammals.
  • RefSeq No. NP_067713 cattle
  • RefSeq No. XP_872829 chimpanzee
  • RefSeq No. XP_525314 dogs
  • their splice variants for example, UniProtKB Nos.
  • Preferred examples of the protein P12 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 24 (RefSeq No. NP_002915), or their orthologs in other mammals (Ref. For example, mice (RefSeq No. NP_036010), rats (RefSeq No. NP_062216), cattle (RefSeq No.
  • NP_776594 chimpanzees
  • chimpanzees RefSeq No. XP_001159739
  • their splice variants for example, UniProtKB Nos. P49802- 1, P49802-2, P49802-3, P49802-4, P49802-5, etc.
  • allelic variants polymorphisms
  • polymorphisms eg refSNP No. rs12746550 (Met / Leu), rs17851953 (Gln / His), etc.
  • Preferred examples of the protein P13 include, for example, a human protein (RefSeq No.
  • NP_001026866 consisting of the amino acid sequence represented by SEQ ID NO: 26, or an ortholog (eg, mouse (RefSeq) in other mammals. No. NP_035246), rat (RefSeq No. NP_001012167), chimpanzee (RefSeq No. XP_524268), dog (RefSeq No. XP_541616) and the like, and their splice variants, allelic variants, and polymorphisms.
  • Preferred examples of the protein P14 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 28 (RefSeq No.
  • NP_003624 or an ortholog thereof in other mammals (for example, , Mice (RefSeq No. NP_031956), rats (RefSeq No. NP_001003401), chimpanzees (RefSeq No. XP_526897), dogs (RefSeq No. XP_544373), etc.) and their splice variants, allelic variants, polymorphisms (for example, RefSNP No. rs11557041 (Phe / Leu), rs11747630 (Thr / Asn), rs16872126 (Ile / Ser), etc.).
  • the protein P15 includes, for example, a human protein (RefSeq No. NP_003170) consisting of the amino acid sequence represented by SEQ ID NO: 30, or an ortholog thereof (eg, mouse (RefSeq No) in other mammals) NP_033331), rats (RefSeq No. NP_036796), cattle (RefSeq No. NP_776388), chimpanzees (RefSeq No. XP_521056), dogs (RefSeq No. XP_855381), etc.) and their splice variants, allelic variants, many Examples include molds.
  • a human protein consisting of the amino acid sequence represented by SEQ ID NO: 30, or an ortholog thereof (eg, mouse (RefSeq No) in other mammals) NP_033331)
  • rats RefSeq No. NP_036796
  • cattle RefSeq No. NP_776388
  • Preferred examples of the protein P16 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 32 (RefSeq No. NP_008862), or other mammals Orthologs of them (eg, mice (RefSeq No. NP_035531), rats (RefSeq No. NP_058798), cattle (RefSeq No. NP_777028), chimpanzees (RefSeq No. XP_508989), dogs (RefSeq No.
  • NP_0010033008 examples include splice variants, allelic variants, and polymorphisms (eg, refSNP No. rs17728193 (Val / Leu)).
  • Preferred examples of the protein P17 (syntaxin binding protein 1) include, for example, a human protein (RefSeq No. NP_001027392) consisting of the amino acid sequence represented by SEQ ID NO: 34, or an ortholog thereof (for example, mouse) in other mammals (RefSeq No. NP_033321), rat (RefSeq No. NP_037170), bovine (RefSeq No.
  • NP_777044 splice variants
  • P61764-1 e.g UniProtKB Nos. P61764-1, P61764-2 etc.
  • allelic variants And polymorphism e.g UniProtKB Nos. P61764-1, P61764-2 etc.
  • Preferred examples of the protein P18 (growth associated protein 43) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 36 (RefSeq No. NP_002036), or an ortholog thereof (for example, mouse) in other mammals (RefSeq No. NP_032109), rat (RefSeq No. NP_058891), cow (RefSeq No. NP_976234), chimpanzee (RefSeq No.
  • NP_001009816 dog (RefSeq No. XP_535747 etc.), and their splice variants and allelic variants And polymorphism (for example, refSNP No. rs6291 (Val / Ile), rs11557762 (Lys / Glu), rs11557763 (Glu / Asp), etc.) and the like.
  • Preferred examples of protein P19 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 38 (RefSeq No. NP_001668), or those in other mammals Orthologs (eg, mice (RefSeq No.
  • NP_033851 rats (RefSeq No. NP_037245), cattle (RefSeq No. NP_001030411), chimpanzees (RefSeq No. NP_001092025), dogs (RefSeq No. NP_001003283), etc.)
  • Splice variants for example, refSNP No. rs11545848 (Lys / Gln), rs1545849 (Met / Val), etc.
  • Preferred examples of the protein P20 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 40 (RefSeq No. NP_000889), or an ortholog thereof in other mammals (For example, mice (RefSeq No. NP_766366), rats (RefSeq No. NP_037330), cattle (RefSeq No. NP_808813), dogs (RefSeq No. XP_855549), etc.), and their splice variants, allelic variants, and polymorphisms (For example, refSNP No. rs12845783 (Ser / Arg)).
  • protein P21 (Muscarinic acetylcholine receptor M1) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 42 (RefSeq No. NP_000729), or an ortholog thereof (for example, mouse) in other mammals (RefSeq No. NP_031724), rat (RefSeq No. NP_542951), cattle (RefSeq No XP_001255658), chimpanzee (RefSeq No. XP_001159938), dog (RefSeq No. XP_540897), etc.) and their splice variants and allelic variants And polymorphism.
  • Preferred examples of the protein P22 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 44 (RefSeq No. NP_066000), or an ortholog thereof in other mammals (for example, Mice (RefSeq No. NP_766449), rats (RefSeq No. NP_001128087), chimpanzees (RefSeq No. XP_526378), dogs (RefSeq No. XP_545285), etc., as well as their splice variants, allelic variants, polymorphisms (for example, refSNP No. rs2276717 (Gly / Arg), etc.).
  • Preferred examples of the protein P23 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 46 (RefSeq No. NP_077718), or an ortholog thereof in other mammals ( For example, mice (RefSeq No. NP_083548), rats (RefSeq No. NP_001008525), cattle (RefSeq No. NP_001033111), dogs (RefSeq No. XP_533960), etc.), as well as their splice variants, allelic variants, polymorphisms ( For example, refSNP No. rs1142530 (Pro / Leu) etc.).
  • Preferred examples of the protein P24 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 48 (RefSeq No. NP_443131), or those in other mammals Orthologs (eg, mouse (RefSeq No. NP_036144), rat (RefSeq No. XP_344572), etc.) and their splice variants (eg, UniProtKB Nos. Q7Z2K8-1, Q7Z2K8-2 etc.), allelic variants, polymorphisms (For example, refSNP No.
  • Preferred examples of the protein P25 include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 50 (RefSeq No. NP_001098999), or an ortholog thereof in other mammals (for example, , Mice (RefSeq No. NP_001034088), cattle (RefSeq No. NP_777263), chimpanzees (RefSeq No.
  • XP_001150768 dogs (RefSeq No. XP_849357), and their splice variants (eg UniProtKB Nos. Q9Y2Q0-1) , Q9Y2Q0-2, etc.), allelic variants, polymorphisms (for example, refSNP No. rs3792687 (Thr / Met) etc.) and the like.
  • the “substance that suppresses the expression of protein Pn” refers to the transcription level of the gene Gn encoding protein Pn, the level of post-transcriptional regulation, the level of translation into protein Pn, the level of post-translational modification, etc. It may act. Therefore, substances that suppress the expression of protein Pn include, for example, substances that inhibit the transcription of gene Gn (eg, antigene), substances that inhibit the processing of early transcription products into mRNA, and transport of mRNA to the cytoplasm.
  • substances that suppress the expression of protein Pn include, for example, substances that inhibit the transcription of gene Gn (eg, antigene), substances that inhibit the processing of early transcription products into mRNA, and transport of mRNA to the cytoplasm.
  • Inhibiting substances inhibiting translation of mRNA to protein Pn (eg, antisense nucleic acid, miRNA) or degrading mRNA (eg, siRNA, ribozyme, miRNA), inhibiting post-translational modification of the initial translation product Substances are included. Any substance that acts at any stage can be preferably used, but a substance that complementarily binds to mRNA and inhibits translation into protein Pn or decomposes mRNA is preferable.
  • a base sequence complementary to or substantially complementary to the base sequence of these mRNAs or one of them A nucleic acid containing a portion.
  • the base sequence substantially complementary to the base sequence of mRNA of gene Gn can bind to the target sequence of mRNA and inhibit its translation under physiological conditions in mammals (or cleave the target sequence).
  • a base sequence having a degree of complementarity specifically, for example, a region that overlaps with a base sequence that is completely complementary to the base sequence of the mRNA (that is, a base sequence of the complementary strand of the mRNA).
  • the base sequence complementary or substantially complementary to the base sequence of the gene Gn mRNA is (a) SEQ ID NO: 2n-1 (n is any integer from 1 to 25) A complementary or substantially complementary nucleotide sequence to the nucleotide sequence represented, or (b) a complementary strand of the nucleotide sequence represented by "SEQ ID NO: 2n-1 (n is any integer from 1 to 25)" Is a base sequence that hybridizes under stringent conditions, and has substantially the same quality of activity as a protein consisting of the amino acid sequence represented by SEQ ID NO: 2n (n is an integer from 1 to 25) Examples of the sequence that encodes a protein include complementary or substantially complementary base sequences.
  • the stringent conditions are, for example, the conditions described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999, for example, 6 ⁇ SSC (sodium chloride / sodium citrate) / 45 ° C. Hybridization, followed by one or more washes at 0.2 ⁇ SSC / 0.1% SDS / 50 to 65 ° C., and those skilled in the art will know the conditions for hybridization that give the same stringency. It can be selected appropriately.
  • mRNA of gene G1 include human ATP9A (RefSeq Accession No. NM_006045) containing the nucleotide sequence represented by SEQ ID NO: 1, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_015731), chimpanzee (RefSeq No. XM_001168714), dog (RefSeq No. XM_534457), etc.), and their splice variants, allelic variants, and polymorphisms.
  • Preferred examples of mRNA of the gene G2 include, for example, human NTRK2 (RefSeq No.
  • NM_006180 consisting of the base acid sequence represented by SEQ ID NO: 3, or their orthologs in other mammals (eg, mouse) (RefSeq No. NM_001025074), rat (RefSeq No. NM_012731), cattle (RefSeq No. NM_001075225), chimpanzee (RefSeq No. XM_001135401), dogs (RefSeq No. XM_851329), etc.) and their splice variants (for example, RefSeq No.
  • mRNA of the gene G3 include, for example, human ELAVL4 (RefSeq No. NM_021952) consisting of the base sequence represented by SEQ ID NO: 5, or their orthologs (eg, mouse ( RefSeq No. NM_010488), rat (RefSeq No. NM_001077651), cattle (RefSeq No. NM_001081606), chimpanzee (RefSeq No.
  • mRNA of the gene G4 include, for example, human NAG6 (RefSeq No. NM_022742) consisting of the base sequence represented by SEQ ID NO: 7, or their orthologs (for example, mouse ( RefSeq No. NM_145574), rat (RefSeq No. XM_342652), cattle (RefSeq No.
  • mRNA of gene G5 include, for example, human HCN2 (RefSeq No. NM_001194) consisting of the base sequence represented by SEQ ID NO: 9, or their orthologs in other mammals (for example, mouse ( RefSeq No. NM_008226), rat (RefSeq No.
  • mRNA of the gene G6 includes, for example, human DNAJA2 (RefSeq No. NM_005880) consisting of the base sequence represented by SEQ ID NO: 11, or their orthologs in other mammals (for example, mouse ( RefSeq No. NM_019794), rat (RefSeq No. NM_032079), cattle (RefSeq No. NM_001040491), chimpanzee (RefSeq No.
  • VAPA VAPA
  • human VAPA RefSeq No. NM_003574
  • SEQ ID NO: 13 the base sequence represented by SEQ ID NO: 13
  • rat RefSeq No. NM_031631
  • cattle RefSeq No.
  • mRNA of the gene G8 include, for example, human SLC2A13 (RefSeq No. NM_052885) consisting of the nucleotide sequence represented by SEQ ID NO: 15, or their orthologs (eg, mouse ( RefSeq No. NM_001033633), rat (RefSeq No.
  • mRNA of the gene G9 include, for example, human CD47 (RefSeq No. NM_001777) consisting of the base sequence represented by SEQ ID NO: 17, or their orthologs in other mammals (eg, mouse ( RefSeq No. NM_010581), rat (RefSeq No.
  • mRNA of the gene G10 include, for example, human FLOT1 (RefSeq No. NM_005803) consisting of the base sequence represented by SEQ ID NO: 19, or their orthologs (for example, mouse ( RefSeq No.
  • NM_008027 rat (RefSeq No. NM_022701), cattle (RefSeq No. NM_001076887), chimpanzee (RefSeq No. NM_001042381), dog (RefSeq No. XM_843685) etc.), and their splice variants and allelic variants And mRNA such as polymorphism (for example, refSNP No. rs1802470 (G / T) etc.).
  • G11 EPB41L1
  • human EPB41L1 RefSeq Accession No.
  • NM_177996 containing the base sequence represented by SEQ ID NO: 21 or an ortholog (eg, rat (RefSeq) in other mammals) No. NM_021681), cattle (RefSeq No. XM_867736), chimpanzee (RefSeq No. XM_525314), dogs (RefSeq No. XM_542979), etc.) and their splice variants, allelic variants, polymorphisms and other mRNAs .
  • Preferred examples of mRNA of the gene G12 (RGS7) include human RGS7 (RefSeq Accession No.
  • NM_002924 containing the nucleotide sequence represented by SEQ ID NO: 23, or their orthologs in other mammals (eg, mouse (RefSeq No. NM_011880), rat (RefSeq No. NM_019343), cattle (RefSeq No. NM_174169), chimpanzee (RefSeq No. XM_001159739)), and their splice variants, allelic variants, and polymorphisms. .
  • mRNA of gene G13 PLD3
  • human PLD3 RefSeq Accession No.
  • NM_001031696 containing the base sequence represented by SEQ ID NO: 25, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_011116), rat (RefSeq No. NM_001012167), chimpanzee (RefSeq No. XM_524268), dog (RefSeq No. XM_541616), etc., and their splice variants, allelic variants, and polymorphisms. .
  • Preferred examples of mRNA of the gene G14 (ENC1) include human ENC1 (RefSeq Accession No.
  • NM_003633 containing the nucleotide sequence represented by SEQ ID NO: 27, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_007930), rat (RefSeq No. NM_001003401), chimpanzee (RefSeq No. XM_526897), dog (RefSeq No. XM_544373), etc., and their splice variants, allelic variants, and polymorphisms .
  • Preferred examples of mRNA of the gene G15 include human SYP (RefSeq Accession No.
  • NM_003179 containing the nucleotide sequence represented by SEQ ID NO: 29, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_009305), rat (RefSeq No. NM_012664), cattle (RefSeq No. NM_173963), chimpanzee (RefSeq No. XM_521056), dog (RefSeq No. XM_850288), etc.), and their splice variants, allelic variants, Examples include polymorphic mRNA. As a preferable example of mRNA of gene G16 (SLC2A3), human SLC2A3 (RefSeq Accession No.
  • NM_006931 containing the base sequence represented by SEQ ID NO: 31 or their orthologs (for example, mouse (RefSeq) in other mammals) No. NM_011401), rat (RefSeq No. NM_017102), cattle (RefSeq No. NM_174603), chimpanzee (RefSeq No. XM_508989), dog (RefSeq No. NM_001003308) etc.), and their splice variants, allelic variants, Examples include polymorphic mRNA. As a preferable example of mRNA of gene G17 (STXBP1), human STXBP1 (RefSeq Accession No.
  • NM_001032221 containing the base sequence represented by SEQ ID NO: 33, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_009295), rat (RefSeq No. NM_013038), bovine (RefSeq No. NM_174619), etc.), and their splice variants, allelic variants, and polymorphisms.
  • mouse RefSeq No. NM_009295
  • rat RefSeq No. NM_013038
  • bovine RefSeq No. NM_174619
  • NM_008083 rat (RefSeq No. NM_017195), cattle (RefSeq No. NM_203358), chimpanzee (RefSeq No. NM_001009816), dog (RefSeq No. XM_535747), etc.), and their splice variants, allelic variants, Examples include polymorphic mRNA.
  • mRNA of gene G19 ATP1B1
  • human ATP1B1 RefSeq Accession No. NM_001677 containing the base sequence represented by SEQ ID NO: 37, or their orthologs in other mammals (for example, mouse (RefSeq No.
  • NM_009721 rat (RefSeq No. NM_013113), cattle (RefSeq No. NM_001035334), chimpanzee (RefSeq No. NM_001098555), dog (RefSeq No. NM_001003283) etc.), and their splice variants, allelic variants, Examples include polymorphic mRNA.
  • mRNA of gene G20 MAOB
  • human MAOB RefSeq Accession No. NM_000898 containing the base sequence represented by SEQ ID NO: 39, or an ortholog thereof (for example, mouse (RefSeq) in other mammals) No.
  • mRNA of the gene G21 include human CHRM1 (RefSeq Accession No. NM_000738) containing the nucleotide sequence represented by SEQ ID NO: 41, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_007698), rat (RefSeq No.
  • NM_080773 cattle (RefSeq No. XM_001255657), chimpanzee (RefSeq No. XM_001159938), dog (RefSeq No. XM_540897), etc.), and their splice variants, allelic variants, Examples include polymorphic mRNA.
  • mRNA of gene G22 SLC7A14
  • human SLC7A14 RefSeq Accession No. NM_020949
  • containing the base sequence represented by SEQ ID NO: 43 or their orthologs in other mammals (for example, mouse (RefSeq No. NM_172861), rat (RefSeq No.
  • NM_001134615 chimpanzee
  • chimpanzee RefSeq No. XM_526378
  • dog RefSeq No. XM_545285
  • mRNA of the gene G23 include human NDUFS7 (RefSeq Accession No. NM_024407) containing the nucleotide sequence represented by SEQ ID NO: 45, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_029272), rat (RefSeq No. NM_001008525), cattle (RefSeq No.
  • NM_001038022 dog (RefSeq No. XM_533960), etc.) and their splice variants, allelic variants, and polymorphisms .
  • GPRIN1 human GPRIN1 (RefSeq Accession No. NM_052899) containing the base sequence represented by SEQ ID NO: 47, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_012014), rat (RefSeq No. XM_344571), etc., and their splice variants, allelic variants, and polymorphisms.
  • mRNA of gene G25 ATP8A1
  • human ATP8A1 (RefSeq Accession No. NM_001105529) containing the base sequence represented by SEQ ID NO: 49, or an ortholog thereof (for example, mouse (RefSeq) in other mammals) No. NM_001038999), cattle (RefSeq No. NM_174838), chimpanzee (RefSeq No. XM_001150768), dogs (RefSeq No. XM_844264), etc.) and their splice variants, allelic variants, polymorphisms and other mRNAs .
  • Part of the base sequence complementary to or substantially complementary to the base sequence of mRNA of gene Gn means that it can specifically bind to the mRNA of gene Gn and translates the protein from the mRNA.
  • the length and the position are not particularly limited as long as they can inhibit (or degrade the mRNA), but at least 10 bases that are complementary or substantially complementary to the target sequence from the viewpoint of sequence specificity. As mentioned above, it contains about 15 bases or more, more preferably about 20 bases or more.
  • the nucleic acid comprising a base sequence complementary to or substantially complementary to the base sequence of the gene Gn mRNA or a part thereof is preferably exemplified by any of the following (a) to (c): Is done.
  • (a) Antisense nucleic acid for gene Gn mRNA (b) Ribozyme nucleic acid for gene Gn mRNA (c) a nucleic acid having RNAi activity against the mRNA of gene Gn or a precursor thereof
  • the “antisense nucleic acid against mRNA of gene Gn” includes a base sequence complementary to or substantially complementary to the base sequence of the mRNA or a part thereof. It is a nucleic acid and has a function of suppressing protein synthesis by forming a specific and stable duplex with a target mRNA.
  • Antisense nucleic acids are polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, Other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence specific nucleic acid polymers) or other polymers containing special linkages, provided that the polymer is a base such as found in DNA or RNA And a nucleotide having a configuration that allows attachment of a base).
  • RNA double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, unmodified polynucleotides (or unmodified oligonucleotides), known modifications Additions, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotide modifications Such as those having uncharged bonds (eg methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.) Things such as proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-rigid Having a side chain group such as sugar (eg, monosaccharide), having an intercurrent compound (eg, acridine, psoralen
  • nucleoside may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also be modified at the sugar moiety, eg, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may have been converted.
  • the antisense nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera.
  • the RNA DNA hybrid formed by the target RNA and the antisense DNA can be recognized by endogenous RNase H and cause selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of gene Gn.
  • the intron sequence can be determined by comparing the genome sequence and the cDNA base sequence of the gene Gn using a homology search program such as BLAST or FASTA.
  • the target region of the antisense nucleic acid of the present invention is not particularly limited in length as long as the antisense nucleic acid hybridizes, and as a result, translation into protein Pn is inhibited.
  • the entire sequence or partial sequence of mRNA may be a short sequence of about 10 bases, and a long sequence of mRNA or the initial transcript.
  • an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferred, but is not limited thereto.
  • 5 'end hairpin loop of gene Gn may be selected as a preferred target region of the antisense nucleic acid, but is not limited thereto.
  • the antisense nucleic acid of the present invention not only hybridizes with the mRNA of the gene Gn and the initial transcription product to inhibit translation into proteins, but also binds to these genes that are double-stranded DNA to form triple strands ( A triplex) that can inhibit transcription to RNA (antigene).
  • the nucleotide molecule constituting the antisense nucleic acid may be natural DNA or RNA, but various chemicals may be used to improve stability (chemical and / or enzyme) and specific activity (affinity with RNA). Modifications can be included.
  • the phosphate residue (phosphate) of each nucleotide constituting the antisense nucleic acid is chemically modified, for example, phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. It can be substituted with a phosphate residue.
  • PS phosphorothioate
  • methylphosphonate methylphosphonate
  • phosphorodithionate etc. It can be substituted with a phosphate residue.
  • the 2′-position hydroxyl group of the sugar (ribose) of each nucleotide is represented by —OR (R ⁇ CH 3 (2′-O-Me), CH 2 CH 2 OCH 3 (2′-O-MOE), CH 2 CH 2 NHC (NH) NH 2 , CH 2 CONHCH 3 , CH 2 CH 2 CN, etc.) may be substituted.
  • the base moiety pyrimidine, purine
  • BNA LNA
  • ENA ENA
  • the antisense oligonucleotide of the present invention determines the target sequence of mRNA or initial transcript based on the cDNA sequence or genomic DNA sequence of gene Gn, and is a commercially available DNA / RNA automatic synthesizer (Applied Biosystems, Beckman) Etc.) can be prepared by synthesizing a complementary sequence thereto.
  • any of the above-described antisense nucleic acids containing various modifications can be chemically synthesized by a method known per se.
  • Ribozyme nucleic acid for gene Gn mRNA As another preferred example of a nucleic acid comprising a base sequence complementary to or substantially complementary to the base sequence of the gene Gn mRNA or a part thereof, the mRNA is used in the coding region. Examples include ribozyme nucleic acids that can be cleaved specifically inside. “Ribozyme”, in a narrow sense, refers to RNA having an enzyme activity for cleaving nucleic acids, but in this specification, it is used as a concept including DNA as long as it has sequence-specific nucleic acid cleaving activity.
  • the most versatile ribozyme nucleic acids include self-splicing RNAs found in infectious RNAs such as viroids and virusoids, and hammerhead and hairpin types are known.
  • the hammerhead type exhibits enzyme activity at about 40 bases, and a few bases at both ends (about 10 bases in total) adjacent to the part that takes the hammerhead structure are made complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA.
  • This type of ribozyme nucleic acid has the additional advantage of not attacking genomic DNA because it uses only RNA as a substrate.
  • the target sequence is made single-stranded by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase.
  • a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)].
  • ribozymes are used in the form of expression vectors containing the DNA that encodes them, in order to promote the transfer of transcripts to the cytoplasm, the ribozyme should be a hybrid ribozyme further linked to a tRNA-modified sequence. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].
  • siRNA siRNA or miRNA for gene Gn mRNA
  • a double-stranded RNA consisting of an oligo RNA complementary to the mRNA of gene Gn and its complementary strand, so-called siRNA, is also complementary or substantially complementary to the base sequence of the gene Gn mRNA. Defined as encompassed by nucleic acid containing base sequence or part thereof.
  • RNAi RNA interference
  • So-called RNA interference (RNAi) has long been known in nematodes, insects, plants, etc. Since this phenomenon has been confirmed to occur widely in animal cells [Nature, 411 (6836): 494-498 (2001)], it has been widely used as an alternative to ribozyme.
  • siRNA can be designed according to the rules proposed by Elbashir et al. (Genes Dev., 15, 188-200 (2001)) based on the cDNA sequence information of the target gene.
  • Examples of the target sequence of siRNA include, but are not limited to, AA + (N) 19 , AA + (N) 21 or NA + (N) 21 (N is an arbitrary base).
  • the position of the target sequence is not particularly limited.
  • the selected candidate target sequence group whether or not there is homology in the 16-17 base sequence in the mRNA other than the target is determined by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ ) And the like, and the specificity of the selected target sequence is confirmed.
  • AA + (N) 19 AA + (N) 21 or NA + (N) 21 (N is an arbitrary base) is used as the target sequence
  • the target sequence whose specificity has been confirmed is AA (or NA) or later.
  • Two strands consisting of a sense strand having a TT or UU 3 'end overhang at 19-21 bases and an antisense strand having a sequence complementary to the 19-21 base and a TT or UU 3' end overhang Strand RNA may be designed as siRNA.
  • siRNA short hairpin RNA
  • an arbitrary linker sequence for example, about 5-25 bases
  • the sense strand and the antisense strand are combined with the short strand. It can be designed by linking via a linker sequence.
  • siRNA Target Finder http://www.ambion.com/jp/techlib/misc/siRNA_finder.html
  • insert design tool for pSilencer TM Expression Vector http: / /www.ambion.com/techlib/misc/psilencer_converter.html
  • GeneSeer provided by RNAi Codex (http://codex.cshl.edu/scripts/newsearchhairpin.cgi) .
  • the ribonucleoside molecule constituting siRNA may also be modified in the same manner as in the above-described antisense nucleic acid in order to improve stability, specific activity and the like.
  • the siRNA is synthesized by synthesizing a sense strand and an antisense strand of a target sequence on mRNA with a DNA / RNA automatic synthesizer, denatured at about 90 to about 95 ° C. for about 1 minute in an appropriate annealing buffer, It can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. Alternatively, it can be prepared by synthesizing a single hairpin RNA (shRNA) serving as a siRNA precursor and cleaving it with a dicer.
  • shRNA single hairpin RNA
  • a nucleic acid designed to generate siRNA for the gene Gn mRNA in vivo also refers to a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the gene Gn mRNA or one of the nucleotide sequences. Defined as encompassed by a nucleic acid containing a moiety. Examples of such nucleic acids include expression vectors constructed so as to express the above-mentioned shRNA and siRNA.
  • shRNA is an oligo containing a base sequence in which the sense strand and the antisense strand of the target sequence on mRNA are linked by inserting a spacer sequence (for example, about 5 to 25 bases) long enough to form an appropriate loop structure.
  • Vectors expressing shRNA include tandem type and stem loop (hairpin) type.
  • the former is a tandem-linked siRNA sense cassette and antisense strand expression cassette. Each strand is expressed in a cell and annealed to form a double-stranded siRNA (dsRNA). It is.
  • dsRNA double-stranded siRNA
  • the latter is one in which an shRNA expression cassette is inserted into a vector, in which shRNA is expressed in cells and processed by dicer to form dsRNA.
  • a pol II promoter for example, a CMV immediate early promoter
  • a pol III promoter is generally used.
  • the polIII promoter include mouse and human U6-snRNA promoter, human H1-RNase P RNA promoter, human valine-tRNA promoter, and the like.
  • a sequence in which 4 or more Ts are continuous is used as a transcription termination signal.
  • the siRNA or shRNA expression cassette thus constructed is then inserted into a plasmid vector or viral vector.
  • vectors include retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, Sendai virus and other viral vectors, animal cell expression plasmids, and the like.
  • Nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of mRNA of gene Gn or a part thereof is provided in a special form such as a liposome or a microsphere, applied to gene therapy, It can be given in an added form.
  • the additional form includes polycationic substances such as polylysine that acts to neutralize the charge of the phosphate group skeleton, lipids that enhance interaction with cell membranes and increase nucleic acid uptake ( Examples include hydrophobic ones such as phospholipid and cholesterol.
  • Preferred lipids for addition include cholesterol and derivatives thereof (eg, cholesteryl chloroformate, cholic acid, etc.).
  • nucleic acids can be attached via bases, sugars, intramolecular nucleoside linkages.
  • examples of the other group include a cap group specifically arranged at the 3 'end or 5' end of a nucleic acid, which prevents degradation by nucleases such as exonuclease and RNase.
  • capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
  • the protein Pn expression inhibitory activity of these nucleic acids can be examined using a transformant into which the gene Gn has been introduced, a gene Gn expression system in vivo or in vitro, or a protein Pn translation system in vivo or in vitro.
  • the substance that suppresses the expression of protein Pn in the present invention is not limited to a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of the mRNA of gene Gn as described above or a part thereof, but includes protein Pn Other substances such as low molecular weight compounds may be used as long as they directly or indirectly inhibit the production of.
  • the substance may be one that suppresses the expression of any one protein selected from proteins P1 to P25, or may be one that can suppress the expression of two or more proteins. Such a substance can be obtained, for example, by the screening method of the present invention described later.
  • the “substance that suppresses the function of protein Pn” may be any substance as long as the protein Pn once functionally produced suppresses the promotion of A ⁇ production. For example, it binds to protein Pn.
  • a substance that suppresses A ⁇ production a substance that inhibits the binding activity between protein Pn and ⁇ -secretase (for example, a substance that inhibits or modulates the promotion of ⁇ -secretase activity due to the binding between protein Pn and ⁇ -secretase) And substances that inhibit the binding or complex formation of protein Pn and ⁇ -secretase), substances that inhibit the transfer of protein Pn to the cell membrane, and the like.
  • examples of the substance that suppresses the function of protein Pn include an antibody against protein Pn.
  • the antibody may be a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to per se known methods for producing antibodies or antisera.
  • the isotype of the antibody is not particularly limited, but preferably IgG, IgM or IgA, particularly preferably IgG.
  • the antibody is not particularly limited as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding a target antigen.
  • CDR complementarity determining region
  • Fab, Fab ′, F (ab ') 2 such as fragments, scFv, scFv-Fc, conjugation molecules prepared by genetic engineering such as minibodies and diabodies, or molecules having a protein stabilizing action such as polyethylene glycol (PEG)
  • PEG polyethylene glycol
  • the antibody preferably a monoclonal antibody
  • the antibody has a reduced risk of being antigenic when administered to a human.
  • Antibodies specifically, fully human antibodies, humanized antibodies, mouse-human chimeric antibodies, etc., particularly preferably fully human antibodies.
  • Humanized antibodies and chimeric antibodies can be produced by genetic engineering according to a conventional method.
  • fully human antibodies can be produced from human-human (or mouse) hybridomas, but in order to provide a large amount of antibodies stably and at low cost, human antibody-producing mice and phage display methods are used. It is desirable to manufacture using.
  • a more preferable substance that suppresses the function of protein Pn is a low-molecular compound suitable for Lipinski's Rule.
  • the low molecular weight compound may be one that suppresses the function of any one protein selected from the proteins P1 to P25, and may be one that can suppress the function of two or more proteins. Such a compound can be obtained, for example, using the screening method of the present invention described later.
  • a substance that suppresses the expression or function of protein Pn suppresses A ⁇ production, and thus is useful for improving the pathology of Alzheimer's disease (AD) or cancer.
  • the substance since the substance may not inhibit the cleavage of Notch protein, the substance has a further advantageous effect that it has a low risk of causing peripheral side effects such as intestinal epithelialization disorder and immune abnormality. Therefore, drugs containing substances that suppress the expression or function of protein Pn are ⁇ -secretase inhibitors or modulators, A ⁇ production inhibitors, diseases involving A ⁇ (eg, AD), and diseases involving Notch protein cleavage. It can be used as a preventive and / or therapeutic agent for (eg, cancer).
  • Only one type of substance that suppresses the expression or function of protein Pn may be used, or two or more types may be used in combination.
  • these substances may suppress the expression or function of the same protein, or two or more kinds of substances may be suppressed. It may be one that suppresses the expression or function of different proteins.
  • two or more substances that suppress the expression or function of protein Pn may be formulated as separate medicaments, or may be blended in the same pharmaceutical composition.
  • the respective preparations may be administered simultaneously or at intervals.
  • the administration route may be the same or different.
  • the dose described below indicates the dose of a single substance that suppresses the expression or function of protein Pn. However, even when two or more substances are used in combination, the dose does not adversely affect the administration subject. Similar dosages can be used for each substance.
  • An antisense nucleic acid of the present invention capable of binding to a transcription product of a gene Gn complementary to an antisense nucleic acid, a ribozyme nucleic acid, siRNA and a precursor thereof, and inhibiting protein translation from the transcription product SiRNAs (or ribozymes) that can cleave the transcripts of nucleic acids and gene Gn transcripts that are homologous (or complementary), and shRNA that is the precursor of the siRNA (hereinafter, comprehensive) May be referred to as “the nucleic acid of the present invention”, an agent for inhibiting and / or treating an A ⁇ production inhibitor, a disease involving A ⁇ (eg, AD) and a disease involving cleavage of Notch protein (eg, cancer) Can be used as The pharmaceutical containing the nucleic acid of the present invention is used as it is, or as a pharmaceutical composition of an appropriate dosage form, as a human or non-human mammal (eg, rat, rabbit, sheep, pig, cow, cat,
  • the nucleic acid of the present invention When used as an A ⁇ production inhibitor or a preventive and / or therapeutic agent for a disease involving A ⁇ , it can be formulated and administered according to a method known per se. That is, the nucleic acid of the present invention may be used alone or inserted in a functional manner into an appropriate expression vector for mammalian cells such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector. You can also.
  • the nucleic acid can be administered as it is or together with an auxiliary agent for promoting intake by a catheter such as a gene gun or a hydrogel catheter. Alternatively, it can be aerosolized and locally administered into the trachea as an inhalant.
  • the nucleic acid may be formulated (injection) alone or with a carrier such as liposome and administered intravenously, subcutaneously, etc. .
  • the nucleic acid of the present invention may be administered per se or as an appropriate pharmaceutical composition.
  • the pharmaceutical composition used for administration may contain the nucleic acid of the present invention and a pharmacologically acceptable carrier, diluent or excipient.
  • Such pharmaceutical compositions are provided as dosage forms suitable for oral or parenteral administration.
  • composition for parenteral administration for example, injection, suppository, intranasal administration, etc. are used, and the injection is intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip injection. You may include dosage forms, such as an agent.
  • Such an injection can be prepared according to a known method. As a method for preparing an injection, it can be prepared, for example, by dissolving, suspending or emulsifying the nucleic acid of the present invention in a sterile aqueous liquid or oily liquid usually used for injection.
  • an aqueous solution for injection for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)) and the like may be used in combination.
  • alcohol eg, ethanol
  • polyalcohol eg, Propylene glycol, polyethylene glycol
  • nonionic surfactants eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)
  • oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solub
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like.
  • Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field.
  • a carrier and excipient for tablets for example, lactose, starch, sucrose, and magnesium stearate are used.
  • the above parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dose of the active ingredient.
  • dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), and suppositories.
  • the nucleic acid of the present invention is preferably contained, for example, usually in an amount of about 0.01 to 500 mg per dosage unit dosage form.
  • the dosage of the above-mentioned medicament containing the nucleic acid of the present invention varies depending on the administration subject, target disease, symptom, administration route, etc., but for example, when used for treatment / prevention of AD, the nucleic acid of the present invention It is convenient to administer about 0.0001 to 20 mg / kg body weight by intravenous injection once a day to about 6 months. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.
  • compositions may contain other active ingredients as long as an undesirable interaction is not caused by blending with the nucleic acid of the present invention.
  • active ingredients include AD therapeutic agents typified by acetylcholinesterase inhibitors, ⁇ -secretase modulators such as nonsteroidal anti-inflammatory drugs (NSAIDs), ⁇ -secretase inhibitors, ⁇ -secretase inhibitors (eg, Statin that can function as a transition state mimic, hydroxyethylene, peptidic inhibitor with hydroxyethylcarbonyl structure (J. Med. Chem., 46 (22), 4625-4630 ⁇ ⁇ ⁇ ⁇ (2003)), non-peptidic small molecule Type inhibitors (WO 01/87293, WO 02/88101, WO 02/96897)) and the like.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • ⁇ -secretase inhibitors eg, Statin that can function as a transition state mimic, hydroxyethylene, peptidic inhibitor with hydroxyethylcarbonyl structure
  • An antibody against protein Pn, or a low molecular compound that suppresses the expression or function of protein Pn The activity can be inhibited, or the interaction (complex formation) between protein Pn and ⁇ -secretase can be inhibited. Therefore, these substances suppress the expression or function of protein Pn in vivo and inhibit ⁇ -secretase activity. Therefore, A ⁇ production inhibitors, diseases involving A ⁇ (eg, AD) and Notch protein cleavage It can be used as a preventive and / or therapeutic agent for a disease involved (eg, cancer).
  • the medicament containing the above antibody or low molecular weight compound can be used as a liquid or as a pharmaceutical composition of an appropriate dosage form as a human or mammal (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey). Etc.) orally or parenterally (eg, intravascular administration, subcutaneous administration, etc.).
  • a human or mammal eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey.
  • Etc. orally or parenterally (eg, intravascular administration, subcutaneous administration, etc.).
  • the above-described antibodies and low-molecular compounds may be administered per se, or may be administered as an appropriate pharmaceutical composition.
  • the pharmaceutical composition used for administration may contain the above antibody or low molecular compound or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient.
  • Such pharmaceutical compositions are provided as dosage forms suitable for oral or parenteral administration.
  • composition for parenteral administration for example, injection, suppository, intranasal administration, etc. are used, and the injection is intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip injection. You may include dosage forms, such as an agent.
  • Such an injection can be prepared according to a known method. As a method for preparing an injection, it can be prepared by, for example, dissolving, suspending or emulsifying the antibody or low molecular compound of the present invention or a salt thereof in a sterile aqueous liquid or oily liquid usually used for injection.
  • an aqueous solution for injection for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)) and the like may be used in combination.
  • alcohol eg, ethanol
  • polyalcohol eg, Propylene glycol, polyethylene glycol
  • nonionic surfactants eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)
  • oily liquid for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solub
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like.
  • Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field.
  • a carrier and excipient for tablets for example, lactose, starch, sucrose, and magnesium stearate are used.
  • the above parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dose of the active ingredient.
  • dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), and suppositories.
  • the antibody or low molecular weight compound is preferably contained in an amount of usually 0.1 to 500 mg, particularly 5 to 100 mg for injections and 10 to 250 mg for other dosage forms per dosage unit form.
  • the dose of the above-mentioned medicament containing the above-mentioned antibody or low-molecular compound or a salt thereof varies depending on the administration subject, target disease, symptom, administration route, etc., but for example, when used for the treatment / prevention of AD Is usually about 0.0001 to 20 mg / kg body weight for a single dose of antibody or low molecular weight compound, about 1 to 5 times a day for low molecular weight compound, orally or parenterally, 1 to several days for antibody Conveniently administered once a month by intravenous injection. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.
  • compositions may contain other active ingredients as long as an undesirable interaction is not caused by blending with the above-described antibody or low-molecular compound.
  • active ingredients include, for example, AD therapeutic agents typified by acetylcholinesterase inhibitors, ⁇ -secretase modulators such as nonsteroidal anti-inflammatory drugs (NSAIDs), ⁇ -secretase inhibitors, ⁇ -secretase inhibitors (eg, Statin that can function as a transition state mimic, hydroxyethylene, peptidic inhibitor with hydroxyethylcarbonyl structure (J. Med. Chem., 46 (22), 4625-4630 ⁇ (2003)), non-peptidic small molecule Type inhibitors (WO 01/87293, WO 02/88101, WO 02/96897)) and the like.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • ⁇ -secretase inhibitors eg, Statin that can function as a transition state mimic, hydroxyethylene, peptidic inhibitor with hydroxyethylcarbony
  • a compound or a salt thereof that suppresses the expression and / or function of protein Pn is an A ⁇ production inhibitor, a disease involving A ⁇ (eg, AD, etc.) or a disease involving cleavage of Notch protein (eg, cancer). It can be used as a prophylactic and / or therapeutic agent. Therefore, protein Pn or a partial peptide thereof, or a cell producing the same can be used as a tool for screening a substance having an inhibitory effect on A ⁇ production by using the expression level and / or activity of the protein (gene) as an index. Can be used.
  • Screening method (I) When screening for a compound or a salt thereof that suppresses the function of protein Pn, the screening method is used in the presence and absence of a test substance for protein Pn or a partial peptide thereof or a cell capable of producing the same. Incubating (culturing) and comparing the activity of protein Pn under both conditions.
  • Cells having the ability to produce the protein Pn used in the above screening methods include human or other mammalian cells that naturally express them or biological samples containing them (eg, blood, tissues, organs, etc.) If there is no particular limitation. In the case of blood, tissues, organs, etc. derived from non-human animals, they may be isolated from the living body and cultured, or the test substance is administered to the living body itself, and these biological samples are isolated after a certain period of time. May be. Examples of cells having the ability to produce protein Pn or a partial peptide thereof include various transformants prepared by known and commonly used genetic engineering techniques. As the host, for example, animal cells such as H4IIE-C3 cells, HepG2 cells, HEK293 cells, COS7 cells, CHO cells are preferably used.
  • a base sequence represented by DNA encoding protein Pn or a partial peptide thereof that is, SEQ ID NO: 2n-1 (n is any integer of 1 to 25) or a complementary strand of the base sequence
  • a DNA containing a base sequence encoding a polypeptide that hybridizes under stringent conditions and has the same quality of activity as the protein consisting of the amino acid sequence represented by SEQ ID NO: 2n) in a promoter in an appropriate expression vector And can be prepared by introducing it into a host animal cell.
  • a DNA encoding the protein Pn or a partial peptide thereof is synthesized, for example, based on the base sequence represented by SEQ ID NO: 2n-1, using a suitable oligonucleotide as a probe or primer, and a cell that produces the protein Pn described above -It can be cloned from tissue-derived cDNA or cDNA library using hybridization or PCR.
  • Hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.
  • the cloned DNA is obtained using a known kit such as Mutan TM -super Express Km (Takara Shuzo), Mutan TM -K (Takara Shuzo), etc.
  • the nucleotide sequence can be converted according to a method known per se such as the Kunkel method or a method analogous thereto.
  • the cloned DNA can be used as it is depending on the purpose, or after digestion with a restriction enzyme or addition of a linker as desired.
  • the DNA may have ATG as a translation initiation codon on the 5 'end side and TAA, TGA or TAG as a translation termination codon on the 3' end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
  • Expression vectors include animal cell expression plasmids (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo); bacteriophages such as ⁇ phage; animal viruses such as retrovirus, vaccinia virus, adenovirus A vector or the like is used.
  • the promoter may be any promoter as long as it is appropriate for the host used for gene expression.
  • SR ⁇ promoter For example, SR ⁇ promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter It is done. Of these, CMV promoter, SR ⁇ promoter and the like are preferable.
  • an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selectable marker, an SV40 origin of replication (hereinafter sometimes abbreviated as SV40 ori), etc. is used as desired. Can do.
  • Selectable markers include, for example, dihydrofolate reductase gene (hereinafter abbreviated as dhfr, methotrexate (MTX) resistance), ampicillin resistance gene (hereinafter abbreviated as amp r ), neomycin resistance gene ( hereinafter sometimes abbreviated as neo r, include G418 resistance) and the like.
  • dhfr dihydrofolate reductase gene
  • amp r ampicillin resistance gene
  • neomycin resistance gene hereinafter sometimes abbreviated as neo r, include G418 resistance
  • the target gene can also be selected using a medium that does not contain thymidine.
  • Protein Pn-expressing cells can be produced by transforming a host with an expression vector containing the DNA encoding the protein Pn described above.
  • Hosts include mammalian cells such as HepG2 cells, HEK293 cells, HeLa cells, human FL cells, monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary cells (hereinafter abbreviated as CHO cells), dhfr gene-deficient CHO Cells (hereinafter abbreviated as CHO (dhfr ⁇ ) cells), mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat H4IIE-C3 cells, rat GH3 cells and the like can be used.
  • mammalian cells such as HepG2 cells, HEK293 cells, HeLa cells, human FL cells, monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary cells (hereinafter abbreviated as CHO cells), dhfr gene-deficient CHO Cells (
  • Transformation can be performed by calcium phosphate coprecipitation method, PEG method, electroporation method, microinjection method, lipofection method and the like.
  • PEG method New Cell Engineering Experiment Protocol
  • electroporation method microinjection method
  • lipofection method lipofection method and the like.
  • the methods described in Cell Engineering Supplement 8, New Cell Engineering Experiment Protocol, 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973) can be used.
  • mammalian cells having the ability to produce the native protein Pn, or tissues / organs containing the cells are, for example, a minimum essential medium containing about 5 to 20% fetal calf serum ( MEM) [Science, 122, 501 (1952)], Dulbecco's Modified Eagle Medium (DMEM) [Virology, 8, 396 (1959)], RPMI 1640 Medium [The Journal of the American Medical Association, 199,519 ( 1967)], 199 medium [Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)].
  • MEM fetal calf serum
  • DMEM Dulbecco's Modified Eagle Medium
  • RPMI 1640 Medium RPMI 1640 Medium
  • the pH of the medium is preferably about 6-8. Cultivation is usually carried out at about 30-40 ° C, with aeration and agitation as necessary.
  • Protein Pn or a partial peptide thereof may be the aforementioned transformed cells (or non-mammalian cells obtained by introducing the gene Gn into bacteria such as Escherichia coli and Bacillus subtilis, yeast and insect cells), or the native protein Pn. It can be separated and purified from a culture obtained by culturing mammalian cells having the ability to produce according to a method known per se.
  • the cells or cells collected from the culture by a known method are suspended in an appropriate buffer, and are subjected to ultrasound, lysozyme and / or
  • a method of obtaining a crude extract of soluble protein by centrifugation or filtration after disrupting cells or cells by freeze-thawing or the like is appropriately used.
  • extracting protein Pn or its partial peptide from the membrane fraction after disrupting the cells or cells in the same manner as described above, cell debris is precipitated and removed by low-speed centrifugation, and the supernatant is centrifuged at high speed to obtain a fraction containing cell membrane.
  • the cell membrane fraction is purified by density gradient centrifugation or the like.
  • methods such as fractionating a culture supernatant from a culture by centrifugation or filtration, are used. Isolation and purification of the protein Pn or its partial peptide contained in the thus obtained soluble fraction, membrane fraction or culture supernatant can be performed according to a method known per se. Examples of such methods include the use of solubilities such as salting out and solvent precipitation; mainly the differences in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
  • a method utilizing a difference in charge such as ion exchange chromatography; a method utilizing a specific affinity such as affinity chromatography; a method utilizing a difference in hydrophobicity such as reverse phase high performance liquid chromatography; A method using a difference in isoelectric point such as point electrophoresis is used. These methods can be combined as appropriate.
  • the free form can be converted into a salt by a method known per se or a method analogous thereto, and when the protein or peptide is obtained as a salt, The salt can be converted into a free form or other salt by a method known per se or a method analogous thereto.
  • the protein Pn produced by the transformed cell or its partial peptide can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification.
  • the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.
  • protein Pn or a partial peptide thereof can be synthesized in vitro using a cell-free protein translation system comprising rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate, etc., using the RNA encoding it as a template.
  • a cell-free transcription / translation system further containing RNA polymerase can be used to synthesize DNA encoding the protein Pn or a partial peptide thereof as a template.
  • any one protein selected from (P1) to (P25) may be used, or two or more proteins may be combined. It may be used.
  • two or more types of proteins are provided in the form of cells, it is preferable to use one type of cell that produces all of the two or more types of proteins.
  • test substances include proteins, peptides, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and these substances are novel. Alternatively, a known one may be used.
  • an A ⁇ production system Preferably coexist.
  • the A ⁇ production system cells expressing APP, which is a precursor of A ⁇ , are preferably used.
  • protein Pn or its partial peptide is provided as a cell which has the capability to produce it, it is preferable that the cell itself expresses APP.
  • the contact of the test substance with the cells may be performed by, for example, the above-mentioned medium or various buffers (for example, HEPES buffer, phosphate buffer, phosphate buffered saline, Tris-HCl buffer, borate buffer, acetic acid).
  • the test substance can be added to a buffer solution or the like, and the cells can be incubated for a certain time.
  • concentration of the test substance to be added varies depending on the type of compound (solubility, toxicity, etc.), but is appropriately selected within the range of about 0.1 nM to about 100 nM, for example. Examples of the incubation time include about 10 minutes to about 24 hours.
  • the contact between the protein or peptide and the test substance is performed by adding both to the culture medium of the cell expressing APP and incubating the cell for a certain period of time.
  • the concentration of the protein Pn or a partial peptide to be added is appropriately selected for each protein or peptide, for example, in the range of about 0.1 to about 100 ⁇ g / ml.
  • cells that produce protein Pn are provided in the form of a non-human mammal individual, an animal that naturally expresses protein Pn may be used, or a gene that incorporates gene Gn so that Pn is overexpressed Recombinant animals (transgenic (Tg) animals) may be used.
  • Tg transgenic
  • a knock-in animal in which the endogenous gene Gn of the animal is replaced with the human gene Gn can also be used.
  • cells expressing APP are inherent in the animal individual.
  • the state of the individual animal is not particularly limited, and may be an AD model animal such as Tg2576 mouse or APP23 Tg mouse.
  • the administration route is not particularly limited, and examples thereof include intravenous administration, intraarterial administration, subcutaneous administration, intradermal administration, intraperitoneal administration, oral administration, intratracheal administration, rectal administration, intranasal administration, intraventricular administration and the like.
  • a dose of about 0.5 to 20 mg / kg can be administered 1 to 5 times a day for about 1 to 6 months.
  • the protein Pn activity in the above screening method can be measured by measuring the production amount of A ⁇ by a method known per se.
  • Test samples for measuring activity include cultured cells that express APP (including tissue culture and organ culture), protein Pn or its partial peptide, or cells that have the ability to produce it (cells that express APP).
  • APP tissue culture and organ culture
  • cells that have the ability to produce it cells that express APP.
  • the test substance is administered to a non-human mammal individual in which the culture supernatant of the cultured cells produces the proteins Pn and APP
  • Examples thereof include bodily fluids collected from the individual, such as blood, cerebrospinal fluid, and the like, or cell, tissue, and organ extracts, such as the brain or homogenates of tissue sections thereof.
  • the production amount of A ⁇ is the amount of A ⁇ (eg, A ⁇ 38, A ⁇ 40, A ⁇ 42, etc., preferably A ⁇ 40 and / or A ⁇ 42) in the test sample, for example, various immunological methods such as ELISA using anti-A ⁇ antibody and Western blotting. It can be performed by measuring using a technique, mass spectrometry or the like.
  • the activity of protein Pn in the presence of the test substance is, for example, about 10% or more compared to the activity in the absence of the test substance
  • the substance is inhibited, preferably about 20% or more, more preferably 30% or more, and even more preferably about 50% or more
  • the test substance is treated as a protein Pn function inhibitory substance, and thus a substance having an A ⁇ production inhibitory action. Can be selected as a candidate.
  • Screening method (Ia) in a non-human animal into which at least one gene selected from (G1) to (G25) has been introduced, screening for a therapeutic or prophylactic agent for AD using an improvement in phenotype reflecting the pathological condition of AD as an index, The efficacy of the drug obtained by the above screening method or other screening methods against AD can be evaluated. Specifically, the method includes the following steps (a) to (c).
  • the method can be carried out in the same manner as in the case of administering the test substance to a non-human mammal individual producing the proteins Pn and APP.
  • phenotypes reflecting the pathology of AD include, for example, (1) A ⁇ amount in tissues such as brain tissue, cerebrospinal fluid, blood, (2) neuronal cell death, (3) central nervous system Inflammatory response of the system, (4) reduced cognitive ability, (5) accumulation of amyloid plaques, (6) deterioration of cerebral blood flow, (7) reduction of cerebral glucose metabolism, but not limited thereto .
  • tissues such as brain tissue, cerebrospinal fluid, blood
  • neuronal cell death e.g., central nervous system Inflammatory response of the system
  • reduced cognitive ability e.g., those known per se can be used (for example, as a method for evaluating cognitive ability, a Y-shaped maze test or the like can be mentioned).
  • the activity of protein Pn can be measured using the increased activity of ⁇ -secretase as an indicator.
  • the screening method includes: (A) contacting the protein Pn and ⁇ -secretase with a test substance; (B) measuring ⁇ -secretase activity, and (c) selecting a substance that reduces or modulates ⁇ -secretase activity compared to when measured in the absence of the test substance. .
  • ⁇ -secretase for example, a cell expressing it or a cell membrane fraction thereof can be used.
  • the protein Pn may be provided by a cell that expresses ⁇ -secretase itself.
  • ⁇ -secretase activity is determined by adding APP or Notch as a substrate, APP metabolite fragments (A ⁇ 38, A ⁇ 40, A ⁇ 42, AICD, etc.) or Notch metabolites (NICD), ELISA, Western blotting, reporter gene assay It can be measured by detecting using mass spectrometry or the like.
  • the ⁇ -secretase activity in the presence of the test substance is about 10% or more, preferably about 20% or more, more preferably 30%, compared to the activity in the absence of the test substance. %, More preferably about 50% or more, the test substance can be selected as a protein Pn function-inhibiting substance, and thus a candidate for an A ⁇ production-inhibiting substance.
  • a control a cell or an animal in which the gene Gn is knocked out or knocked down is used as a control, and in the presence of a test substance in the test cell or animal and the control.
  • An A ⁇ production inhibitor can also be selected by measuring and comparing the activity of the protein Pn.
  • the present invention also relates to a method for screening an A ⁇ production inhibitor, comprising comparing the expression of the protein (gene) in a cell having the ability to produce protein Pn in the presence and absence of the test substance.
  • the cells used in this method, the type of test substance, the mode of contact between the test substance and the cells, and the like are the same as in the method using the activity of the protein Pn as an index.
  • the expression level of protein Pn is a nucleic acid that can hybridize with the above-described DNA encoding protein Pn under highly stringent conditions, that is, the nucleotide sequence represented by SEQ ID NO: n or a complementary nucleotide sequence and string It can be measured at the RNA level by detecting the mRNA of the gene Gn using a nucleic acid that can hybridize under gentle conditions (hereinafter sometimes referred to as “the nucleic acid for detection of the present invention”). Alternatively, the expression level can be measured at the protein level by detecting these proteins using an antibody against the above-described protein Pn (hereinafter sometimes referred to as “the detection antibody of the present invention”). .
  • the present invention (A) Cells having the ability to produce protein Pn are cultured in the presence and absence of a test substance, and the amount of mRNA encoding the protein under both conditions is determined using the nucleic acid for detection of the present invention.
  • a method for screening an A ⁇ production inhibitor characterized by measuring and comparing, and (b) culturing cells capable of producing protein Pn in the presence and absence of a test substance, under both conditions
  • Provided is a method for screening an A ⁇ production inhibitor, characterized in that the amount of the protein is measured and compared using the detection antibody of the present invention.
  • the measurement of the amount of mRNA or protein of protein Pn can be specifically performed as follows.
  • blood, a specific organ (for example, brain), or a tissue or cell isolated from the organ is obtained.
  • the mRNA of the protein Pn contained in the obtained cell can be quantified by, for example, extracting mRNA from the cell etc. by a normal method and using, for example, a technique such as RT-PCR, or a known Northern Northern It can also be quantified by blot analysis.
  • the amount of protein Pn can be quantified using Western blot analysis or various immunoassay methods described in detail below.
  • a transformant introduced with the gene Gn is prepared according to the above method, and the protein Pn contained in the transformant or mRNA encoding the same can be quantified and analyzed in the same manner as in (i) above. it can.
  • the measurement of the amount of protein Pn in the screening method of (b) above is, for example, (I) The detection antibody of the present invention, the sample solution and the labeled protein Pn are reacted competitively, and the protein Pn in the sample solution is detected by detecting the labeled protein bound to the antibody. How to quantify, (Ii) The sample solution is reacted with the detection antibody of the present invention insolubilized on the carrier and another labeled detection antibody of the present invention simultaneously or continuously, and then the labeling agent on the insolubilized carrier. For example, a method of quantifying C protein Pn in a sample solution by measuring the amount (activity) of.
  • the two types of antibodies recognize different portions of the protein Pn.
  • the other antibody can react with the C-terminal part of the protein.
  • a labeling agent used in a measurement method using a labeling substance for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used.
  • the radioisotope for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used.
  • the enzyme is preferably stable and has a large specific activity.
  • ⁇ -galactosidase ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • fluorescent material for example, fluorescamine, fluorescein isothiocyanate and the like are used.
  • luminescent substance for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
  • a biotin- (strept) avidin system can be used for binding of an antibody or antigen to a labeling agent.
  • the method for quantifying protein Pn using the detection antibody of the present invention is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the sample solution is chemically or physically determined. Any measurement method may be used as long as it is a measurement method that is detected by a standard means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used. In view of sensitivity and specificity, for example, the sandwich method described later is preferably used.
  • the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
  • a sample solution is reacted with the insolubilized detection antibody of the present invention (primary reaction), and another labeled detection antibody of the present invention is reacted (secondary reaction), and then on the insolubilized carrier.
  • the protein Pn in the sample solution can be quantified by measuring the amount or activity of the labeling agent.
  • the primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at different times.
  • the labeling agent and the insolubilization method can be the same as those described above.
  • the antibody used for the immobilized antibody or the labeled antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity. May be.
  • the detection antibody of the present invention can also be used in measurement systems other than the sandwich method, such as a competitive method, an immunometric method, or nephrometry.
  • a competitive method the protein Pn in the sample solution and the labeled protein Pn are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody. Separation (B / F separation) and the amount of labeling of either B or F is measured to quantify the protein Pn in the sample solution.
  • a soluble antibody is used as an antibody
  • B / F separation is performed using polyethylene glycol or a secondary antibody against the antibody (primary antibody)
  • a solid phase is used as the primary antibody.
  • Either an antibody is used (direct method), or a primary antibody is soluble, and a solid phase antibody is used as a secondary antibody (indirect method).
  • the protein Pn in the sample solution and the immobilized protein Pn are allowed to compete with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the sample solution in the sample solution is separated. After reacting protein Pn with an excess amount of labeled antibody, and then adding immobilized protein Pn to bind unreacted labeled antibody to the solid phase, the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to quantify the amount of antigen in the sample solution.
  • nephrometry the amount of insoluble precipitate produced as a result of antigen-antibody reaction in a gel or solution is measured.
  • Laser nephrometry using laser scattering is preferably used even when the amount of protein Pn in the sample solution is very small and only a small amount of precipitate can be obtained.
  • the expression level of protein Pn (mRNA amount or protein amount) in the presence of the test substance is about 10% less than that in the absence of the test substance.
  • the test substance is inhibited by 10% or more, preferably about 20% or more, more preferably about 30% or more, and even more preferably about 50% or more, the test substance is expressed as a protein Pn expression-suppressing substance, and thus has an effect of inhibiting A ⁇ production.
  • a cell containing a reporter gene under the control of the transcriptional regulatory region in the gene Gn can be used instead of the cell expressing the gene Gn.
  • a cell may be a cell, tissue, organ or individual of a transgenic animal into which a reporter gene (eg, luciferase, GFP, etc.) under the control of the transcriptional regulatory region of gene Gn is introduced.
  • a reporter gene eg, luciferase, GFP, etc.
  • the expression level of protein Pn can be evaluated by measuring the expression level of the reporter gene using a conventional method.
  • a protein or gene in which the gene Gn is knocked out or knocked down is used as a control, and the protein in the presence of the test substance in the test cell or animal and the control
  • An A ⁇ production inhibitor can also be selected by measuring and comparing the expression level of Pn.
  • the present invention also provides a method for screening an A ⁇ production inhibitor comprising the following steps (a) to (c).
  • a step of bringing protein Pn and ⁇ -secretase into contact with a test substance (b) A step of measuring the binding activity between protein Pn and ⁇ -secretase (c) When measured in the absence of the test substance
  • the step of selecting the substance that decreases the binding activity compared to the above may be exemplified by measuring the amount of binding as shown in the following, as an indicator of binding activity, or by measuring the increased activity of ⁇ -secretase (the former Can be performed according to the above screening method (Ib)).
  • the screening method includes (1) Measuring and comparing the amount of labeled protein Pn bound to ⁇ -secretase when the labeled protein Pn and ⁇ -secretase are contacted in the presence and absence of the test substance. Or (2) the labeled protein Pn and the cell or membrane of the labeled protein Pn when the ⁇ -secretase producing cell or its membrane fraction is contacted in the presence and absence of the test substance The amount of binding to the fraction is measured and compared.
  • Protein Pn can be labeled with a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, and the like according to a conventional method.
  • radioisotope for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used.
  • enzyme those which are stable and have high specific activity are preferable.
  • ⁇ -galactosidase, ⁇ -glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used.
  • fluorescent material for example, fluorescamine, fluorescein isothiocyanate and the like are used.
  • luminescent substance for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
  • the amount of label (protein Pn) that binds to ⁇ -secretase in the presence of the test substance is larger than that in the absence of the test substance.
  • the test substance suppresses the binding activity between protein Pn and ⁇ -secretase.
  • the substance can be selected as a candidate of a substance having an A ⁇ production inhibitory action.
  • a substance that suppresses the expression or function of protein Pn obtained by using any one of the screening methods of the present invention is a disease involving A ⁇ (eg, AD) or a disease involving cleavage of Notch protein (eg, cancer). ) Is useful as a preventive and / or therapeutic agent.
  • the substance obtained using the screening method of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated in the same manner as a low molecular weight compound that suppresses the expression or function of the above protein Pn, and the same administration route And administered orally or parenterally to humans or mammals (eg, mice, rats, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.) Can do.
  • mammals eg, mice, rats, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.
  • the present invention relates to A ⁇ -related diseases (eg, AD) or Notch protein cleavage characterized by measuring the expression level of protein Pn in a sample collected from a test animal
  • test animals include humans and other mammals, preferably humans, mice, rats, rabbits, dogs, monkeys and the like that are widely used as experimental animals.
  • the measurement target sample include blood, plasma, serum, cerebrospinal fluid, lymph fluid, saliva, mucous membrane, urine, tears, semen, joint fluid, biopsy sample, and the like.
  • the expression level of gene Gn and the amount of protein Pn in the sample can be measured by the same method as described in the above screening method using the expression level of the gene or the protein as an index.
  • the expression level of the gene Gn or the protein Pn in the sample collected from the test animal is significantly higher than the expression level of the gene Gn or the protein Pn in the sample collected from the normal animal. If the test animal has a disease associated with A ⁇ (eg, AD) or a disease associated with Notch protein cleavage (eg, cancer), or can be determined to have a high risk of developing in the future. .
  • a ⁇ eg, AD
  • Notch protein cleavage eg, cancer
  • the expression level in a normal animal is identified in advance, for example, the average value + 2SD is defined as a cutoff value, and the expression level of gene Gn or the amount of protein Pn in a sample collected from the test animal When the off-value is exceeded, the test animal has developed a disease involving A ⁇ (eg, AD), a disease involving cleavage of Notch protein (eg, cancer), or has a high risk of developing in the future It can also be determined.
  • a ⁇ eg, AD
  • Notch protein eg, cancer
  • an animal having the gene Gn knocked out or knocked down may be used as a control.
  • An animal in which the gene Gn is knocked out or knocked down is also useful as a tool for analyzing the function of the protein Pn.
  • Example 1 Identification of brain ⁇ -secretase binding protein (1)
  • ⁇ -secretase activity is strongly detected in the microsomal fraction, surfactant-insoluble membrane fraction (DRM), synaptic membranes, and synaptic vesicles. Therefore, each of these fractions was prepared, and the ⁇ -secretase complex was purified by an inhibitor pull-down method.
  • DRM surfactant-insoluble membrane fraction
  • synaptic membranes synaptic membranes
  • synaptic vesicles synaptic vesicles. Therefore, each of these fractions was prepared, and the ⁇ -secretase complex was purified by an inhibitor pull-down method.
  • Preparation of fraction containing brain ⁇ -secretase SD rats were obtained from B & K Universal (Sollentuna, Sweden), and human brain tissue was obtained from Huddinge Brain Bank (Huddinge, Sweden).
  • Rat or human brain tissue was homogenized in buffer A (20 mM Hepes, 50 mM KCl, 2 mM EGTA, pH 7.5 plus complete protease inhibitor mixture (Roche Applied Science, Indianapolis, IN, USA)) Thereafter, cell debris and nuclear components were removed by centrifugation at 1,000 xg and 10,000 xg, and then a microsomal fraction was prepared from the precipitate obtained by centrifugation at 100,000 xg. The prepared microsomal fraction was solubilized with buffer A containing 1% (w / v) CHAPSO, and then the solubilized ⁇ -secretase complex was prepared by centrifugation at 100,000 ⁇ g.
  • the DRM fraction was prepared according to the method of Hur et al. (Hur JY et al., FEBS J. (2008) 275: 1174-87). That is, brain tissue was homogenized in Buffer B (20 mM Hepes (pH 7.5), 50 mM KCl, 2 mM EGTA added with complete protease inhibitor mixture), and the microsomal fraction was obtained by centrifugation as described above. Prepared. Resuspend the obtained microsomal fraction in Buffer C (20 mM Tris / HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 2.0% CHAPSO), and prepare the DRM fraction by the sucrose density gradient method. .
  • Synaptic membranes and synaptic vesicles were prepared according to the method of Cohen et al. (Cohen J. et al., Org Chem (2004), 69, 7344-7347). That is, brain tissue was homogenized in Buffer D (0.32 M sucrose, 1 mM NaHCO 3 , 1 mM MgCl 2 , 0.5 mM CaCl 2 ) and centrifuged at 17,300 ⁇ g. The obtained precipitate is resuspended in Buffer E (1 mM NaHCO 3 , 0.32 M sucrose), and a synaptosome fraction is prepared by a sucrose density gradient method.
  • the synaptosome fraction is solubilized with 6 mM Tris-HCl pH 8.1, centrifuged at 48,250 ⁇ g, and separated into a LP1 membrane fraction and a supernatant fraction containing synaptic vesicles.
  • the synaptic membrane is prepared by further separating the LP1 membrane fraction by the sucrose density gradient method.
  • Synaptic vesicles are prepared by centrifuging the supernatant fraction at 100,000 x g.
  • the prepared synaptic membrane and the synaptic vesicle are solubilized with buffer A containing 1% (w / v) CHAPSO to prepare a solubilized ⁇ -secretase complex.
  • Example 2 Suppression of A ⁇ production by siRNA knockdown of a novel binding factor (1) About the thing identified by the said method, the influence with respect to A (beta) production was evaluated by RNAi method.
  • HEK-APP cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal calf serum.
  • the siRNA of the novel factor can be purchased from Ambion etc., or the method reported by Teramoto R. et al. (Teramoto, R. et al., FEBS Lett. (2005) 579, 2878-2882) and It was designed by the method described in Patent Publication 2006-236153 and synthesized from Invitrogen.
  • siRNA was introduced into HEK-APP cells using the siRNA introduction reagent Lipofectamine TM RNAiMAX Transfection Reagent (Invitrogen). 48 hours after introduction, the culture medium was converted to Opti-MEM I (Invitrogen), and further cultured for 24 hours, and the culture supernatant was collected. The number of viable cells was measured by the WST8 method, and siRNAs showing cytotoxicity of 30% or more were excluded from the test. The knockdown efficiency of the target gene was confirmed by real-time PCR. As a target gene primer, Applied Biosystems Pre-Developed TaqMan (registered trademark) Assay Reagents was used, and the GAPDH gene was used as an internal standard.
  • Applied Biosystems Pre-Developed TaqMan registered trademark
  • the amounts of A ⁇ 40 and A ⁇ 42 in the culture supernatant were measured by ELISA (Wako Chemicals, Osaka, Japan). The inhibition rate was calculated with the value in siRNA-untreated cells (MOCK) as 100%. As a result, it was confirmed that the amount of A ⁇ 40 and A ⁇ 42 was decreased by decreasing the expression level of presenilin 1 (PS1), which is a constituent factor of ⁇ -secretase, used as a test system control (FIGS. 3 and 4). Medium “PSEN1”).
  • PS1 presenilin 1
  • ATP2A2 and FLOT2 have been reported to reduce A ⁇ production by siRNA knockdown, confirming that this method is effective as a novel method for identifying ⁇ -secretase-binding A ⁇ production regulators. ing.
  • the knowledge which reduces the amount of A (beta) 40 and A (beta) 42 by reducing the expression level of a novel factor was acquired (FIGS. 3, 4).
  • Example 3 Identification of binding protein of brain ⁇ -secretase (2) Using the microsomal fraction obtained in Example 1, a plurality of novel ⁇ -secretase constituents that specifically bind to novel ⁇ -secretase and are expressed in the brain were found (Table 3).
  • Example 4 Suppression of A ⁇ production by siRNA knockdown of a novel binding factor (2)
  • siRNA knockdown of a novel binding factor (2) For the novel ⁇ -secretase component identified in Example 3 above, the effect on A ⁇ production was evaluated by the RNAi method in the same manner as described in Example 2.
  • Example 5 Identification of binding protein of brain ⁇ -secretase (3) Using the DRM fraction obtained in Example 1, a plurality of novel ⁇ -secretase components that specifically bind to the novel ⁇ -secretase and are expressed in the brain were found (Tables 5, 17 to 21). . Further, from the synaptic membrane and the synaptic vesicle fraction obtained in Example 1, a plurality of novel ⁇ -secretase constituents that were newly specifically bound to ⁇ -secretase and expressed in the brain were found (Tables 5 and 22). To 27).
  • Example 6 Suppression of A ⁇ production by siRNA knockdown of a novel binding factor (3)
  • the effect on A ⁇ production was evaluated by the RNAi method in the same manner as in Example 2.
  • the screening method provided by the present invention can select a substance having an action of suppressing the expression and function of a novel protein that up-regulates the activity of ⁇ -secretase
  • the substance obtained by this screening method is As a ⁇ production and / or Notch cleavage inhibitor or modulator, it is expected as a prophylactic and therapeutic agent for diseases such as AD and cancer.
  • these novel proteins may not affect the Notch cleavage activity of ⁇ -secretase
  • the substance obtained by this screening method does not inhibit the cleavage of other physiological substrates such as Notch, It may be able to inhibit only ⁇ -secretase activity, and is expected as a safe preventive and therapeutic drug for diseases such as AD with few side effects.
  • the present invention is based on a Japanese patent application filed on June 4, 2009, Japanese Patent Application No. 2009-135589, the entire contents of which are included in this specification.

Abstract

Disclosed is a novel and effective therapeutic agent for Alzheimer's disease. Specifically disclosed are: a β-amyloid (Aβ) production inhibitor, which comprises a substance capable of inhibiting the expression or function of a protein selected from probable phospholipid-transporting ATPase IIA; a BDNF/NT-3 growth factors receptor precursor; ELAV-like protein 4; coiled-coil domain-containing protein 136; potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2; DnaJ homolog subfamily A member 2; vesicle-associated membrane protein-associated protein A; a proton myo-inositol cotransporter; leukocyte surface antigen CD47; flotillin-1; band 4.1 like 1; a regulator of G-protein signaling 7; phospholipase D3; ectoderm-neural cortex protein 1; synaptophysin; solute carrier family 2 (a facilitated glucose transporter), member 3; syntaxin binding protein 1; growth associated protein 43; ATPase, Na+/K+ transporting, beta 1 polypeptide; amine oxidase [flavin-containing] B; muscarinic acetylcholine receptor M1; a probable cationic amino acid transporter; NADH dehydrogenase iron-sulfur protein 7; a G-protein-regulated inducer of neurite outgrowth; and probable phospholipid-transporting ATPase 1A; and a method for screening an Aβ production-inhibiting substance or a prophylactic/therapeutic agent for Alzheimer's disease, which uses the protein.

Description

アミロイドベータペプチドの産生を促進する因子を用いた阻害剤のスクリーニング方法及びそれによって得られる阻害剤Inhibitor screening method using factor that promotes production of amyloid beta peptide and inhibitor obtained thereby
　本発明は、アルツハイマー病の原因であるβアミロイドの産生抑制剤、アルツハイマー病の予防及び治療剤、並びにそのような薬剤のスクリーニング方法に関する。 The present invention relates to a β-amyloid production inhibitor that is the cause of Alzheimer's disease, a prophylactic and therapeutic agent for Alzheimer's disease, and a screening method for such a drug.
　アルツハイマー病（AD）の脳に蓄積するβアミロイド（Aβ）ペプチドは、前駆体タンパク質であるアミロイド前駆タンパク質（APP）からβ－セクレターゼおよびγ－セクレターゼにより切り出されて生成し、これが重合化して老人斑が形成される。Aβの蓄積は、AD脳に特異性が高くAD初期に生じる病変である。さらに家族性アルツハイマー病（FAD）では、病因遺伝子APP及びプレセニリン（PS）の変異により凝集性の高いAβ42の産生が亢進する、などの理由から、AβはADの病因物質と考えられ（アミロイド仮説）、ADの根本的治療法の治療ターゲットとして有力視されている（非特許文献１）。 Β-amyloid (Aβ) peptide, which accumulates in the brain of Alzheimer's disease (AD), is produced by being excised from the precursor protein amyloid precursor protein (APP) by β-secretase and γ-secretase, which is polymerized to form senile plaques Is formed. Accumulation of Aβ is a lesion that occurs early in AD with high specificity in AD brain. Furthermore, in familial Alzheimer's disease (FAD), Aβ is considered to be a causative agent of AD because the mutation of the etiological genes APP and presenilin (PS) increases the production of highly aggregated Aβ42 (amyloid hypothesis). It is regarded as a promising therapeutic target for radical treatment of AD (Non-patent Document 1).
　Aβペプチド産生は、その最終段階の切断を行う責任酵素であるγ－セクレターゼを阻害することにより抑制可能である。しかし、これまでに報告されているγ－セクレターゼ阻害剤では、該酵素の別の生理的基質であるNotchの切断も阻害し、発生や分化に関与するシグナルを阻害するため、腸管上皮形成障害や免疫異常などの末梢性副作用が問題になっている（非特許文献２）。一方で、Notchを介したシグナル経路で異常が発生すると、組織や器官の発生・形成に重大な異常がおこるだけでなく、癌の原因となる場合もある。Notch遺伝子は、発ガン遺伝子として明らかにされており、Notchを切断するγ-セクレターゼの活性を調節することが、制癌効果をもたらすことも知られている（非特許文献３）。
　近年、非ステロイド抗炎症薬（NSAIDs）がAβ42の産生に特異的な阻害能（γ－セクレターゼモジュレーター活性）を示すことが報告され、AD治療薬として開発が進められている（非特許文献４）。このように、今後、脳特異的にγ－セクレターゼの活性を阻害する化合物、あるいはγ－セクレターゼモジュレーターが、末梢性副作用を回避した理想的な治療薬となりうると予測されている。 Aβ peptide production can be suppressed by inhibiting γ-secretase, the responsible enzyme responsible for the final cleavage. However, γ-secretase inhibitors that have been reported so far also inhibit cleavage of Notch, which is another physiological substrate of the enzyme, and inhibit signals involved in development and differentiation. Peripheral side effects such as immune abnormalities are a problem (Non-patent Document 2). On the other hand, when an abnormality occurs in the signal pathway via Notch, not only a serious abnormality occurs in the generation and formation of tissues and organs, but it may cause cancer. The Notch gene has been clarified as an oncogenic gene, and it is also known that regulating the activity of γ-secretase that cleaves Notch has an anticancer effect (Non-patent Document 3).
In recent years, it has been reported that non-steroidal anti-inflammatory drugs (NSAIDs) exhibit specific inhibitory ability (γ-secretase modulator activity) for the production of Aβ42 and are being developed as AD therapeutics (Non-patent Document 4). . In this way, it is predicted that a compound or γ-secretase modulator that specifically inhibits the activity of γ-secretase in the brain can be an ideal therapeutic drug that avoids peripheral side effects in the future.
　γ－セクレターゼは、プレセニリン（PS），ニカストリン（NCT），Aph-1およびPen-2により構成される膜貫通型アスパラギン酸プロテアーゼである（非特許文献５)。γ－セクレターゼは、Aβ40およびAβ42を産生するが、その詳細なAβペプチド産生の制御メカニズムについては明確になっていない。最近、TMP21が、γ－セクレターゼに結合し活性を制御する分子として同定された（非特許文献６）。しかし、それ以外のγ－セクレターゼ活性制御因子の存在については全く知られていない。
　一方、膜結合型タンパク質をコードする遺伝子（ATP2A2, FLOT2）をRNAi法でノックダウンしたところ、Aβの産生が減少することが報告されている（非特許文献７、８）。 γ-secretase is a transmembrane aspartic protease composed of presenilin (PS), nicastrin (NCT), Aph-1 and Pen-2 (Non-patent Document 5). γ-secretase produces Aβ40 and Aβ42, but the detailed control mechanism of Aβ peptide production is not clear. Recently, TMP21 was identified as a molecule that binds to γ-secretase and controls its activity (Non-patent Document 6). However, the existence of other γ-secretase activity regulators is not known at all.
On the other hand, when a gene encoding a membrane-bound protein (ATP2A2, FLOT2) is knocked down by the RNAi method, production of Aβ is reported to decrease (Non-patent Documents 7 and 8).
　有効なAD治療薬の開発が切望されている。本発明の目的は、γ－セクレターゼに結合してその活性を促進/増強する新規因子を同定し、該因子の発現や機能を抑制することを機序とするγ－セクレターゼ阻害剤あるいはモジュレーター、Aβ産生抑制剤、AD予防・治療剤を提供することであり、また、該因子を用いてそのような薬剤をスクリーニングする方法を提供することである。 Developed effective AD therapeutics are eagerly desired. An object of the present invention is to identify a novel factor that binds to γ-secretase and promotes / enhances its activity, and to suppress the expression and function of the factor, a γ-secretase inhibitor or modulator, Aβ It is to provide a production inhibitor and an AD preventive / therapeutic agent, and to provide a method for screening such a drug using the factor.
　本発明者らは、TMP21以外にも、脳組織においてγ－セクレターゼに結合しAβの産生を制御する分子が存在するのではないかと着想し、インヒビタープルダウン法を用いてγ－セクレターゼと結合するタンパク質をヒトおよびラット脳より探索し、脳に発現する複数の新規γ－セクレターゼ結合因子を見出した。さらに、Aβの前駆体タンパク質であるAPPを過剰発現するヒト胎児腎臓細胞株（HEK-APP）において、これらの結合因子の発現をRNAi法によりノックダウンし、Aβ産生能への影響をELISA法で評価した。その結果、27因子について、それらの発現量を低下させることによりAβの産生を特異的に抑制できることを確認して、本発明を完成させるに至った。 The present inventors have conceived that there are molecules other than TMP21 that bind to γ-secretase and control Aβ production in brain tissue, and that bind to γ-secretase using an inhibitor pull-down method. Were searched from human and rat brain, and a plurality of novel γ-secretase binding factors expressed in the brain were found. Furthermore, in human embryonic kidney cell line (HEK-APP) overexpressing APP, which is a precursor protein of Aβ, expression of these binding factors was knocked down by RNAi method, and the effect on Aβ production ability was determined by ELISA method. evaluated. As a result, it was confirmed that the production of Aβ could be specifically suppressed by reducing the expression level of 27 factors, and the present invention was completed.
　すなわち、本発明は以下の通りのものである。
［１］以下のタンパク質：
(P1) Probable phospholipid-transporting ATPase IIA；
(P2) BDNF/NT-3 growth factors receptor precursor；
(P3) ELAV-like protein 4；
(P4) Coiled-coil domain-containing protein 136；
(P5) Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2；
(P6) DnaJ homolog subfamily A member 2；
(P7) Vesicle-associated membrane protein-associated protein A；
(P8) Proton myo-inositol cotransporter；
(P9) Leukocyte surface antigen CD47；
(P10) Flotillin-1；
(P11) Band 4.1 like 1；
(P12) Regulator of G-protein signaling 7；
(P13) Phospholipase D3；
(P14) Ectoderm-neural cortex protein 1；
(P15) synaptophysin；
(P16) solute carrier family 2 (facilitated glucose transporter), member 3；
(P17) syntaxin binding protein 1；
(P18) growth associated protein 43；
(P19) ATPase, Na+/K+ transporting, beta 1 polypeptide；
(P20) Amine oxidase [flavin-containing] B；
(P21) Muscarinic acetylcholine receptor M1；
(P22) Probable cationic amino acid transporter；
(P23) NADH dehydrogenase iron-sulfur protein 7；
(P24) G-protein-regulated inducer of neurite outgrowth；および
(P25) Probable phospholipid-transporting ATPase 1A
から選ばれるタンパク質Pn（nは1～25のいずれかの整数）の発現または機能を抑制する、１以上の物質を含有してなる、Aβ産生抑制剤。
［２］タンパク質Pnの発現を抑制する物質が、以下の（ａ）～（ｃ）からなる群より選択される物質である、上記［１］に記載の剤。
（ａ）タンパク質Pnをコードする遺伝子Gn：
(G1) ATP9A；
(G2) NTRK2；
(G3) ELAVL4；
(G4) NAG6；
(G5) HCN2；
(G6) DNAJA2；
(G7) VAPA；
(G8) SLC2A13；
(G9) CD47;
(G10) FLOT1；
(G11) EPB41L1；
(G12) RGS7；
(G13) PLD3；
(G14) ENC1；
(G15) SYP；
(G16) SLC2A3；
(G17) STXBP1；
(G18) GAP43；
(G19) ATP1B1；
(G20) MAOB；
(G21) CHRM1；
(G22) SLC7A14；
(G23) NDUFS7；
(G24) GPRIN1；または
(G25) ATP8A1
の転写産物に対するアンチセンス核酸
（ｂ）遺伝子Gnの転写産物に対するリボザイム核酸
（ｃ）遺伝子Gnの転写産物に対してRNAi活性を有する核酸もしくはその前駆体
［３］タンパク質Pnの機能を抑制する物質が、以下の（ａ）～（ｃ）からなる群より選択される物質である、上記［１］に記載の剤。
（ａ）タンパク質Pnと結合する抗体
（ｂ）タンパク質Pnと結合する低分子化合物
（ｃ）タンパク質Pnとγ－セクレターゼとの結合活性を阻害する化合物
［４］Aβが、Aβ40またはAβ42である、上記［１］～［３］のいずれかに記載の剤。
［５］アルツハイマー病もしくは癌の治療または予防のための、上記［１］～［４］のいずれかに記載の剤。
［６］哺乳動物において、以下のタンパク質：
(P1) Probable phospholipid-transporting ATPase IIA；
(P2) BDNF/NT-3 growth factors receptor precursor；
(P3) ELAV-like protein 4；
(P4) Coiled-coil domain-containing protein 136；
(P5) Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2；
(P6) DnaJ homolog subfamily A member 2；
(P7) Vesicle-associated membrane protein-associated protein A；
(P8) Proton myo-inositol cotransporter；
(P9) Leukocyte surface antigen CD47；
(P10) Flotillin-1；
(P11) Band 4.1 like 1；
(P12) Regulator of G-protein signaling 7；
(P13) Phospholipase D3；
(P14) Ectoderm-neural cortex protein 1；
(P15) synaptophysin；
(P16) solute carrier family 2 (facilitated glucose transporter), member 3；
(P17) syntaxin binding protein 1；
(P18) growth associated protein 43；
(P19) ATPase, Na+/K+ transporting, beta 1 polypeptide；
(P20) Amine oxidase [flavin-containing] B；
(P21) Muscarinic acetylcholine receptor M1；
(P22) Probable cationic amino acid transporter；
(P23) NADH dehydrogenase iron-sulfur protein 7；
(P24) G-protein-regulated inducer of neurite outgrowth；および
(P25) Probable phospholipid-transporting ATPase 1A
から選ばれるタンパク質Pn（nは1～25のいずれかの整数）の発現または機能を抑制することを含む、該哺乳動物におけるAβ産生の抑制方法。
［７］有効量の、タンパク質Pnの発現または機能を抑制する１以上の物質を哺乳動物に投与することを含む、上記［６］に記載の方法。
［８］タンパク質Pnの発現を抑制する物質が、以下の（ａ）～（ｃ）からなる群より選択される物質である、上記［７］に記載の方法。
（ａ）タンパク質Pnをコードする遺伝子Gn：
(G1) ATP9A；
(G2) NTRK2；
(G3) ELAVL4；
(G4) NAG6；
(G5) HCN2；
(G6) DNAJA2；
(G7) VAPA；
(G8) SLC2A13；
(G9) CD47;
(G10) FLOT1；
(G11) EPB41L1；
(G12) RGS7；
(G13) PLD3；
(G14) ENC1；
(G15) SYP；
(G16) SLC2A3；
(G17) STXBP1；
(G18) GAP43；
(G19) ATP1B1；
(G20) MAOB；
(G21) CHRM1；
(G22) SLC7A14；
(G23) NDUFS7；
(G24) GPRIN1；または
(G25) ATP8A1
の転写産物に対するアンチセンス核酸
（ｂ）遺伝子Gnの転写産物に対するリボザイム核酸
（ｃ）遺伝子Gnの転写産物に対してRNAi活性を有する核酸もしくはその前駆体
［９］タンパク質Pnの機能を抑制する物質が、以下の（ａ）～（ｃ）からなる群より選択される物質である、上記［７］に記載の方法。
（ａ）タンパク質Pnと結合する抗体
（ｂ）タンパク質Pnと結合する低分子化合物
（ｃ）タンパク質Pnとγ－セクレターゼとの結合活性を阻害する化合物
［１０］Aβが、Aβ40またはAβ42である、上記［６］～［９］のいずれかに記載の方法。
［１１］アルツハイマー病もしくは癌の治療または予防のための、上記［６］～［１０］のいずれかに記載の方法。
［１２］前記(G1）～(G25)から選ばれる少なくとも１つの遺伝子の発現量もしくは前記(P1)～(P25)から選ばれる少なくとも１つのタンパク質の活性を低下させる物質を選択することを特徴とする、Aβ産生抑制物質のスクリーニング方法。
［１３］以下の（ａ）～（ｃ）の工程を含む、Aβ産生抑制物質のスクリーニング方法。
（ａ）(a1)遺伝子Gn（nは1～25のいずれかの整数）もしくは(a2) 遺伝子Gn（nは1～25のいずれかの整数）の転写調節領域の制御下にあるレポーター遺伝子を発現する細胞に、被検物質を接触させる工程
（ｂ）前記細胞における(b1)遺伝子Gnもしくは(b2)レポーター遺伝子の発現量を測定する工程
（ｃ）被検物質の非存在下において測定した場合と比較して、発現量を低下させる物質をAβ産生抑制物質の候補として選択する工程
［１４］以下の（ａ）～（ｃ）の工程を含む、Aβ産生抑制物質のスクリーニング方法。
（ａ）アミロイド前駆タンパク質（APP）を発現する細胞に、前記(P1)～(P25)から選ばれる少なくとも１つのタンパク質および被検物質を接触させる工程
（ｂ）前記タンパク質の活性を測定する工程
（ｃ）被検物質の非存在下において測定した場合と比較して、前記タンパク質の活性を低下させる物質を選択する工程
［１５］前記タンパク質が前記細胞自体により提供される、上記［１４］に記載の方法。
［１６］前記タンパク質の活性をAβの産生量を指標として測定することを特徴とする、上記［１４］または［１５］に記載の方法。
［１７］以下の（ａ）～（ｃ）の工程を含む、Aβ産生抑制物質のスクリーニング方法。
（ａ）γ－セクレターゼを発現する細胞もしくはその細胞膜画分に、前記(P1)～(P25)から選ばれる少なくとも１つのタンパク質および被検物質を接触させる工程
（ｂ）γ－セクレターゼ活性を測定する工程
（ｃ）被検物質の非存在下において測定した場合と比較して、γ－セクレターゼ活性を低下またはモジュレートさせる物質を選択する工程
［１８］前記タンパク質が前記細胞自体により提供される、上記［１７］に記載の方法。
［１９］タンパク質Pn（nは1～25のいずれかの整数）とγ－セクレターゼとの結合活性を阻害する物質を選択することを特徴とする、Aβ産生抑制物質のスクリーニング方法。
［２０］以下の（ａ）～（ｃ）の工程を含む、Aβ産生抑制物質のスクリーニング方法。
（ａ）タンパク質Pn（nは1～25のいずれかの整数）およびγ－セクレターゼと、被検物質とを接触させる工程
（ｂ）タンパク質Pnとγ－セクレターゼとの結合活性を測定する工程
（ｃ）被検物質の非存在下において測定した場合と比較して、前記結合活性を低下させる物質を選択する工程
［２１］前記(G1）～(G25)から選ばれる少なくとも１つの遺伝子を導入した非ヒト動物において、アルツハイマー病の病態を反映する表現型を改善する物質を選択することを特徴とする、アルツハイマー病の治療または予防薬のスクリーニングまたは薬効評価方法。
［２２］以下の（ａ）～（ｃ）の工程を含む、アルツハイマー病の治療または予防薬のスクリーニングまたは薬効評価方法。
（ａ）前記(G1）～(G25)から選ばれる少なくとも１つの遺伝子を導入した非ヒト動物に被検物質を投与する工程
（ｂ）該動物における、アルツハイマー病の病態を反映する少なくとも１つの表現型を評価する工程
（ｃ）被検物質の非投与時において評価した場合と比較して、前記表現型を改善させる物質を選択する工程
［２３］前記表現型が、脳組織、脳脊髄液、血液中などの組織中のAβ量、神経細胞死、中枢神経系の炎症反応、認知能力、アミロイドプラークの蓄積量、脳内血流量および脳内グルコース代謝量からなる群より選択される、上記［２２］に記載の方法。
［２４］アルツハイマー病または癌の発症または発症リスクの判定方法であって、以下の（ａ）～（ｃ）の工程を含む方法。
（ａ）被験動物由来の試料を提供する工程
（ｂ）該試料中の、前記(G1）～(G25)から選ばれる少なくとも１つの遺伝子の発現量もしくは前記(P1)～(P25)から選ばれる少なくとも１つのタンパク質の活性を測定する工程
（ｃ）正常動物由来の試料において測定した場合と比較して、前記発現量もしくは活性が上昇している被験動物を、アルツハイマー病または癌を発症しているか、将来発症するリスクが高いと判定する工程 That is, the present invention is as follows.
[1] The following proteins:
(P1) Probable phospholipid-transporting ATPase IIA;
(P2) BDNF / NT-3 growth factors receptor precursor;
(P3) ELAV-like protein 4;
(P4) Coiled-coil domain-containing protein 136;
(P5) Potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 2;
(P6) DnaJ homolog subfamily A member 2;
(P7) Vesicle-associated membrane protein-associated protein A;
(P8) Proton myo-inositol cotransporter;
(P9) Leukocyte surface antigen CD47;
(P10) Flotillin-1;
(P11) Band 4.1 like 1;
(P12) Regulator of G-protein signaling 7;
(P13) Phospholipase D3;
(P14) Ectoderm-neural cortex protein 1;
(P15) synaptophysin;
(P16) solute carrier family 2 (facilitated glucose transporter), member 3;
(P17) syntaxin binding protein 1;
(P18) growth associated protein 43;
(P19) ATPase, Na + / K + transporting, beta 1 polypeptide;
(P20) Amine oxidase [flavin-containing] B;
(P21) Muscarinic acetylcholine receptor M1;
(P22) Probable capable amino acid transporter;
(P23) NADH dehydrogenase iron-sulfur protein 7;
(P24) G-protein-regulated inducer of neurite outgrowth; and
(P25) Probable phospholipid-transporting ATPase 1A
An Aβ production inhibitor comprising one or more substances that suppress the expression or function of a protein Pn selected from (n is an integer of 1 to 25).
[2] The agent according to [1] above, wherein the substance that suppresses the expression of protein Pn is a substance selected from the group consisting of the following (a) to (c).
(A) Gene Gn encoding protein Pn:
(G1) ATP9A;
(G2) NTRK2;
(G3) ELAVL4;
(G4) NAG6;
(G5) HCN2;
(G6) DNAJA2;
(G7) VAPA;
(G8) SLC2A13;
(G9) CD47;
(G10) FLOT1;
(G11) EPB41L1;
(G12) RGS7;
(G13) PLD3;
(G14) ENC1;
(G15) SYP;
(G16) SLC2A3;
(G17) STXBP1;
(G18) GAP43;
(G19) ATP1B1;
(G20) MAOB;
(G21) CHRM1;
(G22) SLC7A14;
(G23) NDUFS7;
(G24) GPRIN1; or
(G25) ATP8A1
(B) a ribozyme nucleic acid against the transcription product of gene Gn (c) a nucleic acid having RNAi activity against the transcription product of gene Gn or a precursor thereof [3] a substance that suppresses the function of protein Pn The agent according to [1] above, which is a substance selected from the group consisting of the following (a) to (c):
(A) an antibody that binds to protein Pn (b) a low molecular compound that binds to protein Pn (c) a compound that inhibits the binding activity between protein Pn and γ-secretase [4] Aβ is Aβ40 or Aβ42 [1] The agent according to any one of [3].
[5] The agent according to any one of [1] to [4] above, for treating or preventing Alzheimer's disease or cancer.
[6] In mammals, the following proteins:
(P1) Probable phospholipid-transporting ATPase IIA;
(P2) BDNF / NT-3 growth factors receptor precursor;
(P3) ELAV-like protein 4;
(P4) Coiled-coil domain-containing protein 136;
(P5) Potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 2;
(P6) DnaJ homolog subfamily A member 2;
(P7) Vesicle-associated membrane protein-associated protein A;
(P8) Proton myo-inositol cotransporter;
(P9) Leukocyte surface antigen CD47;
(P10) Flotillin-1;
(P11) Band 4.1 like 1;
(P12) Regulator of G-protein signaling 7;
(P13) Phospholipase D3;
(P14) Ectoderm-neural cortex protein 1;
(P15) synaptophysin;
(P16) solute carrier family 2 (facilitated glucose transporter), member 3;
(P17) syntaxin binding protein 1;
(P18) growth associated protein 43;
(P19) ATPase, Na + / K + transporting, beta 1 polypeptide;
(P20) Amine oxidase [flavin-containing] B;
(P21) Muscarinic acetylcholine receptor M1;
(P22) Probable capable amino acid transporter;
(P23) NADH dehydrogenase iron-sulfur protein 7;
(P24) G-protein-regulated inducer of neurite outgrowth; and
(P25) Probable phospholipid-transporting ATPase 1A
A method for inhibiting Aβ production in a mammal, comprising inhibiting the expression or function of a protein Pn selected from (n is an integer of 1 to 25).
[7] The method of [6] above, comprising administering to the mammal an effective amount of one or more substances that suppress the expression or function of protein Pn.
[8] The method according to [7] above, wherein the substance that suppresses the expression of protein Pn is a substance selected from the group consisting of the following (a) to (c).
(A) Gene Gn encoding protein Pn:
(G1) ATP9A;
(G2) NTRK2;
(G3) ELAVL4;
(G4) NAG6;
(G5) HCN2;
(G6) DNAJA2;
(G7) VAPA;
(G8) SLC2A13;
(G9) CD47;
(G10) FLOT1;
(G11) EPB41L1;
(G12) RGS7;
(G13) PLD3;
(G14) ENC1;
(G15) SYP;
(G16) SLC2A3;
(G17) STXBP1;
(G18) GAP43;
(G19) ATP1B1;
(G20) MAOB;
(G21) CHRM1;
(G22) SLC7A14;
(G23) NDUFS7;
(G24) GPRIN1; or
(G25) ATP8A1
An antisense nucleic acid against the transcription product of (b) a ribozyme nucleic acid against the transcription product of gene Gn (c) a nucleic acid having RNAi activity against the transcription product of gene Gn or a precursor thereof [9] a substance that suppresses the function of protein Pn The method according to [7] above, which is a substance selected from the group consisting of the following (a) to (c):
(A) an antibody that binds to protein Pn (b) a low molecular compound that binds to protein Pn (c) a compound that inhibits the binding activity between protein Pn and γ-secretase [10] Aβ is Aβ40 or Aβ42 [6] The method according to any one of [9].
[11] The method according to any one of [6] to [10] above, for treating or preventing Alzheimer's disease or cancer.
[12] The method comprises selecting a substance that decreases the expression level of at least one gene selected from (G1) to (G25) or the activity of at least one protein selected from (P1) to (P25). A method for screening an Aβ production inhibitor.
[13] A method for screening an Aβ production inhibitor comprising the following steps (a) to (c):
A reporter gene under the control of the transcriptional regulatory region of (a) (a1) gene Gn (n is any integer from 1 to 25) or (a2) gene Gn (n is any integer from 1 to 25) A step of contacting a test substance with a cell to be expressed (b) a step of measuring the expression level of (b1) gene Gn or (b2) reporter gene in the cell (c) a measurement in the absence of the test substance [14] A method for screening an Aβ production inhibitory substance, comprising the following steps (a) to (c): selecting a substance that reduces the expression level as a candidate for an Aβ production inhibitory substance compared to
(A) contacting a cell expressing amyloid precursor protein (APP) with at least one protein selected from the above (P1) to (P25) and a test substance (b) measuring the activity of the protein ( c) The step of selecting a substance that reduces the activity of the protein compared to the case where it is measured in the absence of a test substance [15] The protein is provided by the cell itself, [14] the method of.
[16] The method according to [14] or [15] above, wherein the activity of the protein is measured using the production amount of Aβ as an index.
[17] A method for screening an Aβ production inhibitor comprising the following steps (a) to (c):
(A) contacting at least one protein selected from (P1) to (P25) and a test substance with a cell expressing γ-secretase or a cell membrane fraction thereof; and (b) measuring γ-secretase activity. Step (c) selecting a substance that reduces or modulates γ-secretase activity compared to that measured in the absence of the test substance [18] The protein is provided by the cell itself, The method according to [17].
[19] A screening method for an Aβ production inhibitor, which comprises selecting a substance that inhibits the binding activity between protein Pn (n is an integer of 1 to 25) and γ-secretase.
[20] A screening method for an Aβ production inhibitor comprising the following steps (a) to (c):
(A) contacting protein Pn (n is an integer of 1 to 25) and γ-secretase with a test substance (b) measuring the binding activity between protein Pn and γ-secretase (c ) A step of selecting a substance that reduces the binding activity as compared with the case where it is measured in the absence of the test substance [21] non-introducing at least one gene selected from (G1) to (G25) A method for screening or evaluating an efficacy of a therapeutic or prophylactic agent for Alzheimer's disease, comprising selecting a substance that improves the phenotype reflecting the pathological condition of Alzheimer's disease in a human animal.
[22] A screening or drug efficacy evaluation method for a therapeutic or prophylactic agent for Alzheimer's disease comprising the following steps (a) to (c):
(A) a step of administering a test substance to a non-human animal into which at least one gene selected from the above (G1) to (G25) has been introduced; (b) at least one expression reflecting the pathological condition of Alzheimer's disease in the animal A step of evaluating the type (c) a step of selecting a substance that improves the phenotype as compared with the case where the test substance is not administered, [23] the phenotype is brain tissue, cerebrospinal fluid, Selected from the group consisting of Aβ amount in tissues such as blood, neuronal cell death, central nervous system inflammatory response, cognitive ability, amyloid plaque accumulation, cerebral blood flow and cerebral glucose metabolism 22].
[24] A method for determining the onset or risk of developing Alzheimer's disease or cancer, comprising the following steps (a) to (c):
(A) Providing a sample derived from a test animal (b) Expression level of at least one gene selected from (G1) to (G25) in the sample or selected from (P1) to (P25) A step of measuring the activity of at least one protein (c) is the subject animal having an increased expression level or activity compared to the case of measuring in a sample derived from a normal animal developing Alzheimer's disease or cancer? The process of determining that the risk of developing in the future is high
　本発明に従って、γ-セクレターゼの活性を調節する新規タンパク質を利用することにより、Aβの産生を阻害する化合物のスクリーニング、抗体や核酸を用いた医療の開発が可能となる。これより、ADやあるいは癌などの疾患の予防および治療に有用である。 According to the present invention, by using a novel protein that regulates the activity of γ-secretase, it becomes possible to screen for compounds that inhibit the production of Aβ and to develop medical treatments using antibodies and nucleic acids. Thus, it is useful for the prevention and treatment of diseases such as AD and cancer.
GCB（γ-secretase inhibitor with a hydrophilic long linker and a cleavable biotin group）の構造を示す図である。It is a figure which shows the structure of GCB (γ-secretase inhibitor with a hydrophilic long linker and a cleavable biotin group). インヒビタープルダウン法の原理の模式図（Ａ）および当該方法によりγ－セクレターゼの既知コンポーネントがプルダウンされたことを示す図である。FIG. 2 is a schematic diagram (A) of the principle of an inhibitor pull-down method and a diagram showing that a known component of γ-secretase is pulled down by the method. HEK-APP細胞に対するsiRNAによる遺伝子ノックダウンによる、内在性Aβ42の産生抑制を示す図である（導入siRNA量：0.1pmol/well）。It is a figure which shows the production suppression of endogenous A (beta) 42 by the gene knockdown by siRNA with respect to a HEK-APP cell (transduction siRNA amount: 0.1pmol / well). HEK-APP細胞に対するsiRNAによる遺伝子ノックダウンによる、内在性Aβ40の産生抑制を示す図である（導入siRNA量：0.1pmol/well）。It is a figure which shows the production suppression of endogenous A (beta) 40 by the gene knockdown by siRNA with respect to HEK-APP cell (introduction siRNA amount: 0.1pmol / well). HEK-APP細胞に対するsiRNAによる遺伝子ノックダウンによる、内在性Aβ42の産生抑制を示す図である（導入siRNA量：0.1pmol/well）。It is a figure which shows the production suppression of endogenous A (beta) 42 by the gene knockdown by siRNA with respect to a HEK-APP cell (transduction siRNA amount: 0.1pmol / well). HEK-APP細胞に対するsiRNAによる遺伝子ノックダウンによる、内在性Aβ40の産生抑制を示す図である（導入siRNA量：0.1pmol/well）。It is a figure which shows the production suppression of endogenous A (beta) 40 by the gene knockdown by siRNA with respect to HEK-APP cell (introduction siRNA amount: 0.1pmol / well). HEK-APP細胞に対するsiRNAによる遺伝子ノックダウンによる、内在性Aβ42の産生抑制を示す図である（導入siRNA量：0.1pmol/well）。It is a figure which shows the production suppression of endogenous A (beta) 42 by the gene knockdown by siRNA with respect to a HEK-APP cell (transduction siRNA amount: 0.1pmol / well). HEK-APP細胞に対するsiRNAによる遺伝子ノックダウンによる、内在性Aβ40の産生抑制を示す図である（導入siRNA量：0.1pmol/well）。It is a figure which shows the production suppression of endogenous A (beta) 40 by the gene knockdown by siRNA with respect to HEK-APP cell (introduction siRNA amount: 0.1pmol / well).
　本発明は、以下のタンパク質：
(P1) Probable phospholipid-transporting ATPase IIA；
(P2) BDNF/NT-3 growth factors receptor precursor；
(P3) ELAV-like protein 4；
(P4) Coiled-coil domain-containing protein 136；
(P5) Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2；
(P6) DnaJ homolog subfamily A member 2；
(P7) Vesicle-associated membrane protein-associated protein A；
(P8) Proton myo-inositol cotransporter；
(P9) Leukocyte surface antigen CD47；
(P10) Flotillin-1；
(P11) Band 4.1 like 1；
(P12) Regulator of G-protein signaling 7；
(P13) Phospholipase D3；
(P14) Ectoderm-neural cortex protein 1；
(P15) synaptophysin；
(P16) solute carrier family 2 (facilitated glucose transporter), member 3；
(P17) syntaxin binding protein 1；
(P18) growth associated protein 43；
(P19) ATPase, Na+/K+ transporting, beta 1 polypeptide；
(P20) Amine oxidase [flavin-containing] B；
(P21) Muscarinic acetylcholine receptor M1；
(P22) Probable cationic amino acid transporter；
(P23) NADH dehydrogenase iron-sulfur protein 7；
(P24) G-protein-regulated inducer of neurite outgrowth；および
(P25) Probable phospholipid-transporting ATPase 1A
から選ばれるタンパク質Pn（nは1～25のいずれかの整数）の発現を抑制する物質または機能を抑制する物質を含有してなる、Ａβ産生抑制剤を提供する。
　本発明におけるタンパク質Pn［(P1) Probable phospholipid-transporting ATPase IIA；(P2) BDNF/NT-3 growth factors receptor precursor；(P3) ELAV-like protein 4；(P4) Coiled-coil domain-containing protein 136；(P5) Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2；(P6) DnaJ homolog subfamily A member 2；(P7) Vesicle-associated membrane protein-associated protein A；(P8) Proton myo-inositol cotransporter；(P9) Leukocyte surface antigen CD47；(P10) Flotillin-1；(P11) Band 4.1 like 1；(P12) Regulator of G-protein signaling 7；(P13) Phospholipase D3；(P14) Ectoderm-neural cortex protein 1；(P15) synaptophysin；(P16) solute carrier family 2 (facilitated glucose transporter), member 3；(P17) syntaxin binding protein 1；(P18) growth associated protein 43；(P19) ATPase, Na+/K+ transporting, beta 1 polypeptide；(P20) Amine oxidase [flavin-containing] B；(P21) Muscarinic acetylcholine receptor M1；(P22) Probable cationic amino acid transporter；(P23) NADH dehydrogenase iron-sulfur protein 7；(P24) G-protein-regulated inducer of neurite outgrowth；または(P25) Probable phospholipid-transporting ATPase 1A］は、配列番号：2n（nは1～25のいずれかの整数）で表されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含むタンパク質である。本明細書において、タンパク質およびペプチドは、ペプチド標記の慣例に従って左端がＮ末端（アミノ末端）、右端がＣ末端（カルボキシル末端）で記載される。
　タンパク質Pnは、ヒトや他の温血動物（例えば、モルモット、ラット、マウス、ニワトリ、ウサギ、イヌ、ブタ、ヒツジ、ウシ、サルなど）の細胞［例えば、肝細胞、脾細胞、神経細胞、グリア細胞、膵臓β細胞、骨髄細胞、メサンギウム細胞、ランゲルハンス細胞、表皮細胞、上皮細胞、肺細胞、内皮細胞、平滑筋細胞、線維芽細胞、線維細胞、筋細胞、脂肪細胞、免疫細胞（例、マクロファージ、Ｔ細胞、Ｂ細胞、ナチュラルキラー細胞、肥満細胞、好中球、好塩基球、好酸球、単球）、巨核球、滑膜細胞、軟骨細胞、骨細胞、骨芽細胞、破骨細胞、乳腺細胞もしくは間質細胞、またはこれら細胞の前駆細胞、幹細胞もしくは癌細胞など］もしくはそれらの細胞が存在するあらゆる組織［例えば、脳、脳の各部位（例、嗅球、扁桃核、大脳基底球、海馬、視床、視床下部、大脳皮質、延髄、小脳）、脊髄、下垂体、胃、膵臓、腎臓、肝臓、生殖腺、甲状腺、胆のう、骨髄、副腎、皮膚、筋肉（例、平滑筋、骨格筋）、肺、消化管（例、大腸、小腸）、血管、心臓、胸腺、脾臓、顎下腺、末梢血、前立腺、睾丸、卵巣、胎盤、子宮、骨、関節、脂肪組織（例、白色脂肪組織、褐色脂肪組織）など］等から、自体公知のタンパク質分離精製技術により単離・精製されるものであってもよい。 The present invention includes the following proteins:
(P1) Probable phospholipid-transporting ATPase IIA;
(P2) BDNF / NT-3 growth factors receptor precursor;
(P3) ELAV-like protein 4;
(P4) Coiled-coil domain-containing protein 136;
(P5) Potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 2;
(P6) DnaJ homolog subfamily A member 2;
(P7) Vesicle-associated membrane protein-associated protein A;
(P8) Proton myo-inositol cotransporter;
(P9) Leukocyte surface antigen CD47;
(P10) Flotillin-1;
(P11) Band 4.1 like 1;
(P12) Regulator of G-protein signaling 7;
(P13) Phospholipase D3;
(P14) Ectoderm-neural cortex protein 1;
(P15) synaptophysin;
(P16) solute carrier family 2 (facilitated glucose transporter), member 3;
(P17) syntaxin binding protein 1;
(P18) growth associated protein 43;
(P19) ATPase, Na + / K + transporting, beta 1 polypeptide;
(P20) Amine oxidase [flavin-containing] B;
(P21) Muscarinic acetylcholine receptor M1;
(P22) Probable capable amino acid transporter;
(P23) NADH dehydrogenase iron-sulfur protein 7;
(P24) G-protein-regulated inducer of neurite outgrowth; and
(P25) Probable phospholipid-transporting ATPase 1A
An Aβ production inhibitor comprising a substance that suppresses expression of a protein Pn selected from (n is an integer of 1 to 25) or a substance that suppresses a function is provided.
Protein Pn [(P1) Probable phospholipid-transporting ATPase IIA; (P2) BDNF / NT-3 growth factors receptor precursor; (P3) ELAV-like protein 4; (P4) Coiled-coil domain-containing protein 136; (P5) Potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 2; (P6) DnaJ homolog subfamily A member 2; (P7) Vesicle-associated membrane protein-associated protein A; (P8) Proton myo-inositol cotransporter; (P9) Leukocyte surface antigen CD47; (P10) Flotillin-1; (P11) Band 4.1 like 1; (P12) Regulator of G-protein signaling 7; (P13) Phospholipase D3; (P14) Ectoderm-neural cortex protein 1; (P15) ) synaptophysin; (P16) solute carrier family 2 (facilitated glucose transporter), member 3; (P17) syntaxin binding protein 1; (P18) growth associated protein 43; (P19) ATPase, Na + / K + transporting, beta 1 polypeptide; P20) Amine oxidase [flavin-containing] B; (P21) Muscarinic acetylcholine receptor M1; (P22) Probable reactive amino acid transp (P23) NADH dehydrogenase iron-sulfur protein 7; (P24) G-protein-regulated inducer of neurite outgrowth; or (P25) Probable phospholipid-transporting ATPase 1A] is SEQ ID NO: 2n (n is 1 to 25) A protein comprising an amino acid sequence identical or substantially identical to the amino acid sequence represented by any integer). In the present specification, proteins and peptides are described with the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide designation.
Protein Pn is a cell of humans and other warm-blooded animals (eg, guinea pigs, rats, mice, chickens, rabbits, dogs, pigs, sheep, cows, monkeys, etc.) [eg, hepatocytes, spleen cells, neurons, glia. Cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, lung cells, endothelial cells, smooth muscle cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg, macrophages) , T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts , Mammary cells or stromal cells, or precursor cells of these cells, stem cells, cancer cells, etc.] or any tissue in which these cells are present [eg, brain, brain regions (eg, olfactory bulb, amygdala, cerebrum) Basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle (eg, smooth muscle, Skeletal muscle), lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, bone, joint, adipose tissue (eg, White adipose tissue, brown adipose tissue), etc.], etc.] may be isolated and purified by protein separation and purification techniques known per se.
　「配列番号：2nで表されるアミノ酸配列と実質的に同一のアミノ酸配列」とは、
(a) 配列番号：2nで表されるアミノ酸配列と約80%以上の相同性を有するアミノ酸配列；
(b) 配列番号：2nで表されるアミノ酸配列において、1～50個のアミノ酸が置換および／または欠失および／または挿入および／または付加されたアミノ酸配列；
(c) 配列番号：2nで表されるアミノ酸配列からなるヒトタンパク質の他の哺乳動物におけるオルソログのアミノ酸配列；または
(d) 配列番号：2nで表されるアミノ酸配列からなるヒトタンパク質もしくは上記(c)のオルソログのスプライスバリアント、アレル変異体もしくは多型におけるアミノ酸配列
を意味する。
　ここで「相同性」とは、当該技術分野において公知の数学的アルゴリズムを用いて２つのアミノ酸配列をアラインさせた場合の、最適なアラインメント（好ましくは、該アルゴリズムは最適なアラインメントのために配列の一方もしくは両方へのギャップの導入を考慮し得るものである）における、オーバーラップする全アミノ酸残基に対する同一アミノ酸および類似アミノ酸残基の割合（％）を意味する。「類似アミノ酸」とは物理化学的性質において類似したアミノ酸を意味し、例えば、芳香族アミノ酸（Phe、Trp、Tyr）、脂肪族アミノ酸（Ala、Leu、Ile、Val）、極性アミノ酸（Gln、Asn）、塩基性アミノ酸（Lys、Arg、His）、酸性アミノ酸（Glu、Asp）、水酸基を有するアミノ酸（Ser、Thr）、側鎖の小さいアミノ酸（Gly、Ala、Ser、Thr、Met）などの同じグループに分類されるアミノ酸が挙げられる。このような類似アミノ酸による置換はタンパク質の表現型に変化をもたらさない（即ち、保存的アミノ酸置換である）ことが予測される。保存的アミノ酸置換の具体例は当該技術分野で周知であり、種々の文献に記載されている（例えば、Bowieら，Science, 247：1306-1310 (1990)を参照）。 “Amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2n”
(a) an amino acid sequence having about 80% or more homology with the amino acid sequence represented by SEQ ID NO: 2n;
(b) in the amino acid sequence represented by SEQ ID NO: 2n, an amino acid sequence in which 1 to 50 amino acids are substituted and / or deleted and / or inserted and / or added;
(c) the amino acid sequence of an ortholog in another mammal of the human protein consisting of the amino acid sequence represented by SEQ ID NO: 2n; or
(d) The amino acid sequence of the human protein consisting of the amino acid sequence represented by SEQ ID NO: 2n or the splice variant, allelic variant or polymorphism of the ortholog of (c) above.
As used herein, “homology” refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm uses a sequence of sequences for optimal alignment). The ratio of the same amino acid residue and similar amino acid residues to all overlapping amino acid residues in the case of introducing a gap into one or both). “Similar amino acids” means amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn). ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is expected that substitution with such similar amino acids will not change the phenotype of the protein (ie, is a conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and are described in various literature (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).
　本明細書におけるアミノ酸配列の相同性は、相同性計算アルゴリズムNCBI BLAST（National Center for Biotechnology Information Basic Local Alignment Search Tool）を用い、以下の条件（期待値=10；ギャップを許す；マトリクス=BLOSUM62；フィルタリング=OFF）にて計算することができる。アミノ酸配列の相同性を決定するための他のアルゴリズムとしては、例えば、Karlinら, Proc. Natl. Acad. Sci. USA, 90： 5873-5877 (1993)に記載のアルゴリズム［該アルゴリズムはNBLASTおよびXBLASTプログラム（version 2.0）に組み込まれている（Altschulら, Nucleic Acids Res., 25： 3389-3402 (1997)）］、Needlemanら, J. Mol. Biol., 48： 444-453 (1970)に記載のアルゴリズム［該アルゴリズムはGCGソフトウェアパッケージ中のGAPプログラムに組み込まれている］、MyersおよびMiller, CABIOS, 4: 11-17 (1988)に記載のアルゴリズム［該アルゴリズムはCGC配列アラインメントソフトウェアパッケージの一部であるALIGNプログラム（version 2.0）に組み込まれている］、Pearsonら, Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988)に記載のアルゴリズム［該アルゴリズムはGCGソフトウェアパッケージ中のFASTAプログラムに組み込まれている］等が挙げられ、それらも同様に好ましく用いられ得る。 The homology of the amino acid sequences in this specification is determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) and the following conditions (expected value = 10; allow gap; matrix = BLOSUM62; filtering) = OFF). Other algorithms for determining amino acid sequence homology include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [the algorithms include NBLAST and XBLAST] Embedded in the program (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 1997 (1997))], Needleman et al., J. Mol. Biol., 48: 444-453 (1970) [The algorithm is incorporated into the GAP program in the GCG software package], Myers and Miller, CABIOS, 4: 11-17 (1988) [The algorithm is part of the CGC sequence alignment software package Embedded in the ALIGN program (version 2.0)], Pearson et al., C Proc. Natl. Acad. Sci. USA, 85: 2444-2448 1988 (1988) [the algorithm is a GCG software package. Embedded in the FASTA program in the cage] and the like, and these can be preferably used as well.
　上記(a)において、より好ましくは、「配列番号：2nで表されるアミノ酸配列と実質的に同一のアミノ酸配列」とは、配列番号：2nで表されるアミノ酸配列と、約80%以上、好ましくは約90%以上、より好ましくは約95%以上、いっそう好ましくは約97%以上、特に好ましくは約98%以上、最も好ましくは約99%以上の同一性を有するアミノ酸配列である。 In the above (a), more preferably, the “amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2n” is about 80% or more of the amino acid sequence represented by SEQ ID NO: 2n, The amino acid sequence preferably has about 90% or more, more preferably about 95% or more, still more preferably about 97% or more, particularly preferably about 98% or more, and most preferably about 99% or more.
　「配列番号：2nで表されるアミノ酸配列と同一もしくは実質的に同一のアミノ酸配列を含むタンパク質」は、配列番号：2nで表されるアミノ酸配列と実質的に同一のアミノ酸配列を含み、かつ配列番号：2nで表されるアミノ酸配列からなるタンパク質と実質的に同質の活性を有するタンパク質である。
　ここで「活性」とは、Ａβの産生を促進する活性をいう。また、「実質的に同質」とは、例えば生理学的に、あるいは薬理学的にみて、その性質が定性的に同じであることを意味する。したがって、Ａβ産生促進活性が同等であることが好ましいが、これらの活性の程度（例、約0.1～約10倍）や、タンパク質の分子量などの量的要素は異なっていてもよい。
　Ａβ産生促進活性の測定は、自体公知の方法に準じて、タンパク質の存在下および非存在下でＡβ産生量（好ましくはＡβ４０またはＡβ４２の産生量）を測定し、比較することによって行うことができる。 “A protein comprising the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 2n” includes the amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2n, and the sequence No .: A protein having substantially the same activity as the protein consisting of the amino acid sequence represented by 2n.
Here, “activity” refers to an activity that promotes the production of Aβ. Further, “substantially the same quality” means that the properties are qualitatively the same, for example, physiologically or pharmacologically. Therefore, it is preferable that the Aβ production promoting activities are equivalent, but quantitative factors such as the degree of these activities (eg, about 0.1 to about 10 times) and the molecular weight of the protein may be different.
Aβ production promoting activity can be measured by measuring and comparing the amount of Aβ produced (preferably the amount of Aβ40 or Aβ42 produced) in the presence and absence of a protein according to a method known per se. .
　また、本発明におけるタンパク質Pnとして、上記(b)に示すとおり、例えば、（i）配列番号：2nで表されるアミノ酸配列中の1～50個、好ましくは1～30個、より好ましくは1～10個、さらに好ましくは1～数（5、4、3もしくは2）個のアミノ酸が欠失したアミノ酸配列、（ii）配列番号：2nで表されるアミノ酸配列に1～50個、好ましくは1～30個、より好ましくは1～10個、さらに好ましくは1～数（5、4、3もしくは2）個のアミノ酸が付加したアミノ酸配列、（iii）配列番号：2nで表されるアミノ酸配列に1～50個、好ましくは1～30個、より好ましくは1～10個、さらに好ましくは1～数（5、4、3もしくは2）個のアミノ酸が挿入されたアミノ酸配列、（iv）配列番号：2nで表されるアミノ酸配列中の1～50個、好ましくは1～30個、より好ましくは1～10個、さらに好ましくは1～数（5、4、3もしくは2）個のアミノ酸が他のアミノ酸で置換されたアミノ酸配列、または（v）それらを組み合わせたアミノ酸配列を含有するタンパク質などのいわゆるムテインも含まれる。
　上記のようにアミノ酸配列が挿入、欠失または置換されている場合、その挿入、欠失または置換の位置は、Ａβ産生促進活性が保持される限り、特に限定されない。 In addition, as the protein Pn in the present invention, as shown in (b) above, for example, (i) 1 to 50, preferably 1 to 30, more preferably 1 in the amino acid sequence represented by SEQ ID NO: 2n ~ 10, more preferably 1 to several (5, 4, 3 or 2) amino acid sequences deleted, (ii) 1 to 50, preferably 1 to the amino acid sequence represented by SEQ ID NO: 2n An amino acid sequence to which 1 to 30, more preferably 1 to 10, more preferably 1 to several (5, 4, 3, or 2) amino acids are added; (iii) an amino acid sequence represented by SEQ ID NO: 2n An amino acid sequence into which 1 to 50, preferably 1 to 30, more preferably 1 to 10, more preferably 1 to several (5, 4, 3 or 2) amino acids are inserted, (iv) a sequence 1 to 50, preferably 1 to 30, more preferably 1 to 10, more preferably in the amino acid sequence represented by the number: 2n It is also included so-called muteins such as proteins containing 1 to several (5, 4, 3 or 2) amino acid sequence number of amino acids are substituted with other amino acids, or (v) amino acid sequence comprising a combination thereof.
When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited as long as the Aβ production promoting activity is retained.
　タンパク質P1（Probable phospholipid-transporting ATPase IIA）の好ましい例としては、例えば、配列番号：２で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_006036）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_056546）、チンパンジー（RefSeq No. XP_001168714）、イヌ（RefSeq No. XP_534457）等）、さらにはそれらのスプライスバリアント（例えば、UniProtKB Nos. O75110-1、O75110-2等）、アレル変異体、多型などがあげられる。
　タンパク質P2（BDNF/NT-3 growth factors receptor precursor）の好ましい例としては、例えば、配列番号：４で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_006171）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_001020245）、ラット（RefSeq No. NP_036863）、ウシ（RefSeq No. NP_001068693）、チンパンジー（RefSeq No. XP_001135401）、イヌ（RefSeq No. XP_856422）等）、さらにはそれらのスプライスバリアント（例えば、RefSeq No. NP_001007098、NP_001018074、NP_001018075、NP_001018076等）、アレル変異体、多型などがあげられる。
　タンパク質P3（ELAV-like protein 4）の好ましい例としては、例えば、配列番号：６で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_068771）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_034618）、ラット（RefSeq No.NP_001071119）、ウシ（RefSeq No. NP_001075075）、チンパンジー（RefSeq No. XP_001135120）、イヌ（RefSeq No. XP_849969）等）、さらにはそれらのスプライスバリアント（例えば、UniProtKB Nos. P26378-1、P26378-2、P26378-3等）、アレル変異体、多型（例えば、refSNP No. rs2494876（Pro/Ser）等）などがあげられる。
　タンパク質P4（Coiled-coil domain-containing protein 136）の好ましい例としては、例えば、配列番号：８で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_073579）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_663549）、ラット（RefSeq No. XP_342653）、ウシ（RefSeq No. XP_872956）、チンパンジー（RefSeq No. XP_519361）等）、さらにはそれらのスプライスバリアント（例えば、UniProtKB Nos. Q96JN2-1、Q96JN2-2、Q96JN2-3、Q96JN2-4等）、アレル変異体、多型（例えば、refSNP Nos. rs3816887（Asp/His）、rs4728137（Glu/Gln）、rs8180870（Thr/Ala）等）などがあげられる。
　タンパク質P5（Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2）の好ましい例としては、例えば、配列番号：１０で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_001185）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_032252）、ラット（RefSeq No. NP_446136）、イヌ（RefSeq No. XP_855233）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などがあげられる。
　タンパク質P6（DnaJ homolog subfamily A member 2）の好ましい例としては、例えば、配列番号：１２で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_005871）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_062768）、ラット（RefSeq No. NP_114468）、ウシ（RefSeq No. NP_001035581）、チンパンジー（RefSeq No. XP_528644）、イヌ（RefSeq No. XP_535319）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型（例えば、refSNP No. rs1065332（Gly/Cys）等）などがあげられる。
　タンパク質P7（Vesicle-associated membrane protein-associated protein A）の好ましい例としては、例えば、配列番号：１４で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_003565）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_038961）、ラット（RefSeq No. NP_113819）、ウシ（RefSeq No. NP_001069201）、チンパンジー（RefSeq No. XP_512022）、イヌ（RefSeq No. XP_537332）等）、さらにはそれらのスプライスバリアント（例えば、RefSeq No. NP_919415）、アレル変異体、多型などがあげられる。
　タンパク質P8（Proton myo-inositol cotransporter）の好ましい例としては、例えば、配列番号：１６で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_443117）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_001028805）、ラット（RefSeq No. NP_598295）、ウシ（RefSeq No. XP_869569）、チンパンジー（RefSeq No. XP_522353）、イヌ（RefSeq No. XP_543735）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などがあげられる。
　タンパク質P9（Leukocyte surface antigen CD47）の好ましい例としては、例えば、配列番号：１８で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_001768）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_034711）、ラット（RefSeq No. NP_062068）、ウシ（RefSeq No. NP_777133）、チンパンジー（RefSeq No. XP_516636）、イヌ（RefSeq No. NP_001074190）等）、さらにはそれらのスプライスバリアント（例えば、UniProtKB Nos. Q08722-1、Q08722-2、Q08722-3、Q08722-4等）、アレル変異体、多型（例えば、refSNP No. rs11546646（Pro/Ala）等）などがあげられる。
　タンパク質P10（Flotillin-1）の好ましい例としては、例えば、配列番号：２０で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_005794）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_032053）、ラット（RefSeq No. NP_073192）、ウシ（RefSeq No. NP_001070355）、チンパンジー（RefSeq No. NP_001035840）、イヌ（RefSeq No. XP_848778）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型（例えば、refSNP No. rs1802470（Asp/Tyr）等）などがあげられる。
　タンパク質P11（Band 4.1 like 1）の好ましい例としては、例えば、配列番号：２２で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_818932）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、ラット（RefSeq No.  NP_067713）、ウシ（RefSeq No. XP_872829）、チンパンジー（RefSeq No. XP_525314）、イヌ（RefSeq No. NP_XP_542979）等）、さらにはそれらのスプライスバリアント（例えば、UniProtKB Nos. Q9H4G0-1、Q9H4G0-2、Q9H4G0-3、Q9H4G0-4等）、アレル変異体、多型（例えば、refSNP No. rs6089009（Lys/Arg）等）などがあげられる。
　タンパク質P12（Regulator of G-protein signaling 7）の好ましい例としては、例えば、配列番号：２４で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_002915）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_036010）、ラット（RefSeq No. NP_062216）、ウシ（RefSeq No. NP_776594）、チンパンジー（RefSeq No. XP_001159739）等）、さらにはそれらのスプライスバリアント（例えば、UniProtKB Nos. P49802-1、P49802-2、P49802-3、P49802-4、P49802-5等）、アレル変異体、多型（例えば、refSNP No. rs12746550（Met/Leu）、rs17851953（Gln/His）等）などがあげられる。
　タンパク質P13（Phospholipase D3）の好ましい例としては、例えば、配列番号：２６で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_001026866）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_035246）、ラット（RefSeq No. NP_001012167）、チンパンジー（RefSeq No. XP_524268）、イヌ（RefSeq No. XP_541616）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などがあげられる。
　タンパク質P14（Ectoderm-neural cortex protein 1）の好ましい例としては、例えば、配列番号：２８で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_003624）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_031956）、ラット（RefSeq No. NP_001003401）、チンパンジー（RefSeq No. XP_526897）、イヌ（RefSeq No. XP_544373）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型（例えば、refSNP No. rs11557041（Phe/Leu）、rs11747630（Thr/Asn）、rs16872126（Ile/Ser）等）などがあげられる。
　タンパク質P15（synaptophysin）の好ましい例としては、例えば、配列番号：３０で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_003170）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_033331）、ラット（RefSeq No. NP_036796）、ウシ（RefSeq No. NP_776388）、チンパンジー（RefSeq No. XP_521056）、イヌ（RefSeq No. XP_855381）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などがあげられる。
　タンパク質P16（solute carrier family 2 (facilitated glucose transporter), member 3）の好ましい例としては、例えば、配列番号：３２で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_008862）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_035531）、ラット（RefSeq No. NP_058798）、ウシ（RefSeq No. NP_777028）、チンパンジー（RefSeq No. XP_508989）、イヌ（RefSeq No. NP_001003308）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型（例えば、refSNP No. rs17728193（Val/Leu）等）などがあげられる。
　タンパク質P17（syntaxin binding protein 1）の好ましい例としては、例えば、配列番号：３４で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_001027392）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_033321）、ラット（RefSeq No. NP_037170）、ウシ（RefSeq No. NP_777044）等）、さらにはそれらのスプライスバリアント（例えば、UniProtKB Nos. P61764-1、P61764-2等）、アレル変異体、多型などがあげられる。
　タンパク質P18（growth associated protein 43）の好ましい例としては、例えば、配列番号：３６で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_002036）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_032109）、ラット（RefSeq No. NP_058891）、ウシ（RefSeq No. NP_976234）、チンパンジー（RefSeq No. NP_001009816）、イヌ（RefSeq No. XP_535747等）、さらにはそれらのスプライスバリアント、アレル変異体、多型（例えば、refSNP No. rs6291（Val/Ile）、rs11557762（Lys/Glu）、rs11557763（Glu/Asp）等）などがあげられる。
　タンパク質P19（ATPase, Na+/K+ transporting, beta 1 polypeptide）の好ましい例としては、例えば、配列番号：３８で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_001668）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_033851）、ラット（RefSeq No. NP_037245）、ウシ（RefSeq No. NP_001030411）、チンパンジー（RefSeq No. NP_001092025）、イヌ（RefSeq No. NP_001003283）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型（例えば、refSNP No. rs11545848（Lys/Gln）、rs1545849（Met/Val）等）などがあげられる。
　タンパク質P20（Amine oxidase [flavin-containing] B）の好ましい例としては、例えば、配列番号：４０で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_000889）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_766366）、ラット（RefSeq No. NP_037330）、ウシ（RefSeq No. NP_808813）、イヌ（RefSeq No. XP_855549）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型（例えば、refSNP No. rs12845783（Ser/Arg）等）などがあげられる。
　タンパク質P21（Muscarinic acetylcholine receptor M1）の好ましい例としては、例えば、配列番号：４２で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_000729）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_031724）、ラット（RefSeq No. NP_542951）、ウシ（RefSeq No XP_001255658）、チンパンジー（RefSeq No. XP_001159938）、イヌ（RefSeq No. XP_540897）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などがあげられる。
　タンパク質P22（Probable cationic amino acid transporter）の好ましい例としては、例えば、配列番号：４４で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_066000）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_766449）、ラット（RefSeq No. NP_001128087）、チンパンジー（RefSeq No. XP_526378）、イヌ（RefSeq No. XP_545285）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型（例えば、refSNP No. rs2276717（Gly/Arg）等）などがあげられる。
　タンパク質P23（NADH dehydrogenase iron-sulfur protein 7）の好ましい例としては、例えば、配列番号：４６で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_077718）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_083548）、ラット（RefSeq No. NP_001008525）、ウシ（RefSeq No. NP_001033111）、イヌ（RefSeq No. XP_533960）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型（例えば、refSNP No. rs1142530（Pro/Leu）等）などがあげられる。
　タンパク質P24（G-protein-regulated inducer of neurite outgrowth）の好ましい例としては、例えば、配列番号：４８で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_443131）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_036144）、ラット（RefSeq No. XP_344572）等）、さらにはそれらのスプライスバリアント（例えば、UniProtKB Nos. Q7Z2K8-1、Q7Z2K8-2等）、アレル変異体、多型（例えば、refSNP No. rs17854765（Pro/Thr）、rs6556276（Met/Val）、refSNP No. rs10037225（Gly/Ala）、rs34285890（Ser/Pro）等）などがあげられる。
　タンパク質P25（Probable phospholipid-transporting ATPase 1A）の好ましい例としては、例えば、配列番号：５０で表されるアミノ酸配列からなるヒトタンパク質（RefSeq No. NP_001098999）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NP_001034088）、ウシ（RefSeq No. NP_777263）、チンパンジー（RefSeq No. XP_001150768）、イヌ（RefSeq No. XP_849357）等）、さらにはそれらのスプライスバリアント（例えば、UniProtKB Nos. Q9Y2Q0-1、Q9Y2Q0-2等）、アレル変異体、多型（例えば、refSNP No. rs３７９２６８７（Thr/Met）等）などがあげられる。 Preferred examples of the protein P1 (Probable phospholipid-transporting ATPase IIA) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (RefSeq No. NP_006036), or their orthologs in other mammals (for example, , Mice (RefSeq No. NP_056546), chimpanzees (RefSeq No. XP_001168714), dogs (RefSeq No. XP_534457), and their splice variants (eg UniProtKB Nos. O75110-1, O75110-2 etc.), alleles Examples include mutants and polymorphisms.
Preferred examples of the protein P2 (BDNF / NT-3 growth factors receptor precursor) include, for example, a human protein (RefSeq No. NP_006171) consisting of the amino acid sequence represented by SEQ ID NO: 4, or those in other mammals. Orthologs (eg, mice (RefSeq No. NP_001020245), rats (RefSeq No. NP_036863), cows (RefSeq No. NP_001068693), chimpanzees (RefSeq No. XP_001135401), dogs (RefSeq No. XP_856422) etc.), and those Examples include splice variants (for example, RefSeq No. NP_001007098, NP_001018074, NP_001018075, NP_001018076, etc.), allelic variants, polymorphisms, and the like.
Preferred examples of the protein P3 (ELAV-like protein 4) include, for example, a human protein (RefSeq No. NP_068771) consisting of the amino acid sequence represented by SEQ ID NO: 6, or an ortholog thereof (for example, Mice (RefSeq No. NP_034618), rats (RefSeq No.NP_001071119), cows (RefSeq No. NP_001075075), chimpanzees (RefSeq No. XP_001135120), dogs (RefSeq No. XP_849969), etc., and their splice variants (for example, UniProtKB Nos. P26378-1, P26378-2, P26378-3, etc.), allelic variants, polymorphisms (for example, refSNP No. rs2494876 (Pro / Ser), etc.).
Preferred examples of protein P4 (Coiled-coil domain-containing protein 136) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 8 (RefSeq No. NP_073579), or an ortholog thereof in other mammals (For example, mouse (RefSeq No. NP_663549), rat (RefSeq No. XP_342653), cattle (RefSeq No. XP_872956), chimpanzee (RefSeq No. XP_519361), etc.) and their splice variants (for example, UniProtKB Nos. Q96JN2 -1, Q96JN2-2, Q96JN2-3, Q96JN2-4, etc.), allelic variants, polymorphisms (eg refSNP Nos. Rs3816887 (Asp / His), rs4728137 (Glu / Gln), rs8180870 (Thr / Ala), etc.) ) Etc.
Preferred examples of the protein P5 (Potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 2) include, for example, a human protein (RefSeq No. NP_001185) consisting of the amino acid sequence represented by SEQ ID NO: 10, or other mammals Orthologs (eg mice (RefSeq No. NP_032252), rats (RefSeq No. NP_446136), dogs (RefSeq No. XP_855233), etc.), and their splice variants, allelic variants, polymorphisms, etc. .
Preferred examples of the protein P6 (DnaJ homolog subfamily A member 2) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 12 (RefSeq No. NP_005871), or an ortholog thereof in other mammals (for example, , Mice (RefSeq No. NP_062768), rats (RefSeq No. NP_114468), cattle (RefSeq No. NP_001035581), chimpanzees (RefSeq No. XP_528644), dogs (RefSeq No. XP_535319)), and their splice variants, Examples include allelic variants and polymorphisms (eg, refSNP No. rs1065332 (Gly / Cys)).
Preferred examples of protein P7 (Vesicle-associated membrane protein-associated protein A) include, for example, a human protein (RefSeq No. NP_003565) consisting of the amino acid sequence represented by SEQ ID NO: 14, or those in other mammals. Orthologs (for example, mice (RefSeq No. NP_038961), rats (RefSeq No. NP_113819), cattle (RefSeq No. NP_001069201), chimpanzees (RefSeq No. XP_512022), dogs (RefSeq No. XP_537332), etc.) Examples include splice variants (for example, RefSeq No. NP_919415), allelic variants, and polymorphisms.
Preferred examples of the protein P8 (Proton myo-inositol cotransporter) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 16 (RefSeq No. NP_443117), or an ortholog thereof in other mammals (for example, Mice (RefSeq No. NP_001028805), rats (RefSeq No. NP_598295), cattle (RefSeq No. XP_869569), chimpanzees (RefSeq No. XP_522353), dogs (RefSeq No. XP_543735), etc., and their splice variants and alleles Examples include mutants and polymorphisms.
Preferred examples of the protein P9 (Leukocyte surface antigen CD47) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 18 (RefSeq No. NP_001768), or an ortholog thereof (for example, mouse) in other mammals (RefSeq No. NP_034711), rat (RefSeq No. NP_062068), cattle (RefSeq No. NP_777133), chimpanzee (RefSeq No. XP_516636), dog (RefSeq No. NP_001074190), etc.) and their splice variants (for example, UniProtKB Nos. Q08722-1, Q08722-2, Q08722-3, Q08722-4, etc.), allelic variants, polymorphisms (for example, refSNP No. rs11546646 (Pro / Ala), etc.).
Preferred examples of the protein P10 (Flotillin-1) include, for example, a human protein (RefSeq No. NP_005794) consisting of the amino acid sequence represented by SEQ ID NO: 20, or an ortholog thereof (for example, mouse ( RefSeq No. NP_032053), rat (RefSeq No. NP_073192), cattle (RefSeq No. NP_001070355), chimpanzee (RefSeq No. NP_001035840), dog (RefSeq No. XP_848778) etc.), and their splice variants and allelic variants And polymorphism (for example, refSNP No. rs1802470 (Asp / Tyr) etc.) and the like.
Preferred examples of the protein P11 (Band 4.1 like 1) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 22 (RefSeq No. NP_818932), or an ortholog thereof (eg, rat) in other mammals. (RefSeq No. NP_067713), cattle (RefSeq No. XP_872829), chimpanzee (RefSeq No. XP_525314), dogs (RefSeq No. NP_XP_542979), etc.) and their splice variants (for example, UniProtKB Nos. Q9H4G0-1, Q9H4G0) -2, Q9H4G0-3, Q9H4G0-4, etc.), allelic variants, polymorphisms (for example, refSNP No. rs6089009 (Lys / Arg), etc.).
Preferred examples of the protein P12 (Regulator of G-protein signaling 7) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 24 (RefSeq No. NP_002915), or their orthologs in other mammals (Ref. For example, mice (RefSeq No. NP_036010), rats (RefSeq No. NP_062216), cattle (RefSeq No. NP_776594), chimpanzees (RefSeq No. XP_001159739), and their splice variants (for example, UniProtKB Nos. P49802- 1, P49802-2, P49802-3, P49802-4, P49802-5, etc.), allelic variants, polymorphisms (eg refSNP No. rs12746550 (Met / Leu), rs17851953 (Gln / His), etc.) It is done.
Preferred examples of the protein P13 (Phospholipase D3) include, for example, a human protein (RefSeq No. NP_001026866) consisting of the amino acid sequence represented by SEQ ID NO: 26, or an ortholog (eg, mouse (RefSeq) in other mammals. No. NP_035246), rat (RefSeq No. NP_001012167), chimpanzee (RefSeq No. XP_524268), dog (RefSeq No. XP_541616) and the like, and their splice variants, allelic variants, and polymorphisms.
Preferred examples of the protein P14 (Ectoderm-neural cortex protein 1) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 28 (RefSeq No. NP_003624), or an ortholog thereof in other mammals (for example, , Mice (RefSeq No. NP_031956), rats (RefSeq No. NP_001003401), chimpanzees (RefSeq No. XP_526897), dogs (RefSeq No. XP_544373), etc.) and their splice variants, allelic variants, polymorphisms (for example, RefSNP No. rs11557041 (Phe / Leu), rs11747630 (Thr / Asn), rs16872126 (Ile / Ser), etc.).
Preferable examples of the protein P15 (synaptophysin) include, for example, a human protein (RefSeq No. NP_003170) consisting of the amino acid sequence represented by SEQ ID NO: 30, or an ortholog thereof (eg, mouse (RefSeq No) in other mammals) NP_033331), rats (RefSeq No. NP_036796), cattle (RefSeq No. NP_776388), chimpanzees (RefSeq No. XP_521056), dogs (RefSeq No. XP_855381), etc.) and their splice variants, allelic variants, many Examples include molds.
Preferred examples of the protein P16 (solute carrier family 2 (facilitated glucose transporter), member 3) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 32 (RefSeq No. NP_008862), or other mammals Orthologs of them (eg, mice (RefSeq No. NP_035531), rats (RefSeq No. NP_058798), cattle (RefSeq No. NP_777028), chimpanzees (RefSeq No. XP_508989), dogs (RefSeq No. NP_001003308), etc.) These include splice variants, allelic variants, and polymorphisms (eg, refSNP No. rs17728193 (Val / Leu)).
Preferred examples of the protein P17 (syntaxin binding protein 1) include, for example, a human protein (RefSeq No. NP_001027392) consisting of the amino acid sequence represented by SEQ ID NO: 34, or an ortholog thereof (for example, mouse) in other mammals (RefSeq No. NP_033321), rat (RefSeq No. NP_037170), bovine (RefSeq No. NP_777044), etc., and their splice variants (eg UniProtKB Nos. P61764-1, P61764-2 etc.), allelic variants And polymorphism.
Preferred examples of the protein P18 (growth associated protein 43) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 36 (RefSeq No. NP_002036), or an ortholog thereof (for example, mouse) in other mammals (RefSeq No. NP_032109), rat (RefSeq No. NP_058891), cow (RefSeq No. NP_976234), chimpanzee (RefSeq No. NP_001009816), dog (RefSeq No. XP_535747 etc.), and their splice variants and allelic variants And polymorphism (for example, refSNP No. rs6291 (Val / Ile), rs11557762 (Lys / Glu), rs11557763 (Glu / Asp), etc.) and the like.
Preferred examples of protein P19 (ATPase, Na + / K + transporting, beta 1 polypeptide) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 38 (RefSeq No. NP_001668), or those in other mammals Orthologs (eg, mice (RefSeq No. NP_033851), rats (RefSeq No. NP_037245), cattle (RefSeq No. NP_001030411), chimpanzees (RefSeq No. NP_001092025), dogs (RefSeq No. NP_001003283), etc.) Splice variants, allelic variants, polymorphisms (for example, refSNP No. rs11545848 (Lys / Gln), rs1545849 (Met / Val), etc.) and the like.
Preferred examples of the protein P20 (Amine oxidase [flavin-containing] B) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 40 (RefSeq No. NP_000889), or an ortholog thereof in other mammals (For example, mice (RefSeq No. NP_766366), rats (RefSeq No. NP_037330), cattle (RefSeq No. NP_808813), dogs (RefSeq No. XP_855549), etc.), and their splice variants, allelic variants, and polymorphisms (For example, refSNP No. rs12845783 (Ser / Arg)).
Preferred examples of protein P21 (Muscarinic acetylcholine receptor M1) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 42 (RefSeq No. NP_000729), or an ortholog thereof (for example, mouse) in other mammals (RefSeq No. NP_031724), rat (RefSeq No. NP_542951), cattle (RefSeq No XP_001255658), chimpanzee (RefSeq No. XP_001159938), dog (RefSeq No. XP_540897), etc.) and their splice variants and allelic variants And polymorphism.
Preferred examples of the protein P22 (Probable reactive amino acid transporter) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 44 (RefSeq No. NP_066000), or an ortholog thereof in other mammals (for example, Mice (RefSeq No. NP_766449), rats (RefSeq No. NP_001128087), chimpanzees (RefSeq No. XP_526378), dogs (RefSeq No. XP_545285), etc., as well as their splice variants, allelic variants, polymorphisms (for example, refSNP No. rs2276717 (Gly / Arg), etc.).
Preferred examples of the protein P23 (NADH dehydrogenase iron-sulfur protein 7) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 46 (RefSeq No. NP_077718), or an ortholog thereof in other mammals ( For example, mice (RefSeq No. NP_083548), rats (RefSeq No. NP_001008525), cattle (RefSeq No. NP_001033111), dogs (RefSeq No. XP_533960), etc.), as well as their splice variants, allelic variants, polymorphisms ( For example, refSNP No. rs1142530 (Pro / Leu) etc.).
Preferred examples of the protein P24 (G-protein-regulated inducer of neurite outgrowth) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 48 (RefSeq No. NP_443131), or those in other mammals Orthologs (eg, mouse (RefSeq No. NP_036144), rat (RefSeq No. XP_344572), etc.) and their splice variants (eg, UniProtKB Nos. Q7Z2K8-1, Q7Z2K8-2 etc.), allelic variants, polymorphisms (For example, refSNP No. rs17854765 (Pro / Thr), rs6556276 (Met / Val), refSNP No. rs10037225 (Gly / Ala), rs34285890 (Ser / Pro), etc.)).
Preferred examples of the protein P25 (Probable phospholipid-transporting ATPase 1A) include, for example, a human protein consisting of the amino acid sequence represented by SEQ ID NO: 50 (RefSeq No. NP_001098999), or an ortholog thereof in other mammals (for example, , Mice (RefSeq No. NP_001034088), cattle (RefSeq No. NP_777263), chimpanzees (RefSeq No. XP_001150768), dogs (RefSeq No. XP_849357), and their splice variants (eg UniProtKB Nos. Q9Y2Q0-1) , Q9Y2Q0-2, etc.), allelic variants, polymorphisms (for example, refSNP No. rs3792687 (Thr / Met) etc.) and the like.
　本発明において「タンパク質Pnの発現を抑制する物質」とは、タンパク質Pnをコードする遺伝子Gnの転写レベル、転写後調節のレベル、タンパク質Pnへの翻訳レベル、翻訳後修飾のレベル等のいかなる段階で作用するものであってもよい。従って、タンパク質Pnの発現を抑制する物質としては、例えば、遺伝子Gnの転写を阻害する物質（例、アンチジーン）、初期転写産物からmRNAへのプロセッシングを阻害する物質、mRNAの細胞質への輸送を阻害する物質、mRNAからタンパク質Pnへの翻訳を阻害するか（例、アンチセンス核酸、miRNA）あるいはmRNAを分解する（例、siRNA、リボザイム、miRNA）物質、初期翻訳産物の翻訳後修飾を阻害する物質などが含まれる。いずれの段階で作用するものであっても好ましく用いることができるが、mRNAに相補的に結合してタンパク質Pnへの翻訳を阻害するかあるいはmRNAを分解する物質が好ましい。 In the present invention, the “substance that suppresses the expression of protein Pn” refers to the transcription level of the gene Gn encoding protein Pn, the level of post-transcriptional regulation, the level of translation into protein Pn, the level of post-translational modification, etc. It may act. Therefore, substances that suppress the expression of protein Pn include, for example, substances that inhibit the transcription of gene Gn (eg, antigene), substances that inhibit the processing of early transcription products into mRNA, and transport of mRNA to the cytoplasm. Inhibiting substances, inhibiting translation of mRNA to protein Pn (eg, antisense nucleic acid, miRNA) or degrading mRNA (eg, siRNA, ribozyme, miRNA), inhibiting post-translational modification of the initial translation product Substances are included. Any substance that acts at any stage can be preferably used, but a substance that complementarily binds to mRNA and inhibits translation into protein Pn or decomposes mRNA is preferable.
　遺伝子GnのmRNAからタンパク質Pnへの翻訳を特異的に阻害する（あるいはmRNAを分解する）物質として、好ましくは、これらのmRNAの塩基配列と相補的もしくは実質的に相補的な塩基配列またはその一部を含む核酸が挙げられる。
　遺伝子GnのmRNAの塩基配列と実質的に相補的な塩基配列とは、哺乳動物の生理的条件下において、該mRNAの標的配列に結合してその翻訳を阻害し得る（あるいは該標的配列を切断する）程度の相補性を有する塩基配列を意味し、具体的には、例えば、該mRNAの塩基配列と完全相補的な塩基配列（すなわち、mRNAの相補鎖の塩基配列）と、オーバーラップする領域に関して、約80％以上、好ましくは約90％以上、より好ましくは約95％以上、特に好ましくは約97％以上の相同性を有する塩基配列である。
　本発明における「塩基配列の相同性」は、相同性計算アルゴリズムNCBI BLAST（National Center for Biotechnology Information Basic Local Alignment Search Tool）を用い、以下の条件（期待値=10；ギャップを許す；フィルタリング=ON；マッチスコア=1；ミスマッチスコア=-3）にて計算することができる。 As a substance that specifically inhibits the translation of mRNA of gene Gn from protein Pn (or degrades mRNA), preferably a base sequence complementary to or substantially complementary to the base sequence of these mRNAs or one of them A nucleic acid containing a portion.
The base sequence substantially complementary to the base sequence of mRNA of gene Gn can bind to the target sequence of mRNA and inhibit its translation under physiological conditions in mammals (or cleave the target sequence). A base sequence having a degree of complementarity, specifically, for example, a region that overlaps with a base sequence that is completely complementary to the base sequence of the mRNA (that is, a base sequence of the complementary strand of the mRNA). The nucleotide sequence having a homology of about 80% or more, preferably about 90% or more, more preferably about 95% or more, and particularly preferably about 97% or more.
The “base sequence homology” in the present invention uses the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) and the following conditions (expected value = 10; allow gaps; filtering = ON; It can be calculated by match score = 1; mismatch score = -3).
　より具体的には、遺伝子GnのmRNAの塩基配列と相補的もしくは実質的に相補的な塩基配列としては、(a)配列番号：2n-1（nは1～25のいずれかの整数）で表される塩基配列と相補的もしくは実質的に相補的な塩基配列、または(b)「配列番号：2n-1（nは1～25のいずれかの整数）で表される塩基配列の相補鎖とストリンジェントな条件下でハイブリダイズする塩基配列であって、配列番号：2n（nは1～25のいずれかの整数）で表されるアミノ酸配列からなるタンパク質と実質的に同質の活性を有するタンパク質をコードする配列」と、相補的もしくは実質的に相補的な塩基配列が挙げられる。ここで「実質的に同質の活性」とは前記と同義である。
　ストリンジェントな条件とは、例えば、Current Protocols in Molecular Biology, John Wiley & Sons,6.3.1-6.3.6, 1999に記載される条件、例えば、6×SSC（sodium chloride/sodium citrate）／45℃でのハイブリダイゼーション、次いで0.2×SSC／0.1％ SDS／50～65℃での一回以上の洗浄等が挙げられるが、当業者であれば、これと同等のストリンジェンシーを与えるハイブリダイゼーションの条件を適宜選択することができる。 More specifically, the base sequence complementary or substantially complementary to the base sequence of the gene Gn mRNA is (a) SEQ ID NO: 2n-1 (n is any integer from 1 to 25) A complementary or substantially complementary nucleotide sequence to the nucleotide sequence represented, or (b) a complementary strand of the nucleotide sequence represented by "SEQ ID NO: 2n-1 (n is any integer from 1 to 25)" Is a base sequence that hybridizes under stringent conditions, and has substantially the same quality of activity as a protein consisting of the amino acid sequence represented by SEQ ID NO: 2n (n is an integer from 1 to 25) Examples of the sequence that encodes a protein include complementary or substantially complementary base sequences. Here, “substantially the same quality of activity” has the same meaning as described above.
The stringent conditions are, for example, the conditions described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999, for example, 6 × SSC (sodium chloride / sodium citrate) / 45 ° C. Hybridization, followed by one or more washes at 0.2 × SSC / 0.1% SDS / 50 to 65 ° C., and those skilled in the art will know the conditions for hybridization that give the same stringency. It can be selected appropriately.
　遺伝子G1（ATP9A）のmRNAの好ましい例としては、配列番号：１で表される塩基配列を含むヒトATP9A（RefSeq Accession No. NM_006045）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_015731）、チンパンジー（RefSeq No. XM_001168714）、イヌ（RefSeq No. XM_534457）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などのmRNAがあげられる。
　遺伝子G2（NTRK2）のmRNAの好ましい例としては、例えば、配列番号：３で表される塩基酸配列からなるヒトNTRK2（RefSeq No. NM_006180）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_001025074）、ラット（RefSeq No. NM_012731）、ウシ（RefSeq No. NM_001075225）、チンパンジー（RefSeq No. XM_001135401）、イヌ（RefSeq No. XM_851329）等）、さらにはそれらのスプライスバリアント（例えば、RefSeq No. NM_001007097、NM_001018064、NM_001018065、NM_001018066等）、アレル変異体、多型などのmRNAがあげられる。
　遺伝子G3（ELAVL4）のmRNAの好ましい例としては、例えば、配列番号：５で表される塩基配列からなるヒトELAVL4（RefSeq No. NM_021952）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_010488）、ラット（RefSeq No. NM_001077651）、ウシ（RefSeq No. NM_001081606）、チンパンジー（RefSeq No. XM_001135120）、イヌ（RefSeq No. XM_844876）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型（例えば、refSNP No. rs2494876（C/T）等）などのmRNAがあげられる。
　遺伝子G4（NAG6）のmRNAの好ましい例としては、例えば、配列番号：７で表される塩基配列からなるヒトNAG6（RefSeq No. NM_022742）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_145574）、ラット（RefSeq No. XM_342652）、ウシ（RefSeq No. XM_867863）、チンパンジー（RefSeq No. XM_519361）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型（例えば、refSNP Nos. rs3816887（C/G）、rs4728137（G/C）、rs8180870（A/G）等）などのmRNAがあげられる。
　遺伝子G5（HCN2）のmRNAの好ましい例としては、例えば、配列番号：９で表される塩基配列からなるヒトHCN2（RefSeq No. NM_001194）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_008226）、ラット（RefSeq No. NM_053684）、イヌ（RefSeq No. XM_850140）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などのmRNAがあげられる。
　遺伝子G6（DNAJA2）のmRNAの好ましい例としては、例えば、配列番号：１１で表される塩基配列からなるヒトDNAJA2（RefSeq No. NM_005880）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_019794）、ラット（RefSeq No. NM_032079）、ウシ（RefSeq No. NM_001040491）、チンパンジー（RefSeq No. XM_528644）、イヌ（RefSeq No. XM_535319）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型（例えば、refSNP No. rs1065332（G/T）等）などのmRNAがあげられる。
　遺伝子G7（VAPA）のmRNAの好ましい例としては、例えば、配列番号：１３で表される塩基配列からなるヒトVAPA（RefSeq No. NM_003574）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_013933）、ラット（RefSeq No. NM_031631）、ウシ（RefSeq No. NM_001075733）、チンパンジー（RefSeq No. XM_512022）、イヌ（RefSeq No. XM_537332）等）、さらにはそれらのスプライスバリアント（例えば、RefSeq No. NM_194434）、アレル変異体、多型などのmRNAがあげられる。
　遺伝子G8（SLC2A13）のmRNAの好ましい例としては、例えば、配列番号：１５で表される塩基配列からなるヒトSLC2A13（RefSeq No. NM_052885）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_001033633）、ラット（RefSeq No. NM_133611）、ウシ（RefSeq No. XM_864476）、チンパンジー（RefSeq No. XM_522353）、イヌ（RefSeq No. XM_543735）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などのmRNAがあげられる。
　遺伝子G9（CD47）のmRNAの好ましい例としては、例えば、配列番号：１７で表される塩基配列からなるヒトCD47（RefSeq No. NM_001777）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_010581）、ラット（RefSeq No. NM_019195）、ウシ（RefSeq No. NM_174708）、チンパンジー（RefSeq No. XM_516636）、イヌ（RefSeq No. NM_001080721）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型（例えば、refSNP No. rs11546646（G/C）等）などのmRNAがあげられる。
　遺伝子G10（FLOT1）のmRNAの好ましい例としては、例えば、配列番号：１９で表される塩基配列からなるヒトFLOT1（RefSeq No. NM_005803）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_008027）、ラット（RefSeq No. NM_022701）、ウシ（RefSeq No. NM_001076887）、チンパンジー（RefSeq No. NM_001042381）、イヌ（RefSeq No. XM_843685）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型（例えば、refSNP No. rs1802470（G/T）等）などのmRNAがあげられる。
　遺伝子G11（EPB41L1）のmRNAの好ましい例としては、配列番号：２１で表される塩基配列を含むヒトEPB41L1（RefSeq Accession No. NM_177996）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、ラット（RefSeq No. NM_021681）、ウシ（RefSeq No. XM_867736）、チンパンジー（RefSeq No. XM_525314）、イヌ（RefSeq No. XM_542979）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などのmRNAがあげられる。
　遺伝子G12（RGS7）のmRNAの好ましい例としては、配列番号：２３で表される塩基配列を含むヒトRGS7（RefSeq Accession No. NM_002924）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_011880）、ラット（RefSeq No. NM_019343）、ウシ（RefSeq No. NM_174169）、チンパンジー（RefSeq No. XM_001159739）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などのmRNAがあげられる。
　遺伝子G13（PLD3）のmRNAの好ましい例としては、配列番号：２５で表される塩基配列を含むヒトPLD3（RefSeq Accession No. NM_001031696）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_011116）、ラット（RefSeq No. NM_001012167）、チンパンジー（RefSeq No. XM_524268）、イヌ（RefSeq No. XM_541616）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などのmRNAがあげられる。
　遺伝子G14（ENC1）のmRNAの好ましい例としては、配列番号：２７で表される塩基配列を含むヒトENC1（RefSeq Accession No. NM_003633）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_007930）、ラット（RefSeq No. NM_001003401）、チンパンジー（RefSeq No. XM_526897）、イヌ（RefSeq No. XM_544373）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などのmRNAがあげられる。
　遺伝子G15（SYP）のmRNAの好ましい例としては、配列番号：２９で表される塩基配列を含むヒトSYP（RefSeq Accession No. NM_003179）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_009305）、ラット（RefSeq No. NM_012664）、ウシ（RefSeq No. NM_173963）、チンパンジー（RefSeq No. XM_521056）、イヌ（RefSeq No. XM_850288）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などのmRNAがあげられる。
　遺伝子G16（SLC2A3）のmRNAの好ましい例としては、配列番号：３１で表される塩基配列を含むヒトSLC2A3（RefSeq Accession No. NM_006931）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_011401）、ラット（RefSeq No. NM_017102）、ウシ（RefSeq No. NM_174603）、チンパンジー（RefSeq No. XM_508989）、イヌ（RefSeq No. NM_001003308）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などのmRNAがあげられる。
　遺伝子G17（STXBP1）のmRNAの好ましい例としては、配列番号：３３で表される塩基配列を含むヒトSTXBP1（RefSeq Accession No. NM_001032221）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_009295）、ラット（RefSeq No. NM_013038）、ウシ（RefSeq No. NM_174619）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などのmRNAがあげられる。
　遺伝子G18（GAP43）のmRNAの好ましい例としては、配列番号：３５で表される塩基配列を含むヒトGAP43（RefSeq Accession No. NM_002045）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_008083）、ラット（RefSeq No. NM_017195）、ウシ（RefSeq No. NM_203358）、チンパンジー（RefSeq No. NM_001009816）、イヌ（RefSeq No. XM_535747）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などのmRNAがあげられる。
　遺伝子G19（ATP1B1）のmRNAの好ましい例としては、配列番号：３７で表される塩基配列を含むヒトATP1B1（RefSeq Accession No. NM_001677）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_009721）、ラット（RefSeq No. NM_013113）、ウシ（RefSeq No. NM_001035334）、チンパンジー（RefSeq No. NM_001098555）、イヌ（RefSeq No. NM_001003283）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などのmRNAがあげられる。
　遺伝子G20（MAOB）のmRNAの好ましい例としては、配列番号：３９で表される塩基配列を含むヒトMAOB（RefSeq Accession No. NM_000898）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_172778）、ラット（RefSeq No. NM_013198）、ウシ（RefSeq No. NM_177944）、イヌ（RefSeq No. XM_850456）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などのmRNAがあげられる。
　遺伝子G21（CHRM1）のmRNAの好ましい例としては、配列番号：４１で表される塩基配列を含むヒトCHRM1（RefSeq Accession No. NM_000738）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_007698）、ラット（RefSeq No. NM_080773）、ウシ（RefSeq No. XM_001255657）、チンパンジー（RefSeq No. XM_001159938）、イヌ（RefSeq No. XM_540897）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などのmRNAがあげられる。
　遺伝子G22（SLC7A14）のmRNAの好ましい例としては、配列番号：４３で表される塩基配列を含むヒトSLC7A14（RefSeq Accession No. NM_020949）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_172861）、ラット（RefSeq No. NM_001134615）、チンパンジー（RefSeq No. XM_526378）、イヌ（RefSeq No. XM_545285）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などのmRNAがあげられる。
　遺伝子G23（NDUFS7）のmRNAの好ましい例としては、配列番号：４５で表される塩基配列を含むヒトNDUFS7（RefSeq Accession No. NM_024407）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_029272）、ラット（RefSeq No. NM_001008525）、ウシ（RefSeq No. NM_001038022）、イヌ（RefSeq No. XM_533960）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などのmRNAがあげられる。
　遺伝子G24（GPRIN1）のmRNAの好ましい例としては、配列番号：４７で表される塩基配列を含むヒトGPRIN1（RefSeq Accession No. NM_052899）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_012014）、ラット（RefSeq No. XM_344571）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などのmRNAがあげられる。
　遺伝子G25（ATP8A1）のmRNAの好ましい例としては、配列番号：４９で表される塩基配列を含むヒトATP8A1（RefSeq Accession No. NM_001105529）、あるいは他の哺乳動物におけるそれらのオルソログ（例えば、マウス（RefSeq No. NM_001038999）、ウシ（RefSeq No. NM_174838）、チンパンジー（RefSeq No. XM_001150768）、イヌ（RefSeq No. XM_844264）等）、さらにはそれらのスプライスバリアント、アレル変異体、多型などのmRNAがあげられる。 Preferred examples of mRNA of gene G1 (ATP9A) include human ATP9A (RefSeq Accession No. NM_006045) containing the nucleotide sequence represented by SEQ ID NO: 1, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_015731), chimpanzee (RefSeq No. XM_001168714), dog (RefSeq No. XM_534457), etc.), and their splice variants, allelic variants, and polymorphisms.
Preferred examples of mRNA of the gene G2 (NTRK2) include, for example, human NTRK2 (RefSeq No. NM_006180) consisting of the base acid sequence represented by SEQ ID NO: 3, or their orthologs in other mammals (eg, mouse) (RefSeq No. NM_001025074), rat (RefSeq No. NM_012731), cattle (RefSeq No. NM_001075225), chimpanzee (RefSeq No. XM_001135401), dogs (RefSeq No. XM_851329), etc.) and their splice variants (for example, RefSeq No. NM_001007097, NM_001018064, NM_001018065, NM_001018066, etc.), allelic variants, and polymorphisms.
Preferred examples of mRNA of the gene G3 (ELAVL4) include, for example, human ELAVL4 (RefSeq No. NM_021952) consisting of the base sequence represented by SEQ ID NO: 5, or their orthologs (eg, mouse ( RefSeq No. NM_010488), rat (RefSeq No. NM_001077651), cattle (RefSeq No. NM_001081606), chimpanzee (RefSeq No. XM_001135120), dog (RefSeq No. XM_844876), etc.), and their splice variants and allelic variants And mRNA such as polymorphism (for example, refSNP No. rs2494876 (C / T), etc.).
Preferred examples of mRNA of the gene G4 (NAG6) include, for example, human NAG6 (RefSeq No. NM_022742) consisting of the base sequence represented by SEQ ID NO: 7, or their orthologs (for example, mouse ( RefSeq No. NM_145574), rat (RefSeq No. XM_342652), cattle (RefSeq No. XM_867863), chimpanzee (RefSeq No. XM_519361), etc.) and their splice variants, allelic variants, polymorphisms (eg, refSNP Nos rs3816887 (C / G), rs4728137 (G / C), rs8180870 (A / G), etc.).
Preferred examples of mRNA of gene G5 (HCN2) include, for example, human HCN2 (RefSeq No. NM_001194) consisting of the base sequence represented by SEQ ID NO: 9, or their orthologs in other mammals (for example, mouse ( RefSeq No. NM_008226), rat (RefSeq No. NM_053684), dog (RefSeq No. XM_850140), etc.) and their splice variants, allelic variants, and polymorphisms.
Preferred examples of mRNA of the gene G6 (DNAJA2) include, for example, human DNAJA2 (RefSeq No. NM_005880) consisting of the base sequence represented by SEQ ID NO: 11, or their orthologs in other mammals (for example, mouse ( RefSeq No. NM_019794), rat (RefSeq No. NM_032079), cattle (RefSeq No. NM_001040491), chimpanzee (RefSeq No. XM_528644), dog (RefSeq No. XM_535319), etc.), and their splice variants and allelic variants And mRNA such as polymorphism (for example, refSNP No. rs1065332 (G / T) etc.).
Preferred examples of mRNA of the gene G7 (VAPA) include, for example, human VAPA (RefSeq No. NM_003574) consisting of the base sequence represented by SEQ ID NO: 13, or their orthologs in other mammals (for example, mouse ( RefSeq No. NM_013933), rat (RefSeq No. NM_031631), cattle (RefSeq No. NM_001075733), chimpanzee (RefSeq No. XM_512022), dog (RefSeq No. XM_537332), etc.) and their splice variants (eg, RefSeq) No. NM_194434), allelic variants, polymorphisms and other mRNAs.
Preferred examples of mRNA of the gene G8 (SLC2A13) include, for example, human SLC2A13 (RefSeq No. NM_052885) consisting of the nucleotide sequence represented by SEQ ID NO: 15, or their orthologs (eg, mouse ( RefSeq No. NM_001033633), rat (RefSeq No. NM_133611), cattle (RefSeq No. XM_864476), chimpanzee (RefSeq No. XM_522353), dog (RefSeq No. XM_543735), etc.) and their splice variants and allelic variants And mRNAs such as polymorphisms.
Preferred examples of mRNA of the gene G9 (CD47) include, for example, human CD47 (RefSeq No. NM_001777) consisting of the base sequence represented by SEQ ID NO: 17, or their orthologs in other mammals (eg, mouse ( RefSeq No. NM_010581), rat (RefSeq No. NM_019195), cattle (RefSeq No. NM_174708), chimpanzee (RefSeq No. XM_516636), dog (RefSeq No. NM_001080721), etc.) and their splice variants and allelic variants And mRNA such as polymorphism (for example, refSNP No. rs11546646 (G / C) and the like).
Preferred examples of mRNA of the gene G10 (FLOT1) include, for example, human FLOT1 (RefSeq No. NM_005803) consisting of the base sequence represented by SEQ ID NO: 19, or their orthologs (for example, mouse ( RefSeq No. NM_008027), rat (RefSeq No. NM_022701), cattle (RefSeq No. NM_001076887), chimpanzee (RefSeq No. NM_001042381), dog (RefSeq No. XM_843685) etc.), and their splice variants and allelic variants And mRNA such as polymorphism (for example, refSNP No. rs1802470 (G / T) etc.).
As a preferable example of mRNA of the gene G11 (EPB41L1), human EPB41L1 (RefSeq Accession No. NM_177996) containing the base sequence represented by SEQ ID NO: 21 or an ortholog (eg, rat (RefSeq) in other mammals) No. NM_021681), cattle (RefSeq No. XM_867736), chimpanzee (RefSeq No. XM_525314), dogs (RefSeq No. XM_542979), etc.) and their splice variants, allelic variants, polymorphisms and other mRNAs .
Preferred examples of mRNA of the gene G12 (RGS7) include human RGS7 (RefSeq Accession No. NM_002924) containing the nucleotide sequence represented by SEQ ID NO: 23, or their orthologs in other mammals (eg, mouse (RefSeq No. NM_011880), rat (RefSeq No. NM_019343), cattle (RefSeq No. NM_174169), chimpanzee (RefSeq No. XM_001159739)), and their splice variants, allelic variants, and polymorphisms. .
As a preferable example of mRNA of gene G13 (PLD3), human PLD3 (RefSeq Accession No. NM_001031696) containing the base sequence represented by SEQ ID NO: 25, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_011116), rat (RefSeq No. NM_001012167), chimpanzee (RefSeq No. XM_524268), dog (RefSeq No. XM_541616), etc., and their splice variants, allelic variants, and polymorphisms. .
Preferred examples of mRNA of the gene G14 (ENC1) include human ENC1 (RefSeq Accession No. NM_003633) containing the nucleotide sequence represented by SEQ ID NO: 27, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_007930), rat (RefSeq No. NM_001003401), chimpanzee (RefSeq No. XM_526897), dog (RefSeq No. XM_544373), etc., and their splice variants, allelic variants, and polymorphisms .
Preferred examples of mRNA of the gene G15 (SYP) include human SYP (RefSeq Accession No. NM_003179) containing the nucleotide sequence represented by SEQ ID NO: 29, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_009305), rat (RefSeq No. NM_012664), cattle (RefSeq No. NM_173963), chimpanzee (RefSeq No. XM_521056), dog (RefSeq No. XM_850288), etc.), and their splice variants, allelic variants, Examples include polymorphic mRNA.
As a preferable example of mRNA of gene G16 (SLC2A3), human SLC2A3 (RefSeq Accession No. NM_006931) containing the base sequence represented by SEQ ID NO: 31 or their orthologs (for example, mouse (RefSeq) in other mammals) No. NM_011401), rat (RefSeq No. NM_017102), cattle (RefSeq No. NM_174603), chimpanzee (RefSeq No. XM_508989), dog (RefSeq No. NM_001003308) etc.), and their splice variants, allelic variants, Examples include polymorphic mRNA.
As a preferable example of mRNA of gene G17 (STXBP1), human STXBP1 (RefSeq Accession No. NM_001032221) containing the base sequence represented by SEQ ID NO: 33, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_009295), rat (RefSeq No. NM_013038), bovine (RefSeq No. NM_174619), etc.), and their splice variants, allelic variants, and polymorphisms.
As a preferable example of mRNA of gene G18 (GAP43), human GAP43 (RefSeq Accession No. NM_002045) containing the base sequence represented by SEQ ID NO: 35, or an ortholog thereof (for example, mouse (RefSeq) in other mammals) No. NM_008083), rat (RefSeq No. NM_017195), cattle (RefSeq No. NM_203358), chimpanzee (RefSeq No. NM_001009816), dog (RefSeq No. XM_535747), etc.), and their splice variants, allelic variants, Examples include polymorphic mRNA.
As a preferable example of mRNA of gene G19 (ATP1B1), human ATP1B1 (RefSeq Accession No. NM_001677) containing the base sequence represented by SEQ ID NO: 37, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_009721), rat (RefSeq No. NM_013113), cattle (RefSeq No. NM_001035334), chimpanzee (RefSeq No. NM_001098555), dog (RefSeq No. NM_001003283) etc.), and their splice variants, allelic variants, Examples include polymorphic mRNA.
As a preferable example of mRNA of gene G20 (MAOB), human MAOB (RefSeq Accession No. NM_000898) containing the base sequence represented by SEQ ID NO: 39, or an ortholog thereof (for example, mouse (RefSeq) in other mammals) No. NM_172778), rat (RefSeq No. NM_013198), cattle (RefSeq No. NM_177944), dogs (RefSeq No. XM_850456), etc.) and their splice variants, allelic variants, polymorphisms and other mRNAs .
Preferred examples of mRNA of the gene G21 (CHRM1) include human CHRM1 (RefSeq Accession No. NM_000738) containing the nucleotide sequence represented by SEQ ID NO: 41, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_007698), rat (RefSeq No. NM_080773), cattle (RefSeq No. XM_001255657), chimpanzee (RefSeq No. XM_001159938), dog (RefSeq No. XM_540897), etc.), and their splice variants, allelic variants, Examples include polymorphic mRNA.
As a preferable example of mRNA of gene G22 (SLC7A14), human SLC7A14 (RefSeq Accession No. NM_020949) containing the base sequence represented by SEQ ID NO: 43, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_172861), rat (RefSeq No. NM_001134615), chimpanzee (RefSeq No. XM_526378), dog (RefSeq No. XM_545285), etc.) and their splice variants, allelic variants, and polymorphisms .
Preferred examples of mRNA of the gene G23 (NDUFS7) include human NDUFS7 (RefSeq Accession No. NM_024407) containing the nucleotide sequence represented by SEQ ID NO: 45, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_029272), rat (RefSeq No. NM_001008525), cattle (RefSeq No. NM_001038022), dog (RefSeq No. XM_533960), etc.) and their splice variants, allelic variants, and polymorphisms .
As a preferable example of mRNA of gene G24 (GPRIN1), human GPRIN1 (RefSeq Accession No. NM_052899) containing the base sequence represented by SEQ ID NO: 47, or their orthologs in other mammals (for example, mouse (RefSeq No. NM_012014), rat (RefSeq No. XM_344571), etc., and their splice variants, allelic variants, and polymorphisms.
As a preferable example of mRNA of gene G25 (ATP8A1), human ATP8A1 (RefSeq Accession No. NM_001105529) containing the base sequence represented by SEQ ID NO: 49, or an ortholog thereof (for example, mouse (RefSeq) in other mammals) No. NM_001038999), cattle (RefSeq No. NM_174838), chimpanzee (RefSeq No. XM_001150768), dogs (RefSeq No. XM_844264), etc.) and their splice variants, allelic variants, polymorphisms and other mRNAs .
　「遺伝子GnのmRNAの塩基配列と相補的もしくは実質的に相補的な塩基配列の一部」とは、遺伝子GnのmRNAに特異的に結合することができ、且つ該mRNAからのタンパク質の翻訳を阻害（あるいは該mRNAを分解）し得るものであれば、その長さや位置に特に制限はないが、配列特異性の面から、標的配列に相補的もしくは実質的に相補的な部分を少なくとも10塩基以上、好ましくは約15塩基以上、より好ましくは約20塩基以上含むものである。 “Part of the base sequence complementary to or substantially complementary to the base sequence of mRNA of gene Gn” means that it can specifically bind to the mRNA of gene Gn and translates the protein from the mRNA. The length and the position are not particularly limited as long as they can inhibit (or degrade the mRNA), but at least 10 bases that are complementary or substantially complementary to the target sequence from the viewpoint of sequence specificity. As mentioned above, it contains about 15 bases or more, more preferably about 20 bases or more.
　具体的には、遺伝子GnのmRNAの塩基配列と相補的もしくは実質的に相補的な塩基配列またはその一部を含む核酸として、以下の(a)～(c)のいずれかのものが好ましく例示される。
(a) 遺伝子GnのmRNAに対するアンチセンス核酸
(b) 遺伝子GnのmRNAに対するリボザイム核酸
(c) 遺伝子GnのmRNAに対してRNAi活性を有する核酸もしくはその前駆体 Specifically, the nucleic acid comprising a base sequence complementary to or substantially complementary to the base sequence of the gene Gn mRNA or a part thereof is preferably exemplified by any of the following (a) to (c): Is done.
(a) Antisense nucleic acid for gene Gn mRNA
(b) Ribozyme nucleic acid for gene Gn mRNA
(c) a nucleic acid having RNAi activity against the mRNA of gene Gn or a precursor thereof
(a) 遺伝子GnのmRNAに対するアンチセンス核酸
　本発明における「遺伝子GnのmRNAに対するアンチセンス核酸」とは、該mRNAの塩基配列と相補的もしくは実質的に相補的な塩基配列またはその一部を含む核酸であって、標的mRNAと特異的かつ安定した二重鎖を形成して結合することにより、タンパク質合成を抑制する機能を有するものである。
　アンチセンス核酸は、2-デオキシ-D-リボースを含有しているポリデオキシリボヌクレオチド、D-リボースを含有しているポリリボヌクレオチド、プリンまたはピリミジン塩基のN-グリコシドであるその他のタイプのポリヌクレオチド、非ヌクレオチド骨格を有するその他のポリマー（例えば、市販のタンパク質核酸および合成配列特異的な核酸ポリマー）または特殊な結合を含有するその他のポリマー（但し、該ポリマーはDNAやRNA中に見出されるような塩基のペアリングや塩基の付着を許容する配置をもつヌクレオチドを含有する）などが挙げられる。それらは、二本鎖DNA、一本鎖DNA、二本鎖RNA、一本鎖RNA、DNA:RNAハイブリッドであってもよく、さらに非修飾ポリヌクレオチド（または非修飾オリゴヌクレオチド）、公知の修飾の付加されたもの、例えば当該分野で知られた標識のあるもの、キャップの付いたもの、メチル化されたもの、１個以上の天然のヌクレオチドを類縁物で置換したもの、分子内ヌクレオチド修飾のされたもの、例えば非荷電結合（例えば、メチルホスホネート、ホスホトリエステル、ホスホルアミデート、カルバメートなど）を持つもの、電荷を有する結合または硫黄含有結合（例、ホスホロチオエート、ホスホロジチオエートなど）を持つもの、例えばタンパク質（例、ヌクレアーゼ、ヌクレアーゼ・インヒビター、トキシン、抗体、シグナルペプチド、ポリ-L-リジンなど）や糖（例、モノサッカライドなど）などの側鎖基を有しているもの、インターカレント化合物（例、アクリジン、ソラレンなど）を持つもの、キレート化合物（例えば、金属、放射活性をもつ金属、ホウ素、酸化性の金属など）を含有するもの、アルキル化剤を含有するもの、修飾された結合を持つもの（例えば、αアノマー型の核酸など）であってもよい。ここで「ヌクレオシド」、「ヌクレオチド」および「核酸」とは、プリンおよびピリミジン塩基を含有するのみでなく、修飾されたその他の複素環型塩基をもつようなものを含んでいて良い。このような修飾物は、メチル化されたプリンおよびピリミジン、アシル化されたプリンおよびピリミジン、あるいはその他の複素環を含むものであってよい。修飾されたヌクレオシドおよび修飾されたヌクレオチドはまた糖部分が修飾されていてよく、例えば、１個以上の水酸基がハロゲンとか、脂肪族基などで置換されていたり、またはエーテル、アミンなどの官能基に変換されていてよい。 (a) Antisense nucleic acid against mRNA of gene Gn In the present invention, the “antisense nucleic acid against mRNA of gene Gn” includes a base sequence complementary to or substantially complementary to the base sequence of the mRNA or a part thereof. It is a nucleic acid and has a function of suppressing protein synthesis by forming a specific and stable duplex with a target mRNA.
Antisense nucleic acids are polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, Other polymers with non-nucleotide backbones (eg, commercially available protein nucleic acids and synthetic sequence specific nucleic acid polymers) or other polymers containing special linkages, provided that the polymer is a base such as found in DNA or RNA And a nucleotide having a configuration that allows attachment of a base). They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, unmodified polynucleotides (or unmodified oligonucleotides), known modifications Additions, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotide modifications Such as those having uncharged bonds (eg methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.) Things such as proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-rigid Having a side chain group such as sugar (eg, monosaccharide), having an intercurrent compound (eg, acridine, psoralen), chelate compound (eg, metal, having radioactivity) It may be one containing a metal, boron, oxidizing metal, etc., one containing an alkylating agent, or one having a modified bond (for example, α-anomeric nucleic acid etc.). As used herein, “nucleoside”, “nucleotide” and “nucleic acid” may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also be modified at the sugar moiety, eg, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may have been converted.
　上記の通り、アンチセンス核酸はDNAであってもRNAであってもよく、あるいはDNA/RNAキメラであってもよい。アンチセンス核酸がDNAの場合、標的RNAとアンチセンスDNAとによって形成されるRNA:DNAハイブリッドは、内在性RNase Hに認識されて標的RNAの選択的な分解を引き起こすことができる。したがって、RNase Hによる分解を指向するアンチセンスDNAの場合、標的配列は、mRNA中の配列だけでなく、遺伝子Gnの初期翻訳産物におけるイントロン領域の配列であってもよい。イントロン配列は、ゲノム配列と、遺伝子GnのcDNA塩基配列とをBLAST、FASTA等のホモロジー検索プログラムを用いて比較することにより、決定することができる。 As described above, the antisense nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. When the antisense nucleic acid is DNA, the RNA: DNA hybrid formed by the target RNA and the antisense DNA can be recognized by endogenous RNase H and cause selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only the sequence in mRNA but also the sequence of the intron region in the initial translation product of gene Gn. The intron sequence can be determined by comparing the genome sequence and the cDNA base sequence of the gene Gn using a homology search program such as BLAST or FASTA.
　本発明のアンチセンス核酸の標的領域は、該アンチセンス核酸がハイブリダイズすることにより、結果としてタンパク質Pnへの翻訳が阻害されるものであればその長さに特に制限はなく、タンパク質PnをコードするmRNAの全配列であっても部分配列であってもよく、短いもので約10塩基程度、長いものでmRNAもしくは初期転写産物の全配列が挙げられる。合成の容易さや抗原性、細胞内移行性の問題等を考慮すれば、約10～約40塩基、特に約15～約30塩基からなるオリゴヌクレオチドが好ましいが、それに限定されない。具体的には、遺伝子Gnの5’端ヘアピンループ、5’端６－ベースペア・リピート、5’端非翻訳領域、翻訳開始コドン、タンパク質コード領域、ORF翻訳終止コドン、3’端非翻訳領域、3’端パリンドローム領域または3’端ヘアピンループなどが、アンチセンス核酸の好ましい標的領域として選択しうるが、それらに限定されない。 The target region of the antisense nucleic acid of the present invention is not particularly limited in length as long as the antisense nucleic acid hybridizes, and as a result, translation into protein Pn is inhibited. The entire sequence or partial sequence of mRNA may be a short sequence of about 10 bases, and a long sequence of mRNA or the initial transcript. In view of easiness of synthesis, antigenicity, intracellular migration, etc., an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferred, but is not limited thereto. Specifically, 5 'end hairpin loop of gene Gn, 5' end 6-base pair repeat, 5 'end untranslated region, translation start codon, protein coding region, ORF translation stop codon, 3' end untranslated region , 3 ′ end palindromic region or 3 ′ end hairpin loop, etc. may be selected as a preferred target region of the antisense nucleic acid, but is not limited thereto.
　さらに、本発明のアンチセンス核酸は、遺伝子GnのmRNAや初期転写産物とハイブリダイズしてタンパク質への翻訳を阻害するだけでなく、二本鎖DNAであるこれらの遺伝子と結合して三重鎖（トリプレックス）を形成し、RNAへの転写を阻害し得るもの（アンチジーン）であってもよい。 Furthermore, the antisense nucleic acid of the present invention not only hybridizes with the mRNA of the gene Gn and the initial transcription product to inhibit translation into proteins, but also binds to these genes that are double-stranded DNA to form triple strands ( A triplex) that can inhibit transcription to RNA (antigene).
　アンチセンス核酸を構成するヌクレオチド分子は、天然型のDNAもしくはRNAでもよいが、安定性（化学的および／または対酵素）や比活性（RNAとの親和性）を向上させるために、種々の化学修飾を含むことができる。例えば、ヌクレアーゼなどの加水分解酵素による分解を防ぐために、アンチセンス核酸を構成する各ヌクレオチドのリン酸残基（ホスフェート）を、例えば、ホスホロチオエート（PS)、メチルホスホネート、ホスホロジチオネートなどの化学修飾リン酸残基に置換することができる。また、各ヌクレオチドの糖（リボース）の2’位の水酸基を、-OR（R=CH3（2’-O-Me）、CH2CH2OCH3（2’-O-MOE）、CH2CH2NHC(NH)NH2、CH2CONHCH3、CH2CH2CN等）に置換してもよい。さらに、塩基部分（ピリミジン、プリン）に化学修飾を施してもよく、例えば、ピリミジン塩基の5位へのメチル基やカチオン性官能基の導入、あるいは2位のカルボニル基のチオカルボニルへの置換などが挙げられる。 The nucleotide molecule constituting the antisense nucleic acid may be natural DNA or RNA, but various chemicals may be used to improve stability (chemical and / or enzyme) and specific activity (affinity with RNA). Modifications can be included. For example, in order to prevent degradation by a hydrolase such as nuclease, the phosphate residue (phosphate) of each nucleotide constituting the antisense nucleic acid is chemically modified, for example, phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. It can be substituted with a phosphate residue. In addition, the 2′-position hydroxyl group of the sugar (ribose) of each nucleotide is represented by —OR (R═CH 3 (2′-O-Me), CH 2 CH 2 OCH 3 (2′-O-MOE), CH 2 CH 2 NHC (NH) NH 2 , CH 2 CONHCH 3 , CH 2 CH 2 CN, etc.) may be substituted. Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or a cationic functional group at the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. Is mentioned.
　RNAの糖部のコンフォーメーションはC2’-endo（S型）とC3’-endo（N型）の２つが支配的であり、一本鎖RNAではこの両者の平衡として存在するが、二本鎖を形成するとN型に固定される。したがって、標的RNAに対して強い結合能を付与するために、2’酸素と4’炭素を架橋することにより、糖部のコンフォーメーションをN型に固定したRNA誘導体であるBNA(LNA)（Imanishi, T. et al., Chem. Commun., 1653-9, 2002; Jepsen, J.S. et al., Oligonucleotides, 14, 130-46, 2004）やENA（Morita, K. et al., Nucleosides Nucleotides Nucleic Acids, 22, 1619-21, 2003）もまた、好ましく用いられ得る。 The conformation of the sugar part of RNA is dominated by C2'-endo (S type) and C3'-endo (N type). In single-stranded RNA, it exists as an equilibrium between the two, but double-stranded Is fixed to the N type. Therefore, in order to give strong binding ability to the target RNA, BNA (LNA) (Imanishi) is an RNA derivative in which the conformation of the sugar moiety is fixed to N-type by cross-linking 2 'oxygen and 4' carbon. , T. et al., Chem. Commun., 1653-9, 2002; Jepsen, JS et al., Oligonucleotides, 14, 130-46, 2004) and ENA (Morita, K. et al., Nucleosides Nucleotides Nucleicides Nucleicides , 22, 1619-21, 2003) can also be preferably used.
　本発明のアンチセンスオリゴヌクレオチドは、遺伝子GnのcDNA配列もしくはゲノミックDNA配列に基づいてmRNAもしくは初期転写産物の標的配列を決定し、市販のDNA/RNA自動合成機（アプライド・バイオシステムズ社、ベックマン社等）を用いて、これに相補的な配列を合成することにより調製することができる。また、上記した各種修飾を含むアンチセンス核酸も、いずれも自体公知の手法により、化学的に合成することができる。 The antisense oligonucleotide of the present invention determines the target sequence of mRNA or initial transcript based on the cDNA sequence or genomic DNA sequence of gene Gn, and is a commercially available DNA / RNA automatic synthesizer (Applied Biosystems, Beckman) Etc.) can be prepared by synthesizing a complementary sequence thereto. In addition, any of the above-described antisense nucleic acids containing various modifications can be chemically synthesized by a method known per se.
(b) 遺伝子GnのmRNAに対するリボザイム核酸
　遺伝子GnのmRNAの塩基配列と相補的もしくは実質的に相補的な塩基配列またはその一部を含む核酸の他の好ましい例としては、該mRNAをコード領域の内部で特異的に切断し得るリボザイム核酸が挙げられる。「リボザイム」とは、狭義には、核酸を切断する酵素活性を有するRNAをいうが、本明細書では配列特異的な核酸切断活性を有する限りDNAをも包含する概念として用いるものとする。リボザイム核酸として最も汎用性の高いものとしては、ウイロイドやウイルソイド等の感染性RNAに見られるセルフスプライシングRNAがあり、ハンマーヘッド型やヘアピン型等が知られている。ハンマーヘッド型は約40塩基程度で酵素活性を発揮し、ハンマーヘッド構造をとる部分に隣接する両端の数塩基ずつ（合わせて約10塩基程度）をmRNAの所望の切断部位と相補的な配列にすることにより、標的mRNAのみを特異的に切断することが可能である。このタイプのリボザイム核酸は、RNAのみを基質とするので、ゲノムDNAを攻撃することがないというさらなる利点を有する。遺伝子GnのmRNAが自身で二本鎖構造をとる場合には、RNAヘリカーゼと特異的に結合し得るウイルス核酸由来のRNAモチーフを連結したハイブリッドリボザイムを用いることにより、標的配列を一本鎖にすることができる［Proc. Natl. Acad. Sci. USA, 98(10): 5572-5577 (2001)］。さらに、リボザイムを、それをコードするDNAを含む発現ベクターの形態で使用する場合には、転写産物の細胞質への移行を促進するために、tRNAを改変した配列をさらに連結したハイブリッドリボザイムとすることもできる［Nucleic Acids Res., 29(13): 2780-2788 (2001)］。 (b) Ribozyme nucleic acid for gene Gn mRNA As another preferred example of a nucleic acid comprising a base sequence complementary to or substantially complementary to the base sequence of the gene Gn mRNA or a part thereof, the mRNA is used in the coding region. Examples include ribozyme nucleic acids that can be cleaved specifically inside. “Ribozyme”, in a narrow sense, refers to RNA having an enzyme activity for cleaving nucleic acids, but in this specification, it is used as a concept including DNA as long as it has sequence-specific nucleic acid cleaving activity. The most versatile ribozyme nucleic acids include self-splicing RNAs found in infectious RNAs such as viroids and virusoids, and hammerhead and hairpin types are known. The hammerhead type exhibits enzyme activity at about 40 bases, and a few bases at both ends (about 10 bases in total) adjacent to the part that takes the hammerhead structure are made complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme nucleic acid has the additional advantage of not attacking genomic DNA because it uses only RNA as a substrate. When the mRNA of gene Gn has a double-stranded structure by itself, the target sequence is made single-stranded by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase. [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)]. Furthermore, when ribozymes are used in the form of expression vectors containing the DNA that encodes them, in order to promote the transfer of transcripts to the cytoplasm, the ribozyme should be a hybrid ribozyme further linked to a tRNA-modified sequence. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].
(c) 遺伝子GnのmRNAに対するsiRNA又はmiRNA
　本明細書においては、遺伝子GnのmRNAに相補的なオリゴRNAとその相補鎖とからなる二本鎖RNA、いわゆるsiRNAもまた、遺伝子GnのmRNAの塩基配列と相補的もしくは実質的に相補的な塩基配列またはその一部を含む核酸に包含されるものとして定義される。短い二本鎖RNAを細胞内に導入するとそのRNAに相補的なmRNAが分解される、いわゆるRNA干渉（RNAi）と呼ばれる現象は、以前から線虫、昆虫、植物等で知られていたが、この現象が動物細胞でも広く起こることが確認されて以来［Nature, 411(6836): 494-498 (2001)］、リボザイムの代替技術として汎用されている。 (c) siRNA or miRNA for gene Gn mRNA
In the present specification, a double-stranded RNA consisting of an oligo RNA complementary to the mRNA of gene Gn and its complementary strand, so-called siRNA, is also complementary or substantially complementary to the base sequence of the gene Gn mRNA. Defined as encompassed by nucleic acid containing base sequence or part thereof. When a short double-stranded RNA is introduced into a cell, the mRNA complementary to that RNA is degraded. So-called RNA interference (RNAi) has long been known in nematodes, insects, plants, etc. Since this phenomenon has been confirmed to occur widely in animal cells [Nature, 411 (6836): 494-498 (2001)], it has been widely used as an alternative to ribozyme.
　siRNAは、標的遺伝子のcDNA配列情報に基づいて、例えば、Elbashirら（Genes Dev., 15, 188-200 (2001)）の提唱する規則に従って設計することができる。siRNAの標的配列としては、例えばAA+(N)19、AA+(N)21もしくはNA+(N)21（Nは任意の塩基）等が挙げられるが、それらに限定されない。標的配列の位置も特に制限されるわけではない。選択された標的配列の候補群について、標的以外のmRNAにおいて16-17塩基の連続した配列に相同性がないかどうかを、BLAST（http://www.ncbi.nlm.nih.gov/BLAST/）等のホモロジー検索ソフトを用いて調べ、選択した標的配列の特異性を確認する。例えば、AA+(N)19、AA+(N)21もしくはNA+(N)21（Nは任意の塩基）を標的配列とする場合、特異性の確認された標的配列について、AA（もしくはNA）以降の19-21塩基にTTもしくはUUの3’末端オーバーハングを有するセンス鎖と、該19-21塩基に相補的な配列及びTTもしくはUUの3’末端オーバーハングを有するアンチセンス鎖とからなる2本鎖RNAをsiRNAとして設計してもよい。また、siRNAの前駆体であるショートヘアピンRNA（shRNA）は、ループ構造を形成しうる任意のリンカー配列（例えば、5-25塩基程度）を適宜選択し、上記センス鎖とアンチセンス鎖とを該リンカー配列を介して連結することにより設計することができる。 siRNA can be designed according to the rules proposed by Elbashir et al. (Genes Dev., 15, 188-200 (2001)) based on the cDNA sequence information of the target gene. Examples of the target sequence of siRNA include, but are not limited to, AA + (N) 19 , AA + (N) 21 or NA + (N) 21 (N is an arbitrary base). The position of the target sequence is not particularly limited. Regarding the selected candidate target sequence group, whether or not there is homology in the 16-17 base sequence in the mRNA other than the target is determined by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ ) And the like, and the specificity of the selected target sequence is confirmed. For example, when AA + (N) 19 , AA + (N) 21 or NA + (N) 21 (N is an arbitrary base) is used as the target sequence, the target sequence whose specificity has been confirmed is AA (or NA) or later. Two strands consisting of a sense strand having a TT or UU 3 'end overhang at 19-21 bases and an antisense strand having a sequence complementary to the 19-21 base and a TT or UU 3' end overhang Strand RNA may be designed as siRNA. In addition, for the short hairpin RNA (shRNA) that is the precursor of siRNA, an arbitrary linker sequence (for example, about 5-25 bases) capable of forming a loop structure is appropriately selected, and the sense strand and the antisense strand are combined with the short strand. It can be designed by linking via a linker sequence.
　siRNA及び／又はshRNAの配列は、種々のwebサイト上に無料で提供される検索ソフトを用いて検索が可能である。このようなサイトとしては、例えば、Ambionが提供するsiRNA Target Finder（http://www.ambion.com/jp/techlib/misc/siRNA_finder.html）及びpSilencerTM Expression Vector用インサートデザインツール（http://www.ambion.com/jp/techlib/misc/psilencer_converter.html）、RNAi Codexが提供するGeneSeer（http://codex.cshl.edu/scripts/newsearchhairpin.cgi）が挙げられるが、これらに限定されない。 The sequence of siRNA and / or shRNA can be searched using search software provided free of charge on various websites. Such sites include, for example, siRNA Target Finder (http://www.ambion.com/jp/techlib/misc/siRNA_finder.html) and insert design tool for pSilencer ™ Expression Vector (http: / /www.ambion.com/techlib/misc/psilencer_converter.html), GeneSeer provided by RNAi Codex (http://codex.cshl.edu/scripts/newsearchhairpin.cgi) .
　siRNAを構成するリボヌクレオシド分子もまた、安定性、比活性などを向上させるために、上記のアンチセンス核酸の場合と同様の修飾を受けていてもよい。 The ribonucleoside molecule constituting siRNA may also be modified in the same manner as in the above-described antisense nucleic acid in order to improve stability, specific activity and the like.
　siRNAは、mRNA上の標的配列のセンス鎖及びアンチセンス鎖をDNA/RNA自動合成機でそれぞれ合成し、適当なアニーリング緩衝液中、約90～約95℃で約1分程度変性させた後、約30～約70℃で約1～約8時間アニーリングさせることにより調製することができる。また、siRNAの前駆体となるシングルヘアピンRNA（shRNA）を合成し、これをダイサー（dicer）を用いて切断することにより調製することもできる。 The siRNA is synthesized by synthesizing a sense strand and an antisense strand of a target sequence on mRNA with a DNA / RNA automatic synthesizer, denatured at about 90 to about 95 ° C. for about 1 minute in an appropriate annealing buffer, It can be prepared by annealing at about 30 to about 70 ° C. for about 1 to about 8 hours. Alternatively, it can be prepared by synthesizing a single hairpin RNA (shRNA) serving as a siRNA precursor and cleaving it with a dicer.
　本明細書においては、生体内で遺伝子GnのmRNAに対するsiRNAを生成し得るようにデザインされた核酸もまた、遺伝子GnのmRNAの塩基配列と相補的もしくは実質的に相補的な塩基配列またはその一部を含む核酸に包含されるものとして定義される。そのような核酸としては、上記したshRNAやsiRNAを発現するように構築された発現ベクターなどが挙げられる。shRNAは、mRNA上の標的配列のセンス鎖およびアンチセンス鎖を適当なループ構造を形成しうる長さ（例えば5～25塩基程度）のスペーサー配列を間に挿入して連結した塩基配列を含むオリゴRNAをデザインし、これをDNA/RNA自動合成機で合成することにより調製することができる。shRNAを発現するベクターには、タンデムタイプとステムループ（ヘアピン）タイプとがある。前者はsiRNAのセンス鎖の発現カセットとアンチセンス鎖の発現カセットをタンデムに連結したもので、細胞内で各鎖が発現してアニーリングすることにより2本鎖のsiRNA（dsRNA）を形成するというものである。一方、後者はshRNAの発現カセットをベクターに挿入したもので、細胞内でshRNAが発現しdicerによるプロセシングを受けてdsRNAを形成するというものである。プロモーターとしては、polII系プロモーター（例えば、CMV前初期プロモーター）を使用することもできるが、短いRNAの転写を正確に行わせるために、polIII系プロモーターを使用するのが一般的である。polIII系プロモーターとしては、マウスおよびヒトのU6-snRNAプロモーター、ヒトH1-RNase P RNAプロモーター、ヒトバリン-tRNAプロモーターなどが挙げられる。また、転写終結シグナルとして4個以上Tが連続した配列が用いられる。
　このようにして構築したsiRNAもしくはshRNA発現カセットを、次いでプラスミドベクターやウイルスベクターに挿入する。このようなベクターとしては、レトロウイルス、レンチウイルス、アデノウイルス、アデノ随伴ウイルス、ヘルペスウイルス、センダイウイルスなどのウイルスベクターや、動物細胞発現プラスミドなどが用いられる。 In the present specification, a nucleic acid designed to generate siRNA for the gene Gn mRNA in vivo also refers to a nucleotide sequence complementary to or substantially complementary to the nucleotide sequence of the gene Gn mRNA or one of the nucleotide sequences. Defined as encompassed by a nucleic acid containing a moiety. Examples of such nucleic acids include expression vectors constructed so as to express the above-mentioned shRNA and siRNA. shRNA is an oligo containing a base sequence in which the sense strand and the antisense strand of the target sequence on mRNA are linked by inserting a spacer sequence (for example, about 5 to 25 bases) long enough to form an appropriate loop structure. It can be prepared by designing RNA and synthesizing it with an automatic DNA / RNA synthesizer. Vectors expressing shRNA include tandem type and stem loop (hairpin) type. The former is a tandem-linked siRNA sense cassette and antisense strand expression cassette. Each strand is expressed in a cell and annealed to form a double-stranded siRNA (dsRNA). It is. On the other hand, the latter is one in which an shRNA expression cassette is inserted into a vector, in which shRNA is expressed in cells and processed by dicer to form dsRNA. As a promoter, a pol II promoter (for example, a CMV immediate early promoter) can be used, but in order to perform transcription of a short RNA accurately, a pol III promoter is generally used. Examples of the polIII promoter include mouse and human U6-snRNA promoter, human H1-RNase P RNA promoter, human valine-tRNA promoter, and the like. Further, a sequence in which 4 or more Ts are continuous is used as a transcription termination signal.
The siRNA or shRNA expression cassette thus constructed is then inserted into a plasmid vector or viral vector. Examples of such vectors include retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes virus, Sendai virus and other viral vectors, animal cell expression plasmids, and the like.
　遺伝子GnのmRNAの塩基配列と相補的もしくは実質的に相補的な塩基配列またはその一部を含む核酸は、リポソーム、ミクロスフェアのような特殊な形態で供与されたり、遺伝子治療に適用されたり、付加された形態で与えられることができうる。こうして付加形態で用いられるものとしては、リン酸基骨格の電荷を中和するように働くポリリジンのようなポリカチオン体、細胞膜との相互作用を高めたり、核酸の取込みを増大せしめるような脂質（例、ホスホリピド、コレステロールなど）などの疎水性のものが挙げられる。付加するに好ましい脂質としては、コレステロールやその誘導体（例、コレステリルクロロホルメート、コール酸など）が挙げられる。こうしたものは、核酸の3’端または5’端に付着させることができ、塩基、糖、分子内ヌクレオシド結合を介して付着させることができうる。その他の基としては、核酸の3’端または5’端に特異的に配置されたキャップ用の基で、エキソヌクレアーゼ、RNaseなどのヌクレアーゼによる分解を阻止するためのものが挙げられる。こうしたキャップ用の基としては、ポリエチレングリコール、テトラエチレングリコールなどのグリコールをはじめとした当該分野で知られた水酸基の保護基が挙げられるが、それに限定されるものではない。 Nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of mRNA of gene Gn or a part thereof is provided in a special form such as a liposome or a microsphere, applied to gene therapy, It can be given in an added form. In this way, the additional form includes polycationic substances such as polylysine that acts to neutralize the charge of the phosphate group skeleton, lipids that enhance interaction with cell membranes and increase nucleic acid uptake ( Examples include hydrophobic ones such as phospholipid and cholesterol. Preferred lipids for addition include cholesterol and derivatives thereof (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'or 5' end of nucleic acids and can be attached via bases, sugars, intramolecular nucleoside linkages. Examples of the other group include a cap group specifically arranged at the 3 'end or 5' end of a nucleic acid, which prevents degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.
　これらの核酸のタンパク質Pn発現抑制活性は、遺伝子Gnを導入した形質転換体、生体内や生体外の遺伝子Gn発現系、または生体内や生体外のタンパク質Pn翻訳系を用いて調べることができる。 The protein Pn expression inhibitory activity of these nucleic acids can be examined using a transformant into which the gene Gn has been introduced, a gene Gn expression system in vivo or in vitro, or a protein Pn translation system in vivo or in vitro.
　本発明におけるタンパク質Pnの発現を抑制する物質は、上記のような遺伝子GnのmRNAの塩基配列と相補的もしくは実質的に相補的な塩基配列またはその一部を含む核酸に限定されず、タンパク質Pnの産生を直接的または間接的に阻害する限り、低分子化合物などの他の物質であってもよい。該物質は、タンパク質P1～P25から選ばれるいずれか1種のタンパク質の発現を抑制するものであってもよいし、2種以上のタンパク質の発現を抑制し得るものであってもよい。そのような物質は、例えば、後述する本発明のスクリーニング方法により取得することができる。 The substance that suppresses the expression of protein Pn in the present invention is not limited to a nucleic acid containing a base sequence complementary to or substantially complementary to the base sequence of the mRNA of gene Gn as described above or a part thereof, but includes protein Pn Other substances such as low molecular weight compounds may be used as long as they directly or indirectly inhibit the production of. The substance may be one that suppresses the expression of any one protein selected from proteins P1 to P25, or may be one that can suppress the expression of two or more proteins. Such a substance can be obtained, for example, by the screening method of the present invention described later.
　本発明において「タンパク質Pnの機能を抑制する物質」とは、いったん機能的に産生されたタンパク質Pnが、Ａβの産生を促進するのを抑制する限りいかなるものでもよく、例えば、タンパク質Pnに結合してＡβ産生を抑制する物質、タンパク質Pnとγ－セクレターゼとの結合活性を阻害する物質（たとえば、タンパク質Pnとγ－セクレターゼとの結合によってγ－セクレターゼ活性が促進するのを阻害またはモジュレートする物質、タンパク質Pnとγ－セクレターゼとの結合若しくは複合体形成を阻害する物質等）、タンパク質Pnの細胞膜への移行を阻害する物質等が挙げられる。 In the present invention, the “substance that suppresses the function of protein Pn” may be any substance as long as the protein Pn once functionally produced suppresses the promotion of Aβ production. For example, it binds to protein Pn. A substance that suppresses Aβ production, a substance that inhibits the binding activity between protein Pn and γ-secretase (for example, a substance that inhibits or modulates the promotion of γ-secretase activity due to the binding between protein Pn and γ-secretase) And substances that inhibit the binding or complex formation of protein Pn and γ-secretase), substances that inhibit the transfer of protein Pn to the cell membrane, and the like.
　具体的には、タンパク質Pnの機能を抑制する物質として、例えば、タンパク質Pnに対する抗体が挙げられる。該抗体はポリクローナル抗体、モノクローナル抗体の何れであってもよい。これらの抗体は、自体公知の抗体または抗血清の製造法に従って製造することができる。抗体のアイソタイプは特に限定されないが、好ましくはIgG、IgMまたはIgA、特に好ましくはIgGが挙げられる。また、該抗体は、標的抗原を特異的に認識し結合するための相補性決定領域（CDR）を少なくとも有するものであれば特に制限はなく、完全抗体分子の他、例えばFab、Fab'、F(ab’)2等のフラグメント、scFv、scFv-Fc、ミニボディー、ダイアボディー等の遺伝子工学的に作製されたコンジュゲート分子、あるいはポリエチレングリコール（PEG）等のタンパク質安定化作用を有する分子等で修飾されたそれらの誘導体などであってもよい。
　好ましい一実施態様において、タンパク質Pnに対する抗体はヒトを投与対象とする医薬品として使用されることから、該抗体（好ましくはモノクローナル抗体）はヒトに投与した場合に抗原性を示す危険性が低減された抗体、具体的には、完全ヒト抗体、ヒト化抗体、マウス－ヒトキメラ抗体などであり、特に好ましくは完全ヒト抗体である。ヒト化抗体およびキメラ抗体は、常法に従って遺伝子工学的に作製することができる。また、完全ヒト抗体は、ヒト－ヒト（もしくはマウス）ハイブリドーマより製造することも可能ではあるが、大量の抗体を安定に且つ低コストで提供するためには、ヒト抗体産生マウスやファージディスプレイ法を用いて製造することが望ましい。 Specifically, examples of the substance that suppresses the function of protein Pn include an antibody against protein Pn. The antibody may be a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to per se known methods for producing antibodies or antisera. The isotype of the antibody is not particularly limited, but preferably IgG, IgM or IgA, particularly preferably IgG. The antibody is not particularly limited as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding a target antigen. In addition to a complete antibody molecule, for example, Fab, Fab ′, F (ab ') 2 such as fragments, scFv, scFv-Fc, conjugation molecules prepared by genetic engineering such as minibodies and diabodies, or molecules having a protein stabilizing action such as polyethylene glycol (PEG) They may be modified derivatives thereof.
In a preferred embodiment, since an antibody against the protein Pn is used as a pharmaceutical for human administration, the antibody (preferably a monoclonal antibody) has a reduced risk of being antigenic when administered to a human. Antibodies, specifically, fully human antibodies, humanized antibodies, mouse-human chimeric antibodies, etc., particularly preferably fully human antibodies. Humanized antibodies and chimeric antibodies can be produced by genetic engineering according to a conventional method. In addition, fully human antibodies can be produced from human-human (or mouse) hybridomas, but in order to provide a large amount of antibodies stably and at low cost, human antibody-producing mice and phage display methods are used. It is desirable to manufacture using.
　タンパク質Pnはγ－セクレターゼを介して、Aβペプチドの生成において重要な役割を担っているので、タンパク質Pnの機能を抑制する物質は、脳移行性（血液脳関門通過性）、細胞膜透過性に優れた物質であることが望ましい。したがって、タンパク質Pnの機能を抑制するより好ましい物質は、Lipinski's Ruleに見合った低分子化合物である。該低分子化合物は、タンパク質P1～P25から選ばれるいずれか1種のタンパク質の機能を抑制するものであってもよいし、2種以上のタンパク質の機能を抑制し得るものであってもよい。そのような化合物は、例えば、後述する本発明のスクリーニング法を用いて取得することができる。 Since protein Pn plays an important role in the production of Aβ peptide via γ-secretase, the substance that suppresses the function of protein Pn is excellent in brain migration (blood-brain barrier permeability) and cell membrane permeability. Desirable material. Therefore, a more preferable substance that suppresses the function of protein Pn is a low-molecular compound suitable for Lipinski's Rule. The low molecular weight compound may be one that suppresses the function of any one protein selected from the proteins P1 to P25, and may be one that can suppress the function of two or more proteins. Such a compound can be obtained, for example, using the screening method of the present invention described later.
　タンパク質Pnの発現もしくは機能を抑制する物質は、Aβ産生を抑制するので、アルツハイマー病（AD）の病態または癌の病態の改善に有用である。しかも該物質は、Notchタンパク質の切断を阻害しない可能性があるので、腸管上皮形成障害や免疫異常などの末梢性副作用を生じる危険性が低いというさらなる有利な効果を奏する。
　したがって、タンパク質Pnの発現もしくは機能を抑制する物質を含有する医薬は、γ－セクレターゼ阻害剤あるいはモジュレーター、Aβ産生抑制剤、Aβが関与する疾患（例、AD）やNotchタンパク質の切断が関与する疾患（例、癌）の予防および／または治療剤として使用することができる。
　タンパク質Pnの発現もしくは機能を抑制する物質は1種のみを用いてもよいし、2種以上を組み合わせて用いてもよい。2種以上の、タンパク質Pnの発現もしくは機能を抑制する物質を組み合わせて使用する場合、それらの物質は、同一のタンパク質の発現もしくは機能を抑制するものであってもよいし、あるいは2種以上の異なるタンパク質の発現もしくは機能を抑制するものであってもよい。また、2種以上の、タンパク質Pnの発現もしくは機能を抑制する物質は、それぞれ別個の医薬として製剤化してもよいし、同一の医薬組成物中に配合してもよい。2種以上の、タンパク質Pnの発現もしくは機能を抑制する物質が、それぞれ別個の医薬として製剤化される場合、各製剤を同時に投与してもよいし、時間をおいて投与してもよい。また、投与経路は同一であってもよいし、異なっていてもよい。後述する投与量は、1種の、タンパク質Pnの発現もしくは機能を抑制する物質の投与量を示すが、2種以上の物質を組み合わせて用いる場合でも、投与対象に好ましくない影響を与えない範囲で、それぞれの物質について同様の投与量を用いることができる。 A substance that suppresses the expression or function of protein Pn suppresses Aβ production, and thus is useful for improving the pathology of Alzheimer's disease (AD) or cancer. In addition, since the substance may not inhibit the cleavage of Notch protein, the substance has a further advantageous effect that it has a low risk of causing peripheral side effects such as intestinal epithelialization disorder and immune abnormality.
Therefore, drugs containing substances that suppress the expression or function of protein Pn are γ-secretase inhibitors or modulators, Aβ production inhibitors, diseases involving Aβ (eg, AD), and diseases involving Notch protein cleavage. It can be used as a preventive and / or therapeutic agent for (eg, cancer).
Only one type of substance that suppresses the expression or function of protein Pn may be used, or two or more types may be used in combination. When two or more kinds of substances that suppress the expression or function of protein Pn are used in combination, these substances may suppress the expression or function of the same protein, or two or more kinds of substances may be suppressed. It may be one that suppresses the expression or function of different proteins. In addition, two or more substances that suppress the expression or function of protein Pn may be formulated as separate medicaments, or may be blended in the same pharmaceutical composition. When two or more kinds of substances that suppress the expression or function of protein Pn are formulated as separate medicaments, the respective preparations may be administered simultaneously or at intervals. Moreover, the administration route may be the same or different. The dose described below indicates the dose of a single substance that suppresses the expression or function of protein Pn. However, even when two or more substances are used in combination, the dose does not adversely affect the administration subject. Similar dosages can be used for each substance.
（１）アンチセンス核酸、リボザイム核酸、siRNAおよびその前駆体を含有する医薬
　遺伝子Gnの転写産物に相補的に結合し、該転写産物からのタンパク質の翻訳を抑制することができる本発明のアンチセンス核酸や、遺伝子Gnの転写産物における相同な（もしくは相補的な）塩基配列を標的として該転写産物を切断し得るsiRNA（もしくはリボザイム）、さらに該siRNAの前駆体であるshRNAなど（以下、包括的に「本発明の核酸」という場合がある）は、Aβ産生抑制剤、Aβが関与する疾患（例、AD）やNotchタンパク質の切断が関与する疾患（例、癌）の予防および／または治療剤として使用することができる。
　本発明の核酸を含有する医薬はそのまま液剤として、または適当な剤型の医薬組成物として、ヒトまたは非ヒト哺乳動物（例、ラット、ウサギ、ヒツジ、ブタ、ウシ、ネコ、イヌ、サルなど）に対して経口的または非経口的（例、血管内投与、皮下投与など）に投与することができる。 (1) An antisense nucleic acid of the present invention capable of binding to a transcription product of a gene Gn complementary to an antisense nucleic acid, a ribozyme nucleic acid, siRNA and a precursor thereof, and inhibiting protein translation from the transcription product SiRNAs (or ribozymes) that can cleave the transcripts of nucleic acids and gene Gn transcripts that are homologous (or complementary), and shRNA that is the precursor of the siRNA (hereinafter, comprehensive) May be referred to as “the nucleic acid of the present invention”, an agent for inhibiting and / or treating an Aβ production inhibitor, a disease involving Aβ (eg, AD) and a disease involving cleavage of Notch protein (eg, cancer) Can be used as
The pharmaceutical containing the nucleic acid of the present invention is used as it is, or as a pharmaceutical composition of an appropriate dosage form, as a human or non-human mammal (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.). Can be administered orally or parenterally (eg, intravascular administration, subcutaneous administration, etc.).
　本発明の核酸を上記のAβ産生抑制剤、Aβが関与する疾患の予防および／または治療剤などとして使用する場合、自体公知の方法に従って製剤化し、投与することができる。即ち、本発明の核酸を単独で用いてもよいし、あるいはレトロウイルスベクター、アデノウイルスベクター、アデノウイルスアソシエーテッドウイルスベクターなどの適当な哺乳動物細胞用の発現ベクターに機能可能な態様で挿入することもできる。該核酸は、そのままで、あるいは摂取促進のための補助剤とともに、遺伝子銃やハイドロゲルカテーテルのようなカテーテルによって投与することができる。あるいは、エアロゾル化して吸入剤として気管内に局所投与することもできる。
　さらに、体内動態の改良、半減期の長期化、細胞内取り込み効率の改善を目的に、前記核酸を単独またはリポソームなどの担体とともに製剤（注射剤）化し、静脈、皮下等に投与してもよい。 When the nucleic acid of the present invention is used as an Aβ production inhibitor or a preventive and / or therapeutic agent for a disease involving Aβ, it can be formulated and administered according to a method known per se. That is, the nucleic acid of the present invention may be used alone or inserted in a functional manner into an appropriate expression vector for mammalian cells such as a retrovirus vector, an adenovirus vector, an adenovirus associated virus vector. You can also. The nucleic acid can be administered as it is or together with an auxiliary agent for promoting intake by a catheter such as a gene gun or a hydrogel catheter. Alternatively, it can be aerosolized and locally administered into the trachea as an inhalant.
Furthermore, for the purpose of improving the pharmacokinetics, prolonging the half-life, and improving the efficiency of cellular uptake, the nucleic acid may be formulated (injection) alone or with a carrier such as liposome and administered intravenously, subcutaneously, etc. .
　本発明の核酸は、それ自体を投与してもよいし、または適当な医薬組成物として投与してもよい。投与に用いられる医薬組成物としては、本発明の核酸と薬理学的に許容され得る担体、希釈剤もしくは賦形剤とを含むものであってよい。このような医薬組成物は、経口または非経口投与に適する剤形として提供される。 The nucleic acid of the present invention may be administered per se or as an appropriate pharmaceutical composition. The pharmaceutical composition used for administration may contain the nucleic acid of the present invention and a pharmacologically acceptable carrier, diluent or excipient. Such pharmaceutical compositions are provided as dosage forms suitable for oral or parenteral administration.
　非経口投与のための組成物としては、例えば、注射剤、坐剤、鼻腔内投与剤等が用いられ、注射剤は静脈注射剤、皮下注射剤、皮内注射剤、筋肉注射剤、点滴注射剤等の剤形を包含しても良い。このような注射剤は、公知の方法に従って調製できる。注射剤の調製方法としては、例えば、上記本発明の核酸を通常注射剤に用いられる無菌の水性液、または油性液に溶解、懸濁または乳化することによって調製できる。注射用の水性液としては、例えば、生理食塩水、ブドウ糖やその他の補助薬を含む等張液等が用いられ、適当な溶解補助剤、例えば、アルコール（例、エタノール）、ポリアルコール（例、プロピレングリコール、ポリエチレングリコール）、非イオン界面活性剤〔例、ポリソルベート80、HCO-50（polyoxyethylene（50mol）adduct of hydrogenated castor oil）〕等と併用してもよい。油性液としては、例えば、ゴマ油、大豆油等が用いられ、溶解補助剤として安息香酸ベンジル、ベンジルアルコール等を併用してもよい。調製された注射液は、適当なアンプルに充填されることが好ましい。直腸投与に用いられる坐剤は、上記核酸を通常の坐薬用基剤に混合することによって調製されてもよい。 As a composition for parenteral administration, for example, injection, suppository, intranasal administration, etc. are used, and the injection is intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip injection. You may include dosage forms, such as an agent. Such an injection can be prepared according to a known method. As a method for preparing an injection, it can be prepared, for example, by dissolving, suspending or emulsifying the nucleic acid of the present invention in a sterile aqueous liquid or oily liquid usually used for injection. As an aqueous solution for injection, for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled in a suitable ampoule. Suppositories used for rectal administration may be prepared by mixing the nucleic acid with a normal suppository base.
　経口投与のための組成物としては、固体または液体の剤形、具体的には錠剤（糖衣錠、フィルムコーティング錠を含む）、丸剤、顆粒剤、散剤、カプセル剤（ソフトカプセル剤を含む）、シロップ剤、乳剤、懸濁剤等が挙げられる。このような組成物は公知の方法によって製造され、製剤分野において通常用いられる担体、希釈剤もしくは賦形剤を含有していても良い。錠剤用の担体、賦形剤としては、例えば、乳糖、でんぷん、蔗糖、ステアリン酸マグネシウムが用いられる。 Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like. Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field. As the carrier and excipient for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.
　上記の非経口用または経口用医薬組成物は、活性成分の投与量に適合するような投薬単位の剤形に調製されることが好都合である。このような投薬単位の剤形としては、例えば、錠剤、丸剤、カプセル剤、注射剤（アンプル）、坐剤が挙げられる。本発明の核酸は、例えば、投薬単位剤形当たり通常0.01～500mg程度含有されていることが好ましい。 The above parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dose of the active ingredient. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), and suppositories. The nucleic acid of the present invention is preferably contained, for example, usually in an amount of about 0.01 to 500 mg per dosage unit dosage form.
　本発明の核酸を含有する上記医薬の投与量は、投与対象、対象疾患、症状、投与ルートなどによっても異なるが、例えば、ADの治療・予防のために使用する場合には、本発明の核酸を１回量として、通常0.0001～20mg/kg体重程度を、1日～６ヶ月に１回程度、静脈注射により投与するのが好都合である。他の非経口投与および経口投与の場合もこれに準ずる量を投与することができる。症状が特に重い場合には、その症状に応じて増量してもよい。 The dosage of the above-mentioned medicament containing the nucleic acid of the present invention varies depending on the administration subject, target disease, symptom, administration route, etc., but for example, when used for treatment / prevention of AD, the nucleic acid of the present invention It is convenient to administer about 0.0001 to 20 mg / kg body weight by intravenous injection once a day to about 6 months. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.
　なお前記した各組成物は、本発明の核酸との配合により好ましくない相互作用を生じない限り他の活性成分を含有してもよい。他の活性成分としては、例えば、アセチルコリンエステラーゼ阻害薬に代表されるAD治療薬、非ステロイド抗炎症薬（NSAIDs）などのγ-セクレターゼモジュレーターおよびγ－セクレターゼ阻害剤、β－セクレターゼ阻害剤（例、遷移状態ミミックとして機能し得るスタチン、ヒドロキシエチレン、ヒドロキシエチルカルボニル構造を持ったペプチド性阻害剤（J. Med. Chem., 46(22), 4625-4630 (2003)）、非ペプチド性の低分子型阻害剤（WO 01/87293、WO 02/88101、WO 02/96897））などがあげられる。 Note that each of the above-described compositions may contain other active ingredients as long as an undesirable interaction is not caused by blending with the nucleic acid of the present invention. Examples of other active ingredients include AD therapeutic agents typified by acetylcholinesterase inhibitors, γ-secretase modulators such as nonsteroidal anti-inflammatory drugs (NSAIDs), γ-secretase inhibitors, β-secretase inhibitors (eg, Statin that can function as a transition state mimic, hydroxyethylene, peptidic inhibitor with hydroxyethylcarbonyl structure (J. Med. Chem., 46 (22), 4625-4630 ペ プ チ ド (2003)), non-peptidic small molecule Type inhibitors (WO 01/87293, WO 02/88101, WO 02/96897)) and the like.
（２）タンパク質Pnに対する抗体、タンパク質Pnの発現もしくは機能を抑制する低分子化合物等を含有する医薬
　タンパク質Pnに対する抗体や、タンパク質Pnの発現もしくは機能を抑制する低分子化合物は、タンパク質Pnの産生または活性を阻害したり、あるいはタンパク質Pnとγ－セクレターゼとの相互作用（複合体形成）を阻害することができる。したがって、これらの物質は、生体内におけるタンパク質Pnの発現もしくは機能を抑制し、γ－セクレターゼ活性を阻害するので、Aβ産生抑制剤、Aβが関与する疾患（例、AD）やNotchタンパク質の切断が関与する疾患（例、癌）の予防および／または治療剤として使用することができる。
　上記の抗体や低分子化合物を含有する医薬は、そのまま液剤として、または適当な剤型の医薬組成物として、ヒトまたは哺乳動物（例、ラット、ウサギ、ヒツジ、ブタ、ウシ、ネコ、イヌ、サルなど）に対して経口的または非経口的（例、血管内投与、皮下投与など）に投与することができる。 (2) A drug containing an antibody against protein Pn, a low molecular compound that suppresses the expression or function of protein Pn, etc. An antibody against protein Pn, or a low molecular compound that suppresses the expression or function of protein Pn, The activity can be inhibited, or the interaction (complex formation) between protein Pn and γ-secretase can be inhibited. Therefore, these substances suppress the expression or function of protein Pn in vivo and inhibit γ-secretase activity. Therefore, Aβ production inhibitors, diseases involving Aβ (eg, AD) and Notch protein cleavage It can be used as a preventive and / or therapeutic agent for a disease involved (eg, cancer).
The medicament containing the above antibody or low molecular weight compound can be used as a liquid or as a pharmaceutical composition of an appropriate dosage form as a human or mammal (eg, rat, rabbit, sheep, pig, cow, cat, dog, monkey). Etc.) orally or parenterally (eg, intravascular administration, subcutaneous administration, etc.).
　上記の抗体や低分子化合物は、それ自体を投与してもよいし、または適当な医薬組成物として投与してもよい。投与に用いられる医薬組成物としては、上記の抗体もしくは低分子化合物またはその塩と薬理学的に許容され得る担体、希釈剤もしくは賦形剤とを含むものであってもよい。このような医薬組成物は、経口または非経口投与に適する剤形として提供される。 The above-described antibodies and low-molecular compounds may be administered per se, or may be administered as an appropriate pharmaceutical composition. The pharmaceutical composition used for administration may contain the above antibody or low molecular compound or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such pharmaceutical compositions are provided as dosage forms suitable for oral or parenteral administration.
　非経口投与のための組成物としては、例えば、注射剤、坐剤、鼻腔内投与剤等が用いられ、注射剤は静脈注射剤、皮下注射剤、皮内注射剤、筋肉注射剤、点滴注射剤等の剤形を包含しても良い。このような注射剤は、公知の方法に従って調製できる。注射剤の調製方法としては、例えば、上記本発明の抗体もしくは低分子化合物またはその塩を通常注射剤に用いられる無菌の水性液、または油性液に溶解、懸濁または乳化することによって調製できる。注射用の水性液としては、例えば、生理食塩水、ブドウ糖やその他の補助薬を含む等張液等が用いられ、適当な溶解補助剤、例えば、アルコール（例、エタノール）、ポリアルコール（例、プロピレングリコール、ポリエチレングリコール）、非イオン界面活性剤〔例、ポリソルベート80、HCO-50（polyoxyethylene（50mol）adduct of hydrogenated castor oil）〕等と併用してもよい。油性液としては、例えば、ゴマ油、大豆油等が用いられ、溶解補助剤として安息香酸ベンジル、ベンジルアルコール等を併用してもよい。調製された注射液は、適当なアンプルに充填されることが好ましい。直腸投与に用いられる坐剤は、上記抗体またはその塩を通常の坐薬用基剤に混合することによって調製されても良い。 As a composition for parenteral administration, for example, injection, suppository, intranasal administration, etc. are used, and the injection is intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip injection. You may include dosage forms, such as an agent. Such an injection can be prepared according to a known method. As a method for preparing an injection, it can be prepared by, for example, dissolving, suspending or emulsifying the antibody or low molecular compound of the present invention or a salt thereof in a sterile aqueous liquid or oily liquid usually used for injection. As an aqueous solution for injection, for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct-of-hydrogenated-castor-oil)) and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled in a suitable ampoule. A suppository used for rectal administration may be prepared by mixing the above-described antibody or a salt thereof with an ordinary suppository base.
　経口投与のための組成物としては、固体または液体の剤形、具体的には錠剤（糖衣錠、フィルムコーティング錠を含む）、丸剤、顆粒剤、散剤、カプセル剤（ソフトカプセル剤を含む）、シロップ剤、乳剤、懸濁剤等が挙げられる。このような組成物は公知の方法によって製造され、製剤分野において通常用いられる担体、希釈剤もしくは賦形剤を含有していても良い。錠剤用の担体、賦形剤としては、例えば、乳糖、でんぷん、蔗糖、ステアリン酸マグネシウムが用いられる。 Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like. Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field. As the carrier and excipient for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.
　上記の非経口用または経口用医薬組成物は、活性成分の投与量に適合するような投薬単位の剤形に調製されることが好都合である。このような投薬単位の剤形としては、例えば、錠剤、丸剤、カプセル剤、注射剤（アンプル）、坐剤が挙げられる。抗体や低分子化合物は、投薬単位剤形当たり通常0.1～500mg、とりわけ注射剤では5～100mg、その他の剤形では10～250mg含有されていることが好ましい。 The above parenteral or oral pharmaceutical composition is conveniently prepared in a dosage unit form suitable for the dose of the active ingredient. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), and suppositories. The antibody or low molecular weight compound is preferably contained in an amount of usually 0.1 to 500 mg, particularly 5 to 100 mg for injections and 10 to 250 mg for other dosage forms per dosage unit form.
　上記の抗体もしくは低分子化合物またはその塩を含有する上記医薬の投与量は、投与対象、対象疾患、症状、投与ルートなどによっても異なるが、例えば、ADの治療・予防のために使用する場合には、抗体もしくは低分子化合物を1回量として、通常0.0001～20mg/kg体重程度、低分子化合物であれば１日１～５回程度、経口または非経口で、抗体であれば１日～数ヶ月に１回、静脈注射により投与するのが好都合である。他の非経口投与および経口投与の場合もこれに準ずる量を投与することができる。症状が特に重い場合には、その症状に応じて増量してもよい。 The dose of the above-mentioned medicament containing the above-mentioned antibody or low-molecular compound or a salt thereof varies depending on the administration subject, target disease, symptom, administration route, etc., but for example, when used for the treatment / prevention of AD Is usually about 0.0001 to 20 mg / kg body weight for a single dose of antibody or low molecular weight compound, about 1 to 5 times a day for low molecular weight compound, orally or parenterally, 1 to several days for antibody Conveniently administered once a month by intravenous injection. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.
　なお前記した各組成物は、上記抗体や低分子化合物との配合により好ましくない相互作用を生じない限り他の活性成分を含有してもよい。他の活性成分としては、例えば、アセチルコリンエステラーゼ阻害薬に代表されるAD治療薬、非ステロイド抗炎症薬（NSAIDs）などのγ-セクレターゼモジュレーターおよびγ－セクレターゼ阻害剤、β－セクレターゼ阻害剤（例、遷移状態ミミックとして機能し得るスタチン、ヒドロキシエチレン、ヒドロキシエチルカルボニル構造を持ったペプチド性阻害剤（J. Med. Chem., 46(22), 4625-4630 (2003)）、非ペプチド性の低分子型阻害剤（WO 01/87293、WO 02/88101、WO 02/96897））などがあげられる。 Each of the above-described compositions may contain other active ingredients as long as an undesirable interaction is not caused by blending with the above-described antibody or low-molecular compound. Other active ingredients include, for example, AD therapeutic agents typified by acetylcholinesterase inhibitors, γ-secretase modulators such as nonsteroidal anti-inflammatory drugs (NSAIDs), γ-secretase inhibitors, β-secretase inhibitors (eg, Statin that can function as a transition state mimic, hydroxyethylene, peptidic inhibitor with hydroxyethylcarbonyl structure (J. Med. Chem., 46 (22), 4625-4630 低 (2003)), non-peptidic small molecule Type inhibitors (WO 01/87293, WO 02/88101, WO 02/96897)) and the like.
（３）疾病に対する医薬候補化合物のスクリーニング
　上述の通り、タンパク質Pnの発現および／または機能を抑制すると、Ａβ産生が抑制される。従って、タンパク質Pnの発現および／または機能を抑制する化合物またはその塩は、Aβ産生抑制剤、Aβが関与する疾患（例、AD等）やNotchタンパク質の切断が関与する疾患（例、癌）の予防および／または治療剤として使用することができる。
　したがって、タンパク質Pnまたはその部分ペプチド、あるいはそれを産生する細胞は、該タンパク質（遺伝子）の発現量および／または活性を指標とすることにより、Aβ産生抑制作用を有する物質のスクリーニングのためのツールとして用いることができる。 (3) Screening of drug candidate compounds for diseases As described above, when the expression and / or function of protein Pn is suppressed, Aβ production is suppressed. Therefore, a compound or a salt thereof that suppresses the expression and / or function of protein Pn is an Aβ production inhibitor, a disease involving Aβ (eg, AD, etc.) or a disease involving cleavage of Notch protein (eg, cancer). It can be used as a prophylactic and / or therapeutic agent.
Therefore, protein Pn or a partial peptide thereof, or a cell producing the same can be used as a tool for screening a substance having an inhibitory effect on Aβ production by using the expression level and / or activity of the protein (gene) as an index. Can be used.
スクリーニング法（Ｉ）
　タンパク質Pnの機能を抑制する化合物またはその塩をスクリーニングする場合、該スクリーニング方法は、タンパク質Pnまたはその部分ペプチド、あるいはそれを産生する能力を有する細胞を、被検物質の存在下および非存在下にインキュベート（培養）し、両条件下におけるタンパク質Pnの活性を比較することを含む。 Screening method (I)
When screening for a compound or a salt thereof that suppresses the function of protein Pn, the screening method is used in the presence and absence of a test substance for protein Pn or a partial peptide thereof or a cell capable of producing the same. Incubating (culturing) and comparing the activity of protein Pn under both conditions.
　上記のスクリーニング方法において用いられるタンパク質Pnを産生する能力を有する細胞としては、それらを生来発現しているヒトもしくは他の哺乳動物細胞またはそれを含む生体試料（例：血液、組織、臓器等）であれば特に制限はない。非ヒト動物由来の血液、組織、臓器等の場合は、それらを生体から単離して培養してもよいし、あるいは生体自体に被検物質を投与し、一定時間経過後にそれら生体試料を単離してもよい。
　また、タンパク質Pnまたはその部分ペプチドを産生する能力を有する細胞としては、公知慣用の遺伝子工学的手法により作製された各種の形質転換体が例示される。宿主としては、例えば、H4IIE-C3細胞、HepG2細胞、HEK293細胞、COS7細胞、CHO細胞などの動物細胞が好ましく用いられる。 Cells having the ability to produce the protein Pn used in the above screening methods include human or other mammalian cells that naturally express them or biological samples containing them (eg, blood, tissues, organs, etc.) If there is no particular limitation. In the case of blood, tissues, organs, etc. derived from non-human animals, they may be isolated from the living body and cultured, or the test substance is administered to the living body itself, and these biological samples are isolated after a certain period of time. May be.
Examples of cells having the ability to produce protein Pn or a partial peptide thereof include various transformants prepared by known and commonly used genetic engineering techniques. As the host, for example, animal cells such as H4IIE-C3 cells, HepG2 cells, HEK293 cells, COS7 cells, CHO cells are preferably used.
　具体的には、タンパク質Pnまたはその部分ペプチドをコードするDNA（即ち、配列番号：2n-1（nは1～25の何れかの整数）で表される塩基配列もしくは該塩基配列の相補鎖とストリンジェントな条件下でハイブリダイズし、且つ配列番号：2nで表されるアミノ酸配列からなるタンパク質と同質の活性を有するポリペプチドをコードする塩基配列を含むDNA）を、適当な発現ベクター中のプロモーターの下流に連結して宿主動物細胞に導入することにより調製することができる。
　タンパク質Pnまたはその部分ペプチドをコードするDNAは、例えば、配列番号：2n-1で表される塩基配列に基づいて、適当なオリゴヌクレオチドをプローブもしくはプライマーとして合成し、前記したタンパク質Pnを産生する細胞・組織由来のcDNAもしくはcDNAライブラリーから、ハイブリダイゼーション法やPCR法を用いてクローニングすることができる。ハイブリダイゼーションは、例えば、モレキュラー・クローニング（Molecular Cloning）第２版（J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989）に記載の方法などに従って行なうことができる。また、市販のライブラリーを使用する場合、ハイブリダイゼーションは、該ライブラリーに添付された使用説明書に記載の方法に従って行なうことができる。 Specifically, a base sequence represented by DNA encoding protein Pn or a partial peptide thereof (that is, SEQ ID NO: 2n-1 (n is any integer of 1 to 25) or a complementary strand of the base sequence) A DNA containing a base sequence encoding a polypeptide that hybridizes under stringent conditions and has the same quality of activity as the protein consisting of the amino acid sequence represented by SEQ ID NO: 2n) in a promoter in an appropriate expression vector And can be prepared by introducing it into a host animal cell.
A DNA encoding the protein Pn or a partial peptide thereof is synthesized, for example, based on the base sequence represented by SEQ ID NO: 2n-1, using a suitable oligonucleotide as a probe or primer, and a cell that produces the protein Pn described above -It can be cloned from tissue-derived cDNA or cDNA library using hybridization or PCR. Hybridization can be performed, for example, according to the method described in Molecular Cloning 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.
　クローン化されたDNAは、公知のキット、例えば、MutanTM-super Express Km（宝酒造（株））、MutanTM-K（宝酒造（株））等を用いて、ODA-LA PCR法、Gapped duplex法、Kunkel法等の自体公知の方法あるいはそれらに準じる方法に従って塩基配列の変換をすることができる。 The cloned DNA is obtained using a known kit such as Mutan ™ -super Express Km (Takara Shuzo), Mutan ™ -K (Takara Shuzo), etc. The nucleotide sequence can be converted according to a method known per se such as the Kunkel method or a method analogous thereto.
　クローン化されたDNAは、目的によりそのまま、または所望により制限酵素で消化するか、リンカーを付加した後に、使用することができる。該DNAはその5’末端側に翻訳開始コドンとしてのATGを有し、また3’末端側には翻訳終止コドンとしてのTAA、TGAまたはTAGを有していてもよい。これらの翻訳開始コドンや翻訳終止コドンは、適当な合成DNAアダプターを用いて付加することができる。 The cloned DNA can be used as it is depending on the purpose, or after digestion with a restriction enzyme or addition of a linker as desired. The DNA may have ATG as a translation initiation codon on the 5 'end side and TAA, TGA or TAG as a translation termination codon on the 3' end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.
　発現ベクターとしては、動物細胞発現プラスミド（例：pA1-11、pXT1、pRc/CMV、pRc/RSV、pcDNAI/Neo）；λファージなどのバクテリオファージ；レトロウイルス、ワクシニアウイルス、アデノウイルスなどの動物ウイルスベクターなどが用いられる。プロモーターとしては、遺伝子の発現に用いる宿主に対応して適切なプロモーターであればいかなるものでもよい。例えば、SRαプロモーター、SV40プロモーター、LTRプロモーター、CMV（サイトメガロウイルス）プロモーター、RSV（ラウス肉腫ウイルス）プロモーター、MoMuLV（モロニーマウス白血病ウイルス）LTR、HSV-TK（単純ヘルペスウイルスチミジンキナーゼ）プロモーターなどが用いられる。なかでも、CMVプロモーター、SRαプロモーターなどが好ましい。
　発現ベクターとしては、上記の他に、所望によりエンハンサー、スプライシングシグナル、ポリA付加シグナル、選択マーカー、SV40複製起点（以下、SV40 oriと略称する場合がある）などを含有しているものを用いることができる。選択マーカーとしては、例えば、ジヒドロ葉酸還元酵素遺伝子（以下、dhfrと略称する場合がある、メソトレキセート（MTX）耐性）、アンピシリン耐性遺伝子（以下、amprと略称する場合がある）、ネオマイシン耐性遺伝子（以下、neorと略称する場合がある、G418耐性）等が挙げられる。特に、dhfr遺伝子欠損チャイニーズハムスター細胞を用い、dhfr遺伝子を選択マーカーとして使用する場合、チミジンを含まない培地によって目的遺伝子を選択することもできる。 Expression vectors include animal cell expression plasmids (eg, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo); bacteriophages such as λ phage; animal viruses such as retrovirus, vaccinia virus, adenovirus A vector or the like is used. The promoter may be any promoter as long as it is appropriate for the host used for gene expression. For example, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter It is done. Of these, CMV promoter, SRα promoter and the like are preferable.
In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selectable marker, an SV40 origin of replication (hereinafter sometimes abbreviated as SV40 ori), etc. is used as desired. Can do. Selectable markers include, for example, dihydrofolate reductase gene (hereinafter abbreviated as dhfr, methotrexate (MTX) resistance), ampicillin resistance gene (hereinafter abbreviated as amp r ), neomycin resistance gene ( hereinafter sometimes abbreviated as neo r, include G418 resistance) and the like. In particular, when dhfr gene-deficient Chinese hamster cells are used and the dhfr gene is used as a selection marker, the target gene can also be selected using a medium that does not contain thymidine.
　上記したタンパク質PnをコードするDNAを含む発現ベクターで宿主を形質転換することにより、タンパク質Pn発現細胞を製造することができる。
　宿主としては、哺乳動物細胞、例えば、HepG2細胞、HEK293細胞、HeLa細胞、ヒトFL細胞、サルCOS-7細胞、サルVero細胞、チャイニーズハムスター卵巣細胞（以下、CHO細胞と略記）、dhfr遺伝子欠損CHO細胞（以下、CHO(dhfr-)細胞と略記）、マウスL細胞，マウスAtT-20細胞、マウスミエローマ細胞，ラットH4IIE-C3細胞、ラットGH3細胞などが用いられ得る。 Protein Pn-expressing cells can be produced by transforming a host with an expression vector containing the DNA encoding the protein Pn described above.
Hosts include mammalian cells such as HepG2 cells, HEK293 cells, HeLa cells, human FL cells, monkey COS-7 cells, monkey Vero cells, Chinese hamster ovary cells (hereinafter abbreviated as CHO cells), dhfr gene-deficient CHO Cells (hereinafter abbreviated as CHO (dhfr − ) cells), mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat H4IIE-C3 cells, rat GH3 cells and the like can be used.
　形質転換は、リン酸カルシウム共沈殿法、PEG法、エレクトロポレーション法、マイクロインジェクション法、リポフェクション法などにより行うことができる。例えば、細胞工学別冊８　新細胞工学実験プロトコール，263-267 (1995)（秀潤社発行）、ヴィロロジー（Virology），52巻，456 (1973)に記載の方法を用いることができる。 Transformation can be performed by calcium phosphate coprecipitation method, PEG method, electroporation method, microinjection method, lipofection method and the like. For example, the methods described in Cell Engineering Supplement 8, New Cell Engineering Experiment Protocol, 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973) can be used.
　上記のようにして得られる形質転換細胞や生来タンパク質Pnを産生する能力を有する哺乳動物細胞または該細胞を含む組織・臓器は、例えば、約5～20%の胎仔牛血清を含む最小必須培地（MEM）〔Science，122巻,501(1952)〕，ダルベッコ改変イーグル培地（DMEM）〔Virology,8巻,396(1959)〕，RPMI 1640培地〔The Journal of the American Medical Association,199巻,519(1967)〕，199培地〔Proceeding of the Society for the Biological Medicine,73巻,1(1950)〕などの培地中で培養することができる。培地のpHは約6～8であるのが好ましい。培養は通常約30～40℃で行ない、必要に応じて通気や撹拌を加える。 The transformed cells obtained as described above, mammalian cells having the ability to produce the native protein Pn, or tissues / organs containing the cells are, for example, a minimum essential medium containing about 5 to 20% fetal calf serum ( MEM) [Science, 122, 501 (1952)], Dulbecco's Modified Eagle Medium (DMEM) [Virology, 8, 396 (1959)], RPMI 1640 Medium [The Journal of the American Medical Association, 199,519 ( 1967)], 199 medium [Proceeding of the Society for the Biological Medicine, Vol. 73, 1 (1950)]. The pH of the medium is preferably about 6-8. Cultivation is usually carried out at about 30-40 ° C, with aeration and agitation as necessary.
　タンパク質Pnまたはその部分ペプチドは、前記形質転換細胞（あるいは、大腸菌、枯草菌などの細菌、酵母、昆虫細胞などに遺伝子Gnを導入して得られる非哺乳動物細胞でもよい）や、生来タンパク質Pnを産生する能力を有する哺乳動物細胞を培養して得られる培養物から、自体公知の方法に従って分離精製することができる。
　例えば、タンパク質Pnまたはその部分ペプチドを培養菌体あるいは細胞の細胞質から抽出する場合、培養物から公知の方法で集めた菌体あるいは細胞を適当な緩衝液に懸濁し、超音波、リゾチームおよび／または凍結融解などによって菌体あるいは細胞を破壊した後、遠心分離やろ過により可溶性タンパク質の粗抽出液を得る方法などが適宜用いられる。一方、膜画分からタンパク質Pnまたはその部分ペプチドを抽出する場合は、上記と同様に菌体あるいは細胞を破壊した後、低速遠心で細胞デブリスを沈澱除去し、上清を高速遠心して細胞膜含有画分を沈澱させる（必要に応じて密度勾配遠心などにより細胞膜画分を精製する）などの方法が用いられる。また、タンパク質Pnまたはその部分ペプチドが菌体（細胞）外に分泌される場合には、培養物から遠心分離またはろ過等により培養上清を分取するなどの方法が用いられる。
　このようにして得られた可溶性画分、膜画分あるいは培養上清中に含まれるタンパク質Pnまたはその部分ペプチドの単離精製は、自体公知の方法に従って行うことができる。このような方法としては、塩析や溶媒沈澱法などの溶解度を利用する方法；透析法、限外ろ過法、ゲルろ過法、およびＳＤＳ－ポリアクリルアミドゲル電気泳動法などの主として分子量の差を利用する方法；イオン交換クロマトグラフィーなどの荷電の差を利用する方法；アフィニティークロマトグラフィーなどの特異的親和性を利用する方法；逆相高速液体クロマトグラフィーなどの疎水性の差を利用する方法；等電点電気泳動法などの等電点の差を利用する方法；などが用いられる。これらの方法は、適宜組み合わせることもできる。
　かくして得られるタンパク質またはペプチドが遊離体である場合には、自体公知の方法あるいはそれに準じる方法によって、該遊離体を塩に変換することができ、タンパク質またはペプチドが塩として得られた場合には、自体公知の方法あるいはそれに準じる方法により、該塩を遊離体または他の塩に変換することができる。なお、形質転換細胞が産生するタンパク質Pnまたはその部分ペプチドを、精製前または精製後に適当なタンパク質修飾酵素を作用させることにより、任意に修飾を加えたり、ポリペプチドを部分的に除去することもできる。該タンパク質修飾酵素としては、例えば、トリプシン、キモトリプシン、アルギニルエンドペプチダーゼ、プロテインキナーゼ、グリコシダーゼなどが用いられる。 Protein Pn or a partial peptide thereof may be the aforementioned transformed cells (or non-mammalian cells obtained by introducing the gene Gn into bacteria such as Escherichia coli and Bacillus subtilis, yeast and insect cells), or the native protein Pn. It can be separated and purified from a culture obtained by culturing mammalian cells having the ability to produce according to a method known per se.
For example, when extracting protein Pn or a partial peptide thereof from cultured cells or cytoplasm of cells, the cells or cells collected from the culture by a known method are suspended in an appropriate buffer, and are subjected to ultrasound, lysozyme and / or For example, a method of obtaining a crude extract of soluble protein by centrifugation or filtration after disrupting cells or cells by freeze-thawing or the like is appropriately used. On the other hand, when extracting protein Pn or its partial peptide from the membrane fraction, after disrupting the cells or cells in the same manner as described above, cell debris is precipitated and removed by low-speed centrifugation, and the supernatant is centrifuged at high speed to obtain a fraction containing cell membrane. Is used (if necessary, the cell membrane fraction is purified by density gradient centrifugation or the like). Moreover, when protein Pn or its partial peptide is secreted outside a microbial cell (cell), methods, such as fractionating a culture supernatant from a culture by centrifugation or filtration, are used.
Isolation and purification of the protein Pn or its partial peptide contained in the thus obtained soluble fraction, membrane fraction or culture supernatant can be performed according to a method known per se. Examples of such methods include the use of solubilities such as salting out and solvent precipitation; mainly the differences in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. A method utilizing a difference in charge such as ion exchange chromatography; a method utilizing a specific affinity such as affinity chromatography; a method utilizing a difference in hydrophobicity such as reverse phase high performance liquid chromatography; A method using a difference in isoelectric point such as point electrophoresis is used. These methods can be combined as appropriate.
When the protein or peptide thus obtained is a free form, the free form can be converted into a salt by a method known per se or a method analogous thereto, and when the protein or peptide is obtained as a salt, The salt can be converted into a free form or other salt by a method known per se or a method analogous thereto. In addition, the protein Pn produced by the transformed cell or its partial peptide can be arbitrarily modified or the polypeptide can be partially removed by applying an appropriate protein modifying enzyme before or after purification. . Examples of the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.
　さらに、タンパク質Pnまたはその部分ペプチドは、それをコードするRNAを鋳型として、ウサギ網状赤血球ライセート、コムギ胚芽ライセート、大腸菌ライセートなどからなる無細胞タンパク質翻訳系を用いてインビトロ合成することができる。あるいは、さらにRNAポリメラーゼを含む無細胞転写／翻訳系を用いて、タンパク質Pnまたはその部分ペプチドをコードするDNAを鋳型としても合成することができる。 Furthermore, protein Pn or a partial peptide thereof can be synthesized in vitro using a cell-free protein translation system comprising rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate, etc., using the RNA encoding it as a template. Alternatively, a cell-free transcription / translation system further containing RNA polymerase can be used to synthesize DNA encoding the protein Pn or a partial peptide thereof as a template.
　タンパク質Pnまたはその部分ペプチド、あるいはそれを産生する能力を有する細胞としては、(P1)～(P25)から選ばれるいずれか1種のタンパク質を用いてもよいし、2種以上のタンパク質を組み合わせて用いてもよい。2種以上のタンパク質が細胞の形態で提供される場合、当該2種以上のタンパク質すべてを産生する1種の細胞を用いることが好ましい。 As the protein Pn or a partial peptide thereof, or a cell having the ability to produce it, any one protein selected from (P1) to (P25) may be used, or two or more proteins may be combined. It may be used. When two or more types of proteins are provided in the form of cells, it is preferable to use one type of cell that produces all of the two or more types of proteins.
　被検物質としては、例えばタンパク質、ペプチド、非ペプチド性化合物、合成化合物、発酵生産物、細胞抽出液、植物抽出液、動物組織抽出液などが挙げられ、これらの物質は新規なものであってもよいし、公知のものであってもよい。 Examples of test substances include proteins, peptides, non-peptidic compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, etc., and these substances are novel. Alternatively, a known one may be used.
　タンパク質Pnの活性は、好ましくはAβの産生抑制を指標として測定されるので、タンパク質Pnまたはその部分ペプチド、あるいはそれを産生する能力を有する細胞と、被検物質との接触に際しては、Aβ産生系を共存させることが好ましい。Aβ産生系としては、Aβの前駆体であるAPPを発現する細胞が好ましく用いられる。ここで、タンパク質Pnまたはその部分ペプチドが、それを産生する能力を有する細胞として提供される場合、当該細胞自体がAPPを発現するものであることが好ましい。 Since the activity of protein Pn is preferably measured using the inhibition of Aβ production as an index, when contacting protein Pn or its partial peptide or a cell capable of producing it with a test substance, an Aβ production system Preferably coexist. As the Aβ production system, cells expressing APP, which is a precursor of Aβ, are preferably used. Here, when protein Pn or its partial peptide is provided as a cell which has the capability to produce it, it is preferable that the cell itself expresses APP.
　被検物質の上記細胞との接触は、例えば、上記の培地や各種緩衝液（例えば、HEPES緩衝液、リン酸緩衝液、リン酸緩衝生理食塩水、トリス塩酸緩衝液、ホウ酸緩衝液、酢酸緩衝液など）の中に被検物質を添加して、細胞を一定時間インキュベートすることにより実施することができる。添加される被検物質の濃度は化合物の種類（溶解度、毒性等）により異なるが、例えば、約0.1nM～約100nMの範囲で適宜選択される。インキュベート時間としては、例えば、約10分～約24時間が挙げられる。一方、単離されたタンパク質Pnまたはその部分ペプチドを用いる場合、該タンパク質またはペプチドと被検物質との接触は、両者を、APPを発現する細胞の培地に添加して、細胞を一定時間インキュベートすることにより実施することができる。添加されるタンパク質Pnまたはその部分ペプチドの濃度は、各タンパク質またはペプチドについて、例えば、約0.1～約100μg/mlの範囲で適宜選択される。 The contact of the test substance with the cells may be performed by, for example, the above-mentioned medium or various buffers (for example, HEPES buffer, phosphate buffer, phosphate buffered saline, Tris-HCl buffer, borate buffer, acetic acid). The test substance can be added to a buffer solution or the like, and the cells can be incubated for a certain time. The concentration of the test substance to be added varies depending on the type of compound (solubility, toxicity, etc.), but is appropriately selected within the range of about 0.1 nM to about 100 nM, for example. Examples of the incubation time include about 10 minutes to about 24 hours. On the other hand, when using the isolated protein Pn or a partial peptide thereof, the contact between the protein or peptide and the test substance is performed by adding both to the culture medium of the cell expressing APP and incubating the cell for a certain period of time. Can be implemented. The concentration of the protein Pn or a partial peptide to be added is appropriately selected for each protein or peptide, for example, in the range of about 0.1 to about 100 μg / ml.
　タンパク質Pnを産生する細胞が、非ヒト哺乳動物個体の形態で提供される場合、タンパク質Pnを生来発現している動物を用いてもよいし、Pnが過剰発現するように遺伝子Gnを組み込んだ遺伝子組換え動物（トランスジェニック（Tg）動物）を用いてもよい。特に、ヒトタンパク質Pnに対する被検物質の効果を調べたい場合等には、該動物の内因性遺伝子Gnをヒト遺伝子Gnで置換したノックイン動物を用いることもできる。尚、この場合、APPを発現する細胞は該動物個体に内在する。該動物個体の状態は特に制限されないが、例えば、Tg2576マウス、APP23 Tgマウス等のADモデル動物であってもよい。使用される動物の飼育条件に特に制限はないが、SPFグレード以上の環境下で飼育されたものであることが好ましい。被検物質の該細胞との接触は、該動物個体への被検物質の投与によって行われる。投与経路は特に制限されないが、例えば、静脈内投与、動脈内投与、皮下投与、皮内投与、腹腔内投与、経口投与、気道内投与、直腸投与、鼻腔内投与、脳室内投与等が挙げられる。投与量も特に制限はないが、例えば、１回量として約0.5～20 mg／kgを、1日1～5回、1日～6ヶ月程度投与することができる。 When cells that produce protein Pn are provided in the form of a non-human mammal individual, an animal that naturally expresses protein Pn may be used, or a gene that incorporates gene Gn so that Pn is overexpressed Recombinant animals (transgenic (Tg) animals) may be used. In particular, when it is desired to examine the effect of the test substance on the human protein Pn, a knock-in animal in which the endogenous gene Gn of the animal is replaced with the human gene Gn can also be used. In this case, cells expressing APP are inherent in the animal individual. The state of the individual animal is not particularly limited, and may be an AD model animal such as Tg2576 mouse or APP23 Tg mouse. There are no particular restrictions on the breeding conditions of the animals to be used, but it is preferable that the animals are raised in an environment of SPF grade or higher. Contact of the test substance with the cells is carried out by administering the test substance to the animal individual. The administration route is not particularly limited, and examples thereof include intravenous administration, intraarterial administration, subcutaneous administration, intradermal administration, intraperitoneal administration, oral administration, intratracheal administration, rectal administration, intranasal administration, intraventricular administration and the like. . Although there is no particular limitation on the dose, for example, a dose of about 0.5 to 20 mg / kg can be administered 1 to 5 times a day for about 1 to 6 months.
　上記のスクリーニング方法におけるタンパク質Pnの活性の測定は、自体公知の方法でAβの産生量を測定することにより行うことができる。活性測定のための被験試料としては、APPを発現する培養細胞（組織培養、臓器培養を含む）に、タンパク質Pnまたはその部分ペプチド、あるいはそれを産生する能力を有する細胞等（APPを発現する細胞等自体であってもよい）と、被検物質とを接触させる場合、該培養細胞の培養上清が、タンパク質PnおよびAPPを産生する非ヒト哺乳動物個体に被検物質を投与する場合は、該個体から採取した体液、例えば、血液、脳脊髄液等、あるいは細胞、組織、臓器の抽出物、例えば、脳、あるいはそれらの組織切片等のホモジネートなどが挙げられる。
　Aβの産生量は、被験試料中のAβ（例、Aβ38、Aβ40、Aβ42等、好ましくAβ40および／またはAβ42）量を、例えば、抗Aβ抗体を用いたELISA法やウエスタンブロッティング等の各種免疫学的手法、質量分析法などを用いて測定することにより行うことができる。 The protein Pn activity in the above screening method can be measured by measuring the production amount of Aβ by a method known per se. Test samples for measuring activity include cultured cells that express APP (including tissue culture and organ culture), protein Pn or its partial peptide, or cells that have the ability to produce it (cells that express APP). When the test substance is administered to a non-human mammal individual in which the culture supernatant of the cultured cells produces the proteins Pn and APP, when the test substance is contacted with the test substance, Examples thereof include bodily fluids collected from the individual, such as blood, cerebrospinal fluid, and the like, or cell, tissue, and organ extracts, such as the brain or homogenates of tissue sections thereof.
The production amount of Aβ is the amount of Aβ (eg, Aβ38, Aβ40, Aβ42, etc., preferably Aβ40 and / or Aβ42) in the test sample, for example, various immunological methods such as ELISA using anti-Aβ antibody and Western blotting. It can be performed by measuring using a technique, mass spectrometry or the like.
　例えば、上記のスクリーニング方法において、被検物質の存在下におけるタンパク質Pnの活性（例えば、被験試料中のAβ量）が、被検物質の非存在下における活性に比べて、例えば、約10％以上、好ましくは約20％以上、より好ましくは30％以上、さらに好ましくは約50％以上阻害された場合に、該被検物質を、タンパク質Pnの機能抑制物質、従って、Aβ産生抑制作用を有する物質の候補として選択することができる。 For example, in the above screening method, the activity of protein Pn in the presence of the test substance (for example, the amount of Aβ in the test sample) is, for example, about 10% or more compared to the activity in the absence of the test substance When the substance is inhibited, preferably about 20% or more, more preferably 30% or more, and even more preferably about 50% or more, the test substance is treated as a protein Pn function inhibitory substance, and thus a substance having an Aβ production inhibitory action. Can be selected as a candidate.
スクリーニング法（Ｉａ）
　あるいはまた、(G1）～(G25)から選ばれる少なくとも1つの遺伝子を導入した非ヒト動物において、ADの病態を反映する表現型の改善を指標として、ADの治療または予防薬をスクリーニングしたり、上記スクリーニング法もしくは他のスクリーニング法により得られた薬剤のADに対する薬効を評価したりすることができる。具体的には、当該方法は、以下の（ａ）～（ｃ）の工程を含む。
（ａ）(G1）～(G25)から選ばれる少なくとも１つの遺伝子を導入した非ヒト動物に被検物質を投与する工程
（ｂ）該動物における、ADの病態を反映する少なくとも１つの表現型を評価する工程
（ｃ）被検物質の非投与時において評価した場合と比較して、前記表現型を改善させる物質を選択する工程
　工程（ａ）は、前記タンパク質Pnの活性を指標とするスクリーニング方法において、タンパク質PnおよびAPPを産生する非ヒト哺乳動物個体に被検物質を投与する場合と同様に実施することができる。
　工程（ｂ）において、ADの病態を反映する表現型としては、例えば、(1) 脳組織、脳脊髄液、血液中など組織中のAβ量、（２）神経細胞死、（３）中枢神経系の炎症反応、（４）認知能力の低下、(５)アミロイドプラークの蓄積、(６)脳内血流の悪化、(７)脳内グルコース代謝の低下等が挙げられるが、それらに限定されない。これらの表現型の評価は、それぞれについて自体公知のものを用いることができる（例えば、認知能力の評価法として、Y字型迷路試験などが挙げられる）。 Screening method (Ia)
Alternatively, in a non-human animal into which at least one gene selected from (G1) to (G25) has been introduced, screening for a therapeutic or prophylactic agent for AD using an improvement in phenotype reflecting the pathological condition of AD as an index, The efficacy of the drug obtained by the above screening method or other screening methods against AD can be evaluated. Specifically, the method includes the following steps (a) to (c).
(A) a step of administering a test substance to a non-human animal into which at least one gene selected from (G1) to (G25) has been introduced; (b) at least one phenotype that reflects the pathological condition of AD in the animal; A step of evaluating (c) a step of selecting a substance that improves the phenotype as compared with the case where the test substance is not administered, and the step (a) is a screening method using the activity of the protein Pn as an index The method can be carried out in the same manner as in the case of administering the test substance to a non-human mammal individual producing the proteins Pn and APP.
In the step (b), phenotypes reflecting the pathology of AD include, for example, (1) Aβ amount in tissues such as brain tissue, cerebrospinal fluid, blood, (2) neuronal cell death, (3) central nervous system Inflammatory response of the system, (4) reduced cognitive ability, (5) accumulation of amyloid plaques, (6) deterioration of cerebral blood flow, (7) reduction of cerebral glucose metabolism, but not limited thereto . For these phenotypes, those known per se can be used (for example, as a method for evaluating cognitive ability, a Y-shaped maze test or the like can be mentioned).
スクリーニング法（Ｉｂ）
　別の好ましい一実施態様においては、タンパク質Pnの活性はγ－セクレターゼの活性亢進を指標にして測定することができる。具体的には、当該スクリーニング方法は、
（ａ）タンパク質Pnおよびγ－セクレターゼと、被検物質とを接触させる工程、
（ｂ）γ－セクレターゼ活性を測定する工程、および
（ｃ）被検物質の非存在下において測定した場合と比較して、γ－セクレターゼ活性を低下あるいはモジュレートさせる物質を選択する工程、を含む。 Screening method (Ib)
In another preferred embodiment, the activity of protein Pn can be measured using the increased activity of γ-secretase as an indicator. Specifically, the screening method includes:
(A) contacting the protein Pn and γ-secretase with a test substance;
(B) measuring γ-secretase activity, and (c) selecting a substance that reduces or modulates γ-secretase activity compared to when measured in the absence of the test substance. .
　γ－セクレターゼとしては、例えば、それを発現する細胞もしくはその細胞膜画分を用いることができる。また、タンパク質Pnは、γ－セクレターゼを発現する細胞が自ら産生することによって提供されてもよい。
　γ－セクレターゼ活性は、基質となるAPPもしくはNotchを添加して、APPの代謝物フラグメント（Aβ38、Aβ40、Aβ42、AICD等）もしくはNotchの代謝物（NICD）を、ELISA法、ウエスタンブロッティング、レポータージーンアッセイ、質量分析法などを用いて検出することにより、測定することができる。 As γ-secretase, for example, a cell expressing it or a cell membrane fraction thereof can be used. The protein Pn may be provided by a cell that expresses γ-secretase itself.
γ-secretase activity is determined by adding APP or Notch as a substrate, APP metabolite fragments (Aβ38, Aβ40, Aβ42, AICD, etc.) or Notch metabolites (NICD), ELISA, Western blotting, reporter gene assay It can be measured by detecting using mass spectrometry or the like.
　例えば、上記のスクリーニング方法において、被検物質の存在下におけるγ－セクレターゼ活性が、被検物質の非存在下における活性に比べて、約10％以上、好ましくは約20％以上、より好ましくは30％以上、さらに好ましくは約50％以上阻害された場合に、該被検物質を、タンパク質Pnの機能抑制物質、従って、Aβ産生抑制作用を有する物質の候補として選択することができる。 For example, in the above screening method, the γ-secretase activity in the presence of the test substance is about 10% or more, preferably about 20% or more, more preferably 30%, compared to the activity in the absence of the test substance. %, More preferably about 50% or more, the test substance can be selected as a protein Pn function-inhibiting substance, and thus a candidate for an Aβ production-inhibiting substance.
　上記いずれかのスクリーニング方法において、コントロールとして、常法を用いて作製される、遺伝子Gnがノックアウトまたはノックダウンされた細胞または動物を用い、被験細胞または動物とコントロールとにおける、被検物質存在下でのタンパク質Pnの活性を測定、比較することによってもＡβ産生抑制物質を選択することができる。 In any one of the above screening methods, as a control, a cell or an animal in which the gene Gn is knocked out or knocked down is used as a control, and in the presence of a test substance in the test cell or animal and the control. An Aβ production inhibitor can also be selected by measuring and comparing the activity of the protein Pn.
スクリーニング法（II）
　本発明はまた、タンパク質Pnを産生する能力を有する細胞における該タンパク質（遺伝子）の発現を、被検物質の存在下と非存在下で比較することを特徴とする、Aβ産生抑制物質のスクリーニング方法を提供する。本方法において用いられる細胞、被検物質の種類、被検物質と細胞との接触の態様などは、上記したタンパク質Pnの活性を指標とする方法と同様である。 Screening method (II)
The present invention also relates to a method for screening an Aβ production inhibitor, comprising comparing the expression of the protein (gene) in a cell having the ability to produce protein Pn in the presence and absence of the test substance. I will provide a. The cells used in this method, the type of test substance, the mode of contact between the test substance and the cells, and the like are the same as in the method using the activity of the protein Pn as an index.
　タンパク質Pnの発現量は、前記したタンパク質PnをコードするDNAとハイストリンジェントな条件下でハイブリダイズし得る核酸、即ち、配列番号：nで表される塩基配列もしくはそれと相補的な塩基配列とストリンジェントな条件下でハイブリダイズし得る核酸（以下、「本発明の検出用核酸」という場合がある）を用いて、遺伝子GnのmRNAを検出することにより、RNAレベルで測定することができる。あるいは、該発現量は、前記したタンパク質Pnに対する抗体（以下、「本発明の検出用抗体」という場合がある）を用いて、これらのタンパク質を検出することにより、タンパク質レベルで測定することもできる。
　従って、より具体的には、本発明は、
（a）タンパク質Pnを産生する能力を有する細胞を被検物質の存在下および非存在下に培養し、両条件下における該タンパク質をコードするmRNAの量を、本発明の検出用核酸を用いて測定、比較することを特徴とする、Aβ産生抑制物質のスクリーニング方法、および
（b）タンパク質Pnを産生する能力を有する細胞を被検物質の存在下および非存在下に培養し、両条件下における該タンパク質の量を、本発明の検出用抗体を用いて測定、比較することを特徴とする、Aβ産生抑制物質のスクリーニング方法を提供する。 The expression level of protein Pn is a nucleic acid that can hybridize with the above-described DNA encoding protein Pn under highly stringent conditions, that is, the nucleotide sequence represented by SEQ ID NO: n or a complementary nucleotide sequence and string It can be measured at the RNA level by detecting the mRNA of the gene Gn using a nucleic acid that can hybridize under gentle conditions (hereinafter sometimes referred to as “the nucleic acid for detection of the present invention”). Alternatively, the expression level can be measured at the protein level by detecting these proteins using an antibody against the above-described protein Pn (hereinafter sometimes referred to as “the detection antibody of the present invention”). .
Therefore, more specifically, the present invention
(A) Cells having the ability to produce protein Pn are cultured in the presence and absence of a test substance, and the amount of mRNA encoding the protein under both conditions is determined using the nucleic acid for detection of the present invention. A method for screening an Aβ production inhibitor characterized by measuring and comparing, and (b) culturing cells capable of producing protein Pn in the presence and absence of a test substance, under both conditions Provided is a method for screening an Aβ production inhibitor, characterized in that the amount of the protein is measured and compared using the detection antibody of the present invention.
　例えば、タンパク質PnのmRNA量またはタンパク質量の測定は、具体的には以下のようにして行うことができる。
（i）正常あるいは疾患（例えば、ADなど）モデル非ヒト哺乳動物（例えば、マウス、ラット、ウサギ、ヒツジ、ブタ、ウシ、ネコ、イヌ、サルなど）に対して薬剤などを与え、一定時間経過した後に、血液、あるいは特定の臓器（例えば、脳等）、あるいは臓器から単離した組織または細胞を得る。
　得られた細胞に含まれるタンパク質PnのmRNAは、例えば、通常の方法により細胞等からmRNAを抽出し、例えば、RT-PCRなどの手法を用いることにより定量することができ、あるいは自体公知のノーザンブロット解析により定量することもできる。一方、タンパク質Pnの量は、ウェスタンブロット解析や以下に詳述する各種イムノアッセイ法を用いて定量することができる。
（ii）遺伝子Gnを導入した形質転換体を上記の方法に従って作製し、該形質転換体に含まれるタンパク質PnあるいはそれをコードするmRNAを、上記（i）と同様にして定量、解析することができる。 For example, the measurement of the amount of mRNA or protein of protein Pn can be specifically performed as follows.
(I) Normal or disease (for example, AD) model non-human mammals (for example, mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, etc.) After that, blood, a specific organ (for example, brain), or a tissue or cell isolated from the organ is obtained.
The mRNA of the protein Pn contained in the obtained cell can be quantified by, for example, extracting mRNA from the cell etc. by a normal method and using, for example, a technique such as RT-PCR, or a known Northern Northern It can also be quantified by blot analysis. On the other hand, the amount of protein Pn can be quantified using Western blot analysis or various immunoassay methods described in detail below.
(Ii) A transformant introduced with the gene Gn is prepared according to the above method, and the protein Pn contained in the transformant or mRNA encoding the same can be quantified and analyzed in the same manner as in (i) above. it can.
　タンパク質Pnの発現量を変化させる物質のスクリーニングは、
（i）正常あるいは疾患モデル非ヒト哺乳動物に対して、薬剤などを与える一定時間前（30分前～24時間前、好ましくは30分前～12時間前、より好ましくは1時間前～6時間前）もしくは一定時間後（30分後～3日後、好ましくは1時間後～2日後、より好ましくは1時間後～24時間後）、または薬剤などと同時に被検物質を投与し、投与から一定時間が経過した後（30分後～3日後、好ましくは1時間後～2日後、より好ましくは1時間後～24時間後）、該動物から単離した細胞に含まれるタンパク質PnをコードするmRNA量、あるいはタンパク質Pnの量を定量、解析することにより、あるいは
（ii）形質転換体を常法に従って培養する際に被検物質を培地もしくは緩衝液中に添加し、一定時間インキュベート後（1日後～7日後、好ましくは1日後～3日後、より好ましくは2日後～3日後）、該形質転換体に含まれるタンパク質PnをコードするmRNA量、あるいはタンパク質Pnの量を定量、解析することにより行うことができる。 Screening for substances that alter the expression level of protein Pn
(I) A certain period of time before giving a drug or the like to a normal or disease model non-human mammal (30 minutes to 24 hours, preferably 30 minutes to 12 hours, more preferably 1 hour to 6 hours) Before) or after a certain time (30 minutes to 3 days later, preferably 1 hour to 2 days later, more preferably 1 hour to 24 hours later) After time has elapsed (30 minutes to 3 days later, preferably 1 hour to 2 days later, more preferably 1 hour to 24 hours later), mRNA encoding protein Pn contained in cells isolated from the animal Quantitatively analyze the amount of protein or protein Pn, or (ii) add a test substance to the medium or buffer when culturing the transformant according to a conventional method, and incubate for a certain period of time (after 1 day) ~ 7 days later, preferably 1 day to 3 days later, more preferred After days 2 days ~ 3), mRNA amount encodes a protein Pn included in the transformant, or the amount of protein Pn quantification can be performed by analyzing.
　上記（b）のスクリーニング方法におけるタンパク質Pnの量の測定は、具体的には、例えば、
（i）本発明の検出用抗体と、試料液および標識化されたタンパク質Pnとを競合的に反応させ、該抗体に結合した標識化されたタンパク質を検出することにより試料液中のタンパク質Pnを定量する方法や、
（ii）試料液と、担体上に不溶化した本発明の検出用抗体および標識化された別の本発明の検出用抗体とを、同時あるいは連続的に反応させた後、不溶化担体上の標識剤の量（活性）を測定することにより、試料液中のCタンパク質Pnを定量する方法等が挙げられる。
　上記（ii）の定量法においては、２種の抗体はタンパク質Pnの異なる部分を認識するものであることが望ましい。例えば、一方の抗体がタンパク質PnのＮ端部を認識する抗体であれば、他方の抗体として該タンパク質のＣ端部と反応するものを用いることができる。
　標識物質を用いる測定法に用いられる標識剤としては、例えば、放射性同位元素、酵素、蛍光物質、発光物質などが用いられる。放射性同位元素としては、例えば、〔125I〕、〔131I〕、〔3H〕、〔14C〕などが用いられる。上記酵素としては、安定で比活性の大きなものが好ましく、例えば、β－ガラクトシダーゼ、β－グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素などが用いられる。蛍光物質としては、例えば、フルオレスカミン、フルオレッセンイソチオシアネートなどが用いられる。発光物質としては、例えば、ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニンなどが用いられる。さらに、抗体あるいは抗原と標識剤との結合にビオチン－（ストレプト）アビジン系を用いることもできる。 Specifically, the measurement of the amount of protein Pn in the screening method of (b) above is, for example,
(I) The detection antibody of the present invention, the sample solution and the labeled protein Pn are reacted competitively, and the protein Pn in the sample solution is detected by detecting the labeled protein bound to the antibody. How to quantify,
(Ii) The sample solution is reacted with the detection antibody of the present invention insolubilized on the carrier and another labeled detection antibody of the present invention simultaneously or continuously, and then the labeling agent on the insolubilized carrier. For example, a method of quantifying C protein Pn in a sample solution by measuring the amount (activity) of.
In the above quantification method (ii), it is desirable that the two types of antibodies recognize different portions of the protein Pn. For example, if one antibody recognizes the N-terminal part of protein Pn, the other antibody can react with the C-terminal part of the protein.
As a labeling agent used in a measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. As the radioisotope, for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used. The enzyme is preferably stable and has a large specific activity. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent material, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, a biotin- (strept) avidin system can be used for binding of an antibody or antigen to a labeling agent.
　本発明の検出用抗体を用いるタンパク質Pnの定量法は、特に制限されるべきものではなく、試料液中の抗原量に対応した、抗体、抗原もしくは抗体－抗原複合体の量を化学的または物理的手段により検出し、これを既知量の抗原を含む標準液を用いて作製した標準曲線より算出する測定法であれば、いずれの測定法を用いてもよい。例えば、ネフロメトリー、競合法、イムノメトリック法およびサンドイッチ法が好適に用いられる。感度、特異性の点で、例えば、後述するサンドイッチ法を用いるのが好ましい。 The method for quantifying protein Pn using the detection antibody of the present invention is not particularly limited, and the amount of antibody, antigen or antibody-antigen complex corresponding to the amount of antigen in the sample solution is chemically or physically determined. Any measurement method may be used as long as it is a measurement method that is detected by a standard means and calculated from a standard curve prepared using a standard solution containing a known amount of antigen. For example, nephrometry, competition method, immunometric method and sandwich method are preferably used. In view of sensitivity and specificity, for example, the sandwich method described later is preferably used.
　抗原あるいは抗体の不溶化にあたっては、物理吸着を用いてもよく、また通常タンパク質あるいは酵素等を不溶化・固定化するのに用いられる化学結合を用いてもよい。担体としては、アガロース、デキストラン、セルロースなどの不溶性多糖類、ポリスチレン、ポリアクリルアミド、シリコン等の合成樹脂、あるいはガラス等があげられる。 In the insolubilization of the antigen or antibody, physical adsorption may be used, or a chemical bond usually used for insolubilizing and immobilizing proteins or enzymes may be used. Examples of the carrier include insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass.
　サンドイッチ法においては不溶化した本発明の検出用抗体に試料液を反応させ（１次反応）、さらに標識化した別の本発明の検出用抗体を反応させ（２次反応）た後、不溶化担体上の標識剤の量もしくは活性を測定することにより、試料液中のタンパク質Pnを定量することができる。１次反応と２次反応は逆の順序で行っても、また、同時に行ってもよいし、時間をずらして行ってもよい。標識化剤および不溶化の方法は前記のそれらに準じることができる。また、サンドイッチ法による免疫測定法において、固相化抗体あるいは標識化抗体に用いられる抗体は必ずしも１種類である必要はなく、測定感度を向上させる等の目的で２種類以上の抗体の混合物を用いてもよい。 In the sandwich method, a sample solution is reacted with the insolubilized detection antibody of the present invention (primary reaction), and another labeled detection antibody of the present invention is reacted (secondary reaction), and then on the insolubilized carrier. The protein Pn in the sample solution can be quantified by measuring the amount or activity of the labeling agent. The primary reaction and the secondary reaction may be performed in the reverse order, may be performed simultaneously, or may be performed at different times. The labeling agent and the insolubilization method can be the same as those described above. Further, in the immunoassay by the sandwich method, the antibody used for the immobilized antibody or the labeled antibody is not necessarily one type, and a mixture of two or more types of antibodies is used for the purpose of improving measurement sensitivity. May be.
　本発明の検出用抗体は、サンドイッチ法以外の測定システム、例えば、競合法、イムノメトリック法あるいはネフロメトリーなどにも用いることができる。
　競合法では、試料液中のタンパク質Pnと標識したタンパク質Pnとを抗体に対して競合的に反応させた後、未反応の標識抗原(Ｆ)と、抗体と結合した標識抗原（Ｂ）とを分離し（Ｂ／Ｆ分離）、Ｂ，Ｆいずれかの標識量を測定することにより、試料液中のタンパク質Pnを定量する。本反応法には、抗体として可溶性抗体を用い、ポリエチレングリコールや前記抗体（１次抗体）に対する２次抗体などを用いてＢ／Ｆ分離を行う液相法、および、１次抗体として固相化抗体を用いるか（直接法）、あるいは１次抗体は可溶性のものを用い、２次抗体として固相化抗体を用いる（間接法）固相化法とが用いられる。
　イムノメトリック法では、試料液中のタンパク質Pnと固相化したタンパク質Pnとを一定量の標識化抗体に対して競合反応させた後、固相と液相を分離するか、あるいは試料液中のタンパク質Pnと過剰量の標識化抗体とを反応させ、次に固相化したタンパク質Pnを加えて未反応の標識化抗体を固相に結合させた後、固相と液相を分離する。次に、いずれかの相の標識量を測定し試料液中の抗原量を定量する。
　また、ネフロメトリーでは、ゲル内あるいは溶液中で抗原抗体反応の結果生じた不溶性の沈降物の量を測定する。試料液中のタンパク質Pnの量がわずかであり、少量の沈降物しか得られない場合にもレーザーの散乱を利用するレーザーネフロメトリーなどが好適に用いられる。 The detection antibody of the present invention can also be used in measurement systems other than the sandwich method, such as a competitive method, an immunometric method, or nephrometry.
In the competition method, the protein Pn in the sample solution and the labeled protein Pn are reacted competitively with the antibody, and then the unreacted labeled antigen (F) and the labeled antigen (B) bound to the antibody. Separation (B / F separation) and the amount of labeling of either B or F is measured to quantify the protein Pn in the sample solution. In this reaction method, a soluble antibody is used as an antibody, B / F separation is performed using polyethylene glycol or a secondary antibody against the antibody (primary antibody), and a solid phase is used as the primary antibody. Either an antibody is used (direct method), or a primary antibody is soluble, and a solid phase antibody is used as a secondary antibody (indirect method).
In the immunometric method, the protein Pn in the sample solution and the immobilized protein Pn are allowed to compete with a certain amount of labeled antibody, and then the solid phase and the liquid phase are separated, or the sample solution in the sample solution is separated. After reacting protein Pn with an excess amount of labeled antibody, and then adding immobilized protein Pn to bind unreacted labeled antibody to the solid phase, the solid phase and the liquid phase are separated. Next, the amount of label in any phase is measured to quantify the amount of antigen in the sample solution.
In nephrometry, the amount of insoluble precipitate produced as a result of antigen-antibody reaction in a gel or solution is measured. Laser nephrometry using laser scattering is preferably used even when the amount of protein Pn in the sample solution is very small and only a small amount of precipitate can be obtained.
　これら個々の免疫学的測定法を本発明の定量方法に適用するにあたっては、特別の条件、操作等の設定は必要とされない。それぞれの方法における通常の条件、操作法に当業者の通常の技術的配慮を加えてタンパク質Pnの測定系を構築すればよい。これらの一般的な技術手段の詳細については、総説、成書などを参照することができる。
　例えば、入江　寛編「ラジオイムノアッセイ」（講談社、昭和４９年発行）、入江　寛編「続ラジオイムノアッセイ」（講談社、昭和５４年発行）、石川栄治ら編「酵素免疫測定法」（医学書院、昭和５３年発行）、石川栄治ら編「酵素免疫測定法」（第２版）（医学書院、昭和５７年発行）、石川栄治ら編「酵素免疫測定法」（第３版）（医学書院、昭和６２年発行）、「Methods in ENZYMOLOGY」 Vol. 70 (Immunochemical Techniques (Part A))、同書 Vol. 73 (Immunochemical Techniques (Part B))、同書 Vol. 74 (Immunochemical Techniques (Part C))、同書 Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays))、同書 Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods))、同書 Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies))（以上、アカデミックプレス社発行）などを参照することができる。
　以上のようにして、本発明の検出用抗体を用いることによって、細胞におけるタンパク質Pnの量を感度よく定量することができる。 In applying these individual immunological measurement methods to the quantification method of the present invention, special conditions, operations and the like are not required to be set. What is necessary is just to construct | assemble the measurement system of protein Pn, adding the usual technical consideration of those skilled in the art to the usual conditions and operation methods in each method. For details of these general technical means, it is possible to refer to reviews, books and the like.
For example, Hiroshi Irie “Radioimmunoassay” (Kodansha, published in 1974), Hiroshi Irie “Sequential Radioimmunoassay” (published in Kodansha, 1979), “Enzyme Immunoassay” edited by Eiji Ishikawa et al. 53)), edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (2nd edition) (Medical Shoin, published in 1982), edited by Eiji Ishikawa et al. "Enzyme Immunoassay" (3rd edition) (Medical Shoin, Showa) 62), “Methods in ENZYMOLOGY” Vol. 70 (Immunochemical Techniques (Part A)), Id. Vol. 73 (Immunochemical Techniques (Part B)), Id. Vol. 74 (Immunochemical Techniques (Part C)), Id. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), ibid.Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid.Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies) )) (End of academic program , Inc. issued) and the like can be referred to.
As described above, the amount of protein Pn in cells can be quantified with high sensitivity by using the detection antibody of the present invention.
　例えば、上記（a）および（b）のスクリーニング法において、被検物質の存在下におけるタンパク質Pnの発現量（mRNA量またはタンパク質量）が、被検物質の非存在下における場合に比べて、約10%以上、好ましくは約20%以上、より好ましくは約30％以上、さらに好ましくは約50％以上阻害された場合、該被検物質を、タンパク質Pnの発現抑制物質、従って、Aβ産生抑制作用を有する物質の候補として選択することができる。 For example, in the screening methods (a) and (b) above, the expression level of protein Pn (mRNA amount or protein amount) in the presence of the test substance is about 10% less than that in the absence of the test substance. When the test substance is inhibited by 10% or more, preferably about 20% or more, more preferably about 30% or more, and even more preferably about 50% or more, the test substance is expressed as a protein Pn expression-suppressing substance, and thus has an effect of inhibiting Aβ production. Can be selected as a candidate for a substance having
　あるいは、上記スクリーニング法において、遺伝子Gnを発現する細胞に代えて、遺伝子Gnの内在の転写調節領域の制御下にあるレポーター遺伝子を含む細胞を用いることができる。このような細胞は、遺伝子Gnの転写調節領域の制御下にあるレポーター遺伝子（例、ルシフェラーゼ、GFPなど）を導入したトランスジェニック動物の細胞、組織、臓器もしくは個体であってもよい。かかる細胞を用いる場合には、タンパク質Pnの発現量は、レポーター遺伝子の発現レベルを、常法を用いて測定することにより評価することができる。 Alternatively, in the above screening method, a cell containing a reporter gene under the control of the transcriptional regulatory region in the gene Gn can be used instead of the cell expressing the gene Gn. Such a cell may be a cell, tissue, organ or individual of a transgenic animal into which a reporter gene (eg, luciferase, GFP, etc.) under the control of the transcriptional regulatory region of gene Gn is introduced. When such cells are used, the expression level of protein Pn can be evaluated by measuring the expression level of the reporter gene using a conventional method.
　上記のスクリーニング方法において、コントロールとして、常法を用いて作製される、遺伝子Gnがノックアウトまたはノックダウンされた細胞または動物を用い、被験細胞または動物とコントロールとにおける、被検物質存在下でのタンパク質Pnの発現量を測定、比較することによってもＡβ産生抑制物質を選択することができる。 In the screening method described above, as a control, a protein or gene in which the gene Gn is knocked out or knocked down is used as a control, and the protein in the presence of the test substance in the test cell or animal and the control An Aβ production inhibitor can also be selected by measuring and comparing the expression level of Pn.
スクリーニング法（III）
　本発明はまた、以下の（ａ）～（ｃ）の工程を含む、Ａβ産生抑制物質のスクリーニング方法を提供する。
（ａ）タンパク質Pnおよびγ－セクレターゼと、被検物質とを接触させる工程
（ｂ）タンパク質Pnとγ－セクレターゼとの結合活性を測定する工程
（ｃ）被検物質の非存在下において測定した場合と比較して、前記結合活性を低下させる物質を選択する工程
　結合活性の指標としては、γ－セクレターゼの活性亢進で測定するか、下記に示すごとく、結合量を測定することが例示できる（前者については、上記スクリーニング法（Ｉｂ）に準じて行うことができる）。
　より具体的には、上記スクリーニング方法は、
(1)標識したタンパク質Pnと、γ－セクレターゼとを、被検物質の存在下および非存在下で接触させた場合における、標識したタンパク質Pnのγ－セクレターゼに対する結合量を測定し、比較する、または
(2)標識したタンパク質Pnと、γ－セクレターゼを産生する細胞またはその膜画分とを、被検物質の存在下および非存在下で接触させた場合における、標識したタンパク質Pnの該細胞または膜画分に対する結合量を測定し、比較することを特徴とする。
　タンパク質Pnは、常法に従って、例えば放射性同位元素、酵素、蛍光物質、発光物質などで標識することができる。放射性同位元素としては、例えば、〔125I〕、〔131I〕、〔3H〕、〔14C〕などが用いられる。酵素としては、安定で比活性の大きなものが好ましく、例えば、β－ガラクトシダーゼ、β－グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素などが用いられる。蛍光物質としては、例えば、フルオレスカミン、フルオレッセンイソチオシアネートなどが用いられる。発光物質としては、例えば、ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニンなどが用いられる。 Screening method (III)
The present invention also provides a method for screening an Aβ production inhibitor comprising the following steps (a) to (c).
(A) A step of bringing protein Pn and γ-secretase into contact with a test substance (b) A step of measuring the binding activity between protein Pn and γ-secretase (c) When measured in the absence of the test substance The step of selecting the substance that decreases the binding activity compared to the above may be exemplified by measuring the amount of binding as shown in the following, as an indicator of binding activity, or by measuring the increased activity of γ-secretase (the former Can be performed according to the above screening method (Ib)).
More specifically, the screening method includes
(1) Measuring and comparing the amount of labeled protein Pn bound to γ-secretase when the labeled protein Pn and γ-secretase are contacted in the presence and absence of the test substance. Or
(2) the labeled protein Pn and the cell or membrane of the labeled protein Pn when the γ-secretase producing cell or its membrane fraction is contacted in the presence and absence of the test substance The amount of binding to the fraction is measured and compared.
Protein Pn can be labeled with a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, and the like according to a conventional method. As the radioisotope, for example, [ 125 I], [ 131 I], [ 3 H], [ 14 C] and the like are used. As the enzyme, those which are stable and have high specific activity are preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent material, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.
　例えば、上記（1）および（2）のスクリーニング法において、被検物質の存在下におけるγ－セクレターゼに結合する標識量（タンパク質Pn量）が、被検物質の非存在下における場合に比べて、約10%以上、好ましくは約20%以上、より好ましくは約30％以上、さらに好ましくは約50％以上阻害された場合、該被検物質をタンパク質Pnとγ－セクレターゼとの結合活性を抑制する物質、従って、Aβ産生抑制作用を有する物質の候補として選択することができる。 For example, in the screening methods (1) and (2) above, the amount of label (protein Pn) that binds to γ-secretase in the presence of the test substance is larger than that in the absence of the test substance. When it is inhibited by about 10% or more, preferably about 20% or more, more preferably about 30% or more, more preferably about 50% or more, the test substance suppresses the binding activity between protein Pn and γ-secretase. The substance can be selected as a candidate of a substance having an Aβ production inhibitory action.
　本発明の上記いずれかのスクリーニング方法を用いて得られる、タンパク質Pnの発現または機能を抑制する物質は、Aβが関与する疾患（例、AD）やNotchタンパク質の切断が関与する疾患（例、癌）の予防および／または治療薬として有用である。
　本発明のスクリーニング方法を用いて得られる物質を上述の予防・治療剤として使用する場合、上記タンパク質Pnの発現または機能を抑制する低分子化合物と同様に製剤化することができ、同様の投与経路および投与量で、ヒトまたは哺乳動物（例えば、マウス、ラット、ウサギ、ヒツジ、ブタ、ウシ、ウマ、ネコ、イヌ、サル、チンパンジーなど）に対して、経口的にまたは非経口的に投与することができる。 A substance that suppresses the expression or function of protein Pn obtained by using any one of the screening methods of the present invention is a disease involving Aβ (eg, AD) or a disease involving cleavage of Notch protein (eg, cancer). ) Is useful as a preventive and / or therapeutic agent.
When the substance obtained using the screening method of the present invention is used as the above-mentioned prophylactic / therapeutic agent, it can be formulated in the same manner as a low molecular weight compound that suppresses the expression or function of the above protein Pn, and the same administration route And administered orally or parenterally to humans or mammals (eg, mice, rats, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, etc.) Can do.
（４）診断薬としての利用
　また、本発明は、被験動物より採取した試料中のタンパク質Pnの発現量を測定することを特徴とするAβが関与する疾患（例、AD）やNotchタンパク質の切断が関与する疾患（例、癌）の発症または発症リスクの判定方法を提供する。当該方法は以下の（ａ）～（ｃ）の工程を含む。
（ａ）被験動物由来の試料を提供する工程
（ｂ）該試料中の、遺伝子Gn（n=1～25）から選ばれる少なくとも１つの遺伝子の発現量もしくはタンパク質Pn（n=1～25）から選ばれる少なくとも１つのタンパク質の活性を測定する工程
（ｃ）正常動物由来の試料において測定した場合と比較して、前記発現量もしくは活性が上昇している被験動物を、Aβが関与する疾患（例、AD）やNotchタンパク質の切断が関与する疾患（例、癌）を発症しているか、将来発症するリスクが高いと判定する工程 (4) Use as a diagnostic agent In addition, the present invention relates to Aβ-related diseases (eg, AD) or Notch protein cleavage characterized by measuring the expression level of protein Pn in a sample collected from a test animal A method for determining the onset or risk of developing a disease (eg, cancer) in which is involved. The method includes the following steps (a) to (c).
(A) Providing a test animal-derived sample (b) Expression level of at least one gene selected from gene Gn (n = 1-25) or protein Pn (n = 1-25) in the sample A step of measuring the activity of at least one protein selected (c) a test animal having an increased expression level or activity compared to a case of measuring in a sample derived from a normal animal, , AD) or a disease that involves Notch protein cleavage (eg, cancer) or has a high risk of developing in the future
　被験動物としては、ヒトもしくは他の哺乳動物が挙げられるが、好ましくはヒト、あるいは実験動物として汎用されるマウス、ラット、ウサギ、イヌ、サル等である。測定対象試料としては、血液、血漿、血清、脳脊髄液、リンパ液、唾液、粘膜、尿、涙、精液、関節液、生検サンプル等が挙げられる。 Examples of test animals include humans and other mammals, preferably humans, mice, rats, rabbits, dogs, monkeys and the like that are widely used as experimental animals. Examples of the measurement target sample include blood, plasma, serum, cerebrospinal fluid, lymph fluid, saliva, mucous membrane, urine, tears, semen, joint fluid, biopsy sample, and the like.
　試料中の遺伝子Gnの発現量およびタンパク質Pnの量は、該遺伝子もしくは該タンパク質の発現量を指標とする上記スクリーニング法に記載されたのと同様の方法により測定することができる。
　上記測定の結果、被験動物より採取した試料中の遺伝子Gnの発現量またはタンパク質Pnの量が、正常動物より採取した試料中の遺伝子Gnの発現量またはタンパク質Pnの量と比較して有意に高かった場合、該被験動物は、Aβが関与する疾患（例、AD）やNotchタンパク質の切断が関与する疾患（例、癌）を発症しているか、将来発症するリスクが高いと判定することができる。あるいは、正常動物における発現量を予め同定しておき、例えば、その平均値＋2SDをカットオフ値として規定し、被験動物より採取した試料中の遺伝子Gnの発現量またはタンパク質Pnの量が、当該カットオフ値を超えた場合に、該被験動物は、Aβが関与する疾患（例、AD）やNotchタンパク質の切断が関与する疾患（例、癌）を発症しているか、将来発症するリスクが高いと判定することもできる。 The expression level of gene Gn and the amount of protein Pn in the sample can be measured by the same method as described in the above screening method using the expression level of the gene or the protein as an index.
As a result of the above measurement, the expression level of the gene Gn or the protein Pn in the sample collected from the test animal is significantly higher than the expression level of the gene Gn or the protein Pn in the sample collected from the normal animal. If the test animal has a disease associated with Aβ (eg, AD) or a disease associated with Notch protein cleavage (eg, cancer), or can be determined to have a high risk of developing in the future. . Alternatively, the expression level in a normal animal is identified in advance, for example, the average value + 2SD is defined as a cutoff value, and the expression level of gene Gn or the amount of protein Pn in a sample collected from the test animal When the off-value is exceeded, the test animal has developed a disease involving Aβ (eg, AD), a disease involving cleavage of Notch protein (eg, cancer), or has a high risk of developing in the future It can also be determined.
　非ヒト動物の上記診断においては、コントロールとして、遺伝子Gnをノックアウトまたはノックダウンした動物を用いてもよい。また、遺伝子Gnをノックアウトまたはノックダウンした動物は、タンパク質Pnの機能解析のためのツールとしても有用である。 In the above diagnosis of a non-human animal, an animal having the gene Gn knocked out or knocked down may be used as a control. An animal in which the gene Gn is knocked out or knocked down is also useful as a tool for analyzing the function of the protein Pn.
　以下、実施例により本発明をより詳細に説明するが、本発明はこれらの実施例により何ら限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
実施例１　脳γ－セクレターゼ結合タンパク質の同定（１）
　脳組織では、ミクロゾーム画分、界面活性剤不溶性の膜画分（DRM）、シナプス膜、およびシナプス小胞においてγ－セクレターゼ活性が強く検出されている。そこで、これらの画分をそれぞれ調製し、インヒビタープルダウン法によりγ－セクレターゼ複合体の精製を行った。
（１）脳γ－セクレターゼの含有画分の調製
　SD ラットは B&K Universal (Sollentuna, Sweden)から入手し、ヒト脳組織をHuddinge Brain Bank (Huddinge, Sweden)より入手した。
（ミクロゾーム画分）
　ラットあるいはヒトの脳組織を緩衝液A (20 mM Hepes, 50 mM KCl, 2 mM EGTA, pH 7.5にComplete protease inhibitor mixture(Roche Applied Science, Indianapolis, IN, USA)を添加したもの)中でホモジナイズした後、1,000 x gおよび10,000 x gの遠心分離により細胞片、核成分を取り除いたのち、100,000 x gの遠心分離による沈殿物よりミクロゾーム画分を調製した。調製されたミクロゾーム画分を1% (w/v) CHAPSOを含む緩衝液Aで可溶化した後、100,000 x gの遠心分離により、可溶化γ－セクレターゼ複合体を調製した。
（DRM画分）
　DRM画分の調製はHur らの方法に従った。(Hur J.Y. et al., FEBS J. (2008) 275:1174-87)。すなわち、脳組織をBuffer B (20 mM Hepes (pH 7.5), 50 mM KCl, 2 mM EGTAにComplete protease inhibitor mixtureを添加したもの)中でホモジナイズし、上記と同様の遠心分離法によりミクロゾーム画分を調製した。得られたミクロゾーム画分を、Buffer C (20 mM Tris / HCl(pH 7.4), 150 mM NaCl, 1 mM EDTA, 2.0% CHAPSO) で再懸濁し、ショ糖密度勾配法によりDRM画分を調製する。
（シナプス膜、およびシナプス小胞）
　シナプス膜、およびシナプス小胞の調製はCohen らの方法に従った(Cohen J. et al., Org Chem (2004), 69, 7344-7347)。すなわち、脳組織をBuffer D (0.32 M sucrose, 1 mM NaHCO3, 1 mM MgCl2, 0.5 mM CaCl2)中でホモジナイズし、17,300 x gで遠心分離した。得られた沈殿物を、Buffer E (1 mM NaHCO3, 0.32 M sucrose) で再懸濁し、ショ糖密度勾配法により、シナプトソーム画分を調製する。シナプトソーム画分を6 mM Tris-HCl pH 8.1にて可溶化し、48,250 x g にて遠心分離しLP1膜画分とシナプス小胞を含む上清画分に分離する。シナプス膜は、LP1膜画分をショ糖密度勾配法によりさらに分離することにより調製する。シナプス小胞は上清画分を、100,000 x gで遠心分離し調製する。調製されたシナプス膜、およびシナプス小胞を1% (w/v) CHAPSOを含む緩衝液Aで可溶化し、可溶化γ－セクレターゼ複合体を調製する。
（２）脳γ－セクレターゼの含有画分の精製、および結合タンパク質の同定
　一般的に用いられるγ－セクレターゼ阻害剤（L-685,458）に親水性のリンカーと切断可能なビオチンを持つγ－セクレターゼ複合体の特異的なプルダウンプローブである、GCB（γ-secretase inhibitor with a hydrophilic long linker and a cleavable biotin group）（図１）(GCB法、2008、ICAD報告、P4-201、Inhibitor Pulldown Of γ-Secretase And Associated Components From Human And Rat Brain)を用いて以下の試験を行った。
　上記方法で調製された粗精製γ-セクレターゼを含む画分に200nMのGCBを添加し、阻害剤とγ-セクレターゼを結合させたのち、ストレプトアビジンビーズを添加し、ストレプトアビジンとビオチンの相互作用により、特異的にγ-セクレターゼ複合体をプルダウンした。ストレプトアビジンビーズを、0.5%CHAPSOを含む緩衝液で洗浄した後、DTTを添加することにより、プルダウンされたγ-セクレターゼを特異的に溶出し、精製γ-セクレターゼ複合体を得た。結合の特異性は拮抗阻害剤である10μM　L-685,458をスタート材料である粗精製γ-セクレターゼに添加した条件で、既知のγ-セクレターゼ構成タンパク質がプルダウンされていないことにより確認した(図２)。GCB法により、γ-セクレターゼの既知コンポーネントがヒト死後脳試料（ミクロゾーム画分）よりプルダウンされた。
　得られた、精製γ－セクレターゼ複合体の成分の同定は、公知の方法である、トリプシン消化によりペプチド断片化し、LC-MS/MSで同定する方法で行った。非特異的な結合は、阻害薬L-685,458添加条件で同定されたものの差分をとり排除した。この結果、特異的にγ-セクレターゼに結合し、脳に発現する複数の新規γ-セクレターゼ構成因子を見出した（表１）。 Example 1 Identification of brain γ-secretase binding protein (1)
In brain tissue, γ-secretase activity is strongly detected in the microsomal fraction, surfactant-insoluble membrane fraction (DRM), synaptic membranes, and synaptic vesicles. Therefore, each of these fractions was prepared, and the γ-secretase complex was purified by an inhibitor pull-down method.
(1) Preparation of fraction containing brain γ-secretase SD rats were obtained from B & K Universal (Sollentuna, Sweden), and human brain tissue was obtained from Huddinge Brain Bank (Huddinge, Sweden).
(Microsome fraction)
Rat or human brain tissue was homogenized in buffer A (20 mM Hepes, 50 mM KCl, 2 mM EGTA, pH 7.5 plus complete protease inhibitor mixture (Roche Applied Science, Indianapolis, IN, USA)) Thereafter, cell debris and nuclear components were removed by centrifugation at 1,000 xg and 10,000 xg, and then a microsomal fraction was prepared from the precipitate obtained by centrifugation at 100,000 xg. The prepared microsomal fraction was solubilized with buffer A containing 1% (w / v) CHAPSO, and then the solubilized γ-secretase complex was prepared by centrifugation at 100,000 × g.
(DRM fraction)
The DRM fraction was prepared according to the method of Hur et al. (Hur JY et al., FEBS J. (2008) 275: 1174-87). That is, brain tissue was homogenized in Buffer B (20 mM Hepes (pH 7.5), 50 mM KCl, 2 mM EGTA added with complete protease inhibitor mixture), and the microsomal fraction was obtained by centrifugation as described above. Prepared. Resuspend the obtained microsomal fraction in Buffer C (20 mM Tris / HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 2.0% CHAPSO), and prepare the DRM fraction by the sucrose density gradient method. .
(Synaptic membranes and synaptic vesicles)
Synaptic membranes and synaptic vesicles were prepared according to the method of Cohen et al. (Cohen J. et al., Org Chem (2004), 69, 7344-7347). That is, brain tissue was homogenized in Buffer D (0.32 M sucrose, 1 mM NaHCO 3 , 1 mM MgCl 2 , 0.5 mM CaCl 2 ) and centrifuged at 17,300 × g. The obtained precipitate is resuspended in Buffer E (1 mM NaHCO 3 , 0.32 M sucrose), and a synaptosome fraction is prepared by a sucrose density gradient method. The synaptosome fraction is solubilized with 6 mM Tris-HCl pH 8.1, centrifuged at 48,250 × g, and separated into a LP1 membrane fraction and a supernatant fraction containing synaptic vesicles. The synaptic membrane is prepared by further separating the LP1 membrane fraction by the sucrose density gradient method. Synaptic vesicles are prepared by centrifuging the supernatant fraction at 100,000 x g. The prepared synaptic membrane and the synaptic vesicle are solubilized with buffer A containing 1% (w / v) CHAPSO to prepare a solubilized γ-secretase complex.
(2) Purification of fraction containing brain γ-secretase and identification of binding protein γ-secretase complex with hydrophilic linker and cleavable biotin in commonly used γ-secretase inhibitor (L-685,458) GCB (γ-secretase inhibitor with a hydrophilic long linker and a cleavable biotin group) (Fig. 1) (GCB method, 2008, ICAD report, P4-201, Inhibitor Pulldown Of γ-Secretase The following tests were conducted using And Associated Components From Human And Rat Brain.
Add 200 nM GCB to the fraction containing the crude γ-secretase prepared by the above method, bind the inhibitor and γ-secretase, add streptavidin beads, and interact with streptavidin and biotin. Specifically, the γ-secretase complex was pulled down. The streptavidin beads were washed with a buffer containing 0.5% CHAPSO, and then DTT was added to specifically elute the pulled-down γ-secretase to obtain a purified γ-secretase complex. Specificity of the binding was confirmed by the fact that a known γ-secretase component protein was not pulled down under the condition that 10 μM L-685,458, which is a competitive inhibitor, was added to the crude purified γ-secretase (FIG. 2). . By GCB method, known components of γ-secretase were pulled down from human postmortem brain samples (microsomal fraction).
The components of the purified γ-secretase complex thus obtained were identified by a known method of peptide fragmentation by trypsin digestion and identification by LC-MS / MS. Non-specific binding was eliminated by taking the difference between those identified with the addition of the inhibitor L-685,458. As a result, a plurality of novel γ-secretase components that specifically bind to γ-secretase and are expressed in the brain were found (Table 1).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
実施例２　新規結合因子のsiRNAノックダウンによるAβ産生抑制（１）
　上記方法で同定されたものについて、Aβ産生に対する影響をRNAi法により評価した。
　評価に用いた細胞である、アミロイドプレカーサータンパク（APP）を恒常的に発現するヒト胎児腎臓細胞株（HEK-APP）は、カロリンスカ研究所のDr. Eirikur Benedikz氏より入手した。 (Nilsson et al., Biochem Biophys Res Commun, 341,1294-9.).
　HEK-APP細胞は、10% fetal calf serum を含む Dulbecco’s modified Eagle’s medium (Invitrogen) で培養した。
　新規因子のsiRNAは、Ambion社などから市販されているものを購入するか、Teramoto R.らが報告する方法（Teramoto, R. et al., FEBS Lett. (2005) 579, 2878-2882）および特許公開2006-236153に記載の方法によりデザインしインビトロジェン社より合成された。 Example 2 Suppression of Aβ production by siRNA knockdown of a novel binding factor (1)
About the thing identified by the said method, the influence with respect to A (beta) production was evaluated by RNAi method.
A human embryonic kidney cell line (HEK-APP) that constantly expresses amyloid precursor protein (APP), which is a cell used for evaluation, was obtained from Dr. Eirikur Benedikz of Karolinska Institute. (Nilsson et al., Biochem Biophys Res Commun, 341, 1294-9.).
HEK-APP cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal calf serum.
The siRNA of the novel factor can be purchased from Ambion etc., or the method reported by Teramoto R. et al. (Teramoto, R. et al., FEBS Lett. (2005) 579, 2878-2882) and It was designed by the method described in Patent Publication 2006-236153 and synthesized from Invitrogen.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　siRNAは、siRNA導入試薬LipofectamineTM RNAiMAX Transfection Reagent (Invitrogen) を用い、HEK-APP細胞に導入した。導入48時間後に、培養培地をOpti-MEM I　(Invitrogen)に変換した後、さらに24時間培養し、培養上清を回収した。
　WST8 法により、生細胞数を測定し、30％以上の細胞毒性を示したsiRNAは試験より排除した。標的遺伝子のノックダウン効率は、リアルタイムPCR法により確認した。標的遺伝子のプライマーはアプライドバイオシステムズ社のPre-Developed TaqMan（登録商標） Assay Reagentsを用い、内部標準としてGAPDH遺伝子を用いた。培養上清中のAβ40量およびAβ42量は、ELISA法 (Wako Chemicals, Osaka, Japan)を用いて測定した。siRNA未処理の細胞（MOCK）での値を100％として抑制率を算出した。その結果、試験系のコントロールとして用いた、γ－セクレターゼの構成因子であるプレセニリン1（PS1）の発現量を低下させることにより、Aβ40量およびAβ42量を減少させることを確認した（図３及び４中「PSEN1」）。また、ATP2A2とFLOT2については、siRNAノックダウンによりAβ産生が減少することが既に報告されており、本方法が新規のγ－セクレターゼ結合性のAβ産生制御因子の同定法として有効であることを裏付けている。さらに、新規因子の発現量を低下させることにより、Aβ40量およびAβ42量を減少させる知見を得た(図３，４)。 siRNA was introduced into HEK-APP cells using the siRNA introduction reagent Lipofectamine ™ RNAiMAX Transfection Reagent (Invitrogen). 48 hours after introduction, the culture medium was converted to Opti-MEM I (Invitrogen), and further cultured for 24 hours, and the culture supernatant was collected.
The number of viable cells was measured by the WST8 method, and siRNAs showing cytotoxicity of 30% or more were excluded from the test. The knockdown efficiency of the target gene was confirmed by real-time PCR. As a target gene primer, Applied Biosystems Pre-Developed TaqMan (registered trademark) Assay Reagents was used, and the GAPDH gene was used as an internal standard. The amounts of Aβ40 and Aβ42 in the culture supernatant were measured by ELISA (Wako Chemicals, Osaka, Japan). The inhibition rate was calculated with the value in siRNA-untreated cells (MOCK) as 100%. As a result, it was confirmed that the amount of Aβ40 and Aβ42 was decreased by decreasing the expression level of presenilin 1 (PS1), which is a constituent factor of γ-secretase, used as a test system control (FIGS. 3 and 4). Medium “PSEN1”). In addition, ATP2A2 and FLOT2 have been reported to reduce Aβ production by siRNA knockdown, confirming that this method is effective as a novel method for identifying γ-secretase-binding Aβ production regulators. ing. Furthermore, the knowledge which reduces the amount of A (beta) 40 and A (beta) 42 by reducing the expression level of a novel factor was acquired (FIGS. 3, 4).
実施例３　脳γ－セクレターゼの結合タンパク質の同定（２）
　実施例１で得られたミクロゾーム画分を用いて、更に特異的に新規γ-セクレターゼに結合し、脳に発現する複数の新規γ-セクレターゼ構成因子を見出した（表３）。 Example 3 Identification of binding protein of brain γ-secretase (2)
Using the microsomal fraction obtained in Example 1, a plurality of novel γ-secretase constituents that specifically bind to novel γ-secretase and are expressed in the brain were found (Table 3).
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
実施例４　新規結合因子のsiRNAノックダウンによるAβ産生抑制（２）
　上記実施例３で同定された新規γ-セクレターゼ構成因子について、実施例２に記載の方法と同様にして、Aβ産生に対する影響をRNAi法により評価した。 Example 4 Suppression of Aβ production by siRNA knockdown of a novel binding factor (2)
For the novel γ-secretase component identified in Example 3 above, the effect on Aβ production was evaluated by the RNAi method in the same manner as described in Example 2.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　その結果、新規因子の発現量を低下させることにより、Aβ42量およびAβ40量を減少させる知見を得た(図５，６)。 As a result, it was found that the amount of Aβ42 and Aβ40 was decreased by decreasing the expression level of the novel factor (FIGS. 5 and 6).
実施例５　脳γ－セクレターゼの結合タンパク質の同定（３）
　実施例１で得られたDRM画分を用いて、新たに特異的に新規γ-セクレターゼに結合し、脳に発現する複数の新規γ-セクレターゼ構成因子を見出した（表５、１７～２１）。また、実施例１で得られたシナプス膜およびシナプス小胞画分から、新たに特異的に新規γ-セクレターゼに結合し脳に発現する複数の新規γ-セクレターゼ構成因子を見出した（表５、２２～２７）。 Example 5 Identification of binding protein of brain γ-secretase (3)
Using the DRM fraction obtained in Example 1, a plurality of novel γ-secretase components that specifically bind to the novel γ-secretase and are expressed in the brain were found (Tables 5, 17 to 21). . Further, from the synaptic membrane and the synaptic vesicle fraction obtained in Example 1, a plurality of novel γ-secretase constituents that were newly specifically bound to γ-secretase and expressed in the brain were found (Tables 5 and 22). To 27).
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
実施例６　新規結合因子のsiRNAノックダウンによるAβ産生抑制（３）
　上記実施例５で同定された新規γ-セクレターゼ構成因子について、実施例２に記載の方法と同様にして、Aβ産生に対する影響をRNAi法により評価した。 Example 6 Suppression of Aβ production by siRNA knockdown of a novel binding factor (3)
For the novel γ-secretase component identified in Example 5 above, the effect on Aβ production was evaluated by the RNAi method in the same manner as in Example 2.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
　その結果、新規因子の発現量を低下させることにより、Aβ42量およびAβ40量を減少させる知見を得た(図７，８)。 As a result, it was found that the amount of Aβ42 and Aβ40 decreased by decreasing the expression level of the novel factor (FIGS. 7 and 8).
　本発明により提供されるスクリーニング法は、γ-セクレターゼの活性をアップレギュレートする新規タンパク質の発現や機能を抑制する作用を有する物質を選択することができるので、本スクリーニング法により得られる物質は、Aβの産生および／またはNotchの切断阻害剤あるいはモジュレーターとして、ADや癌などの疾患の予防および治療薬として期待される。あるいは、これらの新規タンパク質は、γ－セクレターゼのNotch切断活性には影響しない可能性があるので、本スクリーニング法により得られる物質は、Notchなどの他の生理的基質の切断を阻害することなく、γ－セクレターゼ活性のみを阻害できる可能性があり、副作用の少ない安全なADなどの疾患の予防および治療薬としても期待される。 Since the screening method provided by the present invention can select a substance having an action of suppressing the expression and function of a novel protein that up-regulates the activity of γ-secretase, the substance obtained by this screening method is As a ββ production and / or Notch cleavage inhibitor or modulator, it is expected as a prophylactic and therapeutic agent for diseases such as AD and cancer. Alternatively, since these novel proteins may not affect the Notch cleavage activity of γ-secretase, the substance obtained by this screening method does not inhibit the cleavage of other physiological substrates such as Notch, It may be able to inhibit only γ-secretase activity, and is expected as a safe preventive and therapeutic drug for diseases such as AD with few side effects.
　本発明は、２００９年６月４日出願の日本国特許出願、特願２００９-１３５５８９を基礎としており、その内容は全て本明細書に包含される。 The present invention is based on a Japanese patent application filed on June 4, 2009, Japanese Patent Application No. 2009-135589, the entire contents of which are included in this specification.

Claims (24)

  1. 　以下のタンパク質：
    (P1) Probable phospholipid-transporting ATPase IIA；
    (P2) BDNF/NT-3 growth factors receptor precursor；
    (P3) ELAV-like protein 4；
    (P4) Coiled-coil domain-containing protein 136；
    (P5) Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2；
    (P6) DnaJ homolog subfamily A member 2；
    (P7) Vesicle-associated membrane protein-associated protein A；
    (P8) Proton myo-inositol cotransporter；
    (P9) Leukocyte surface antigen CD47；
    (P10) Flotillin-1；
    (P11) Band 4.1 like 1；
    (P12) Regulator of G-protein signaling 7；
    (P13) Phospholipase D3；
    (P14) Ectoderm-neural cortex protein 1；
    (P15) synaptophysin；
    (P16) solute carrier family 2 (facilitated glucose transporter), member 3；
    (P17) syntaxin binding protein 1；
    (P18) growth associated protein 43；
    (P19) ATPase, Na+/K+ transporting, beta 1 polypeptide；
    (P20) Amine oxidase [flavin-containing] B；
    (P21) Muscarinic acetylcholine receptor M1；
    (P22) Probable cationic amino acid transporter；
    (P23) NADH dehydrogenase iron-sulfur protein 7；
    (P24) G-protein-regulated inducer of neurite outgrowth；および
    (P25) Probable phospholipid-transporting ATPase 1A
    から選ばれるタンパク質Pn（nは1～25のいずれかの整数）の発現または機能を抑制する、1以上の物質を含有してなる、Aβ産生抑制剤。     The following proteins:
    (P1) Probable phospholipid-transporting ATPase IIA;
    (P2) BDNF / NT-3 growth factors receptor precursor;
    (P3) ELAV-like protein 4;
    (P4) Coiled-coil domain-containing protein 136;
    (P5) Potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 2;
    (P6) DnaJ homolog subfamily A member 2;
    (P7) Vesicle-associated membrane protein-associated protein A;
    (P8) Proton myo-inositol cotransporter;
    (P9) Leukocyte surface antigen CD47;
    (P10) Flotillin-1;
    (P11) Band 4.1 like 1;
    (P12) Regulator of G-protein signaling 7;
    (P13) Phospholipase D3;
    (P14) Ectoderm-neural cortex protein 1;
    (P15) synaptophysin;
    (P16) solute carrier family 2 (facilitated glucose transporter), member 3;
    (P17) syntaxin binding protein 1;
    (P18) growth associated protein 43;
    (P19) ATPase, Na + / K + transporting, beta 1 polypeptide;
    (P20) Amine oxidase [flavin-containing] B;
    (P21) Muscarinic acetylcholine receptor M1;
    (P22) Probable capable amino acid transporter;
    (P23) NADH dehydrogenase iron-sulfur protein 7;
    (P24) G-protein-regulated inducer of neurite outgrowth; and
    (P25) Probable phospholipid-transporting ATPase 1A
    An Aβ production inhibitor comprising one or more substances that suppress the expression or function of a protein Pn selected from (n is an integer of 1 to 25).
  2. 　タンパク質Pnの発現を抑制する物質が、以下の（ａ）～（ｃ）からなる群より選択される物質である、請求項１に記載の剤。
    （ａ）タンパク質Pnをコードする遺伝子Gn：
    (G1) ATP9A；
    (G2) NTRK2；
    (G3) ELAVL4；
    (G4) NAG6；
    (G5) HCN2；
    (G6) DNAJA2；
    (G7) VAPA；
    (G8) SLC2A13；
    (G9) CD47;
    (G10) FLOT1；
    (G11) EPB41L1；
    (G12) RGS7；
    (G13) PLD3；
    (G14) ENC1；
    (G15) SYP；
    (G16) SLC2A3；
    (G17) STXBP1；
    (G18) GAP43；
    (G19) ATP1B1；
    (G20) MAOB；
    (G21) CHRM1；
    (G22) SLC7A14；
    (G23) NDUFS7；
    (G24) GPRIN1；または
    (G25) ATP8A1
    の転写産物に対するアンチセンス核酸
    （ｂ）遺伝子Gnの転写産物に対するリボザイム核酸
    （ｃ）遺伝子Gnの転写産物に対してRNAi活性を有する核酸もしくはその前駆体     The agent according to claim 1, wherein the substance that suppresses the expression of protein Pn is a substance selected from the group consisting of the following (a) to (c).
    (A) Gene Gn encoding protein Pn:
    (G1) ATP9A;
    (G2) NTRK2;
    (G3) ELAVL4;
    (G4) NAG6;
    (G5) HCN2;
    (G6) DNAJA2;
    (G7) VAPA;
    (G8) SLC2A13;
    (G9) CD47;
    (G10) FLOT1;
    (G11) EPB41L1;
    (G12) RGS7;
    (G13) PLD3;
    (G14) ENC1;
    (G15) SYP;
    (G16) SLC2A3;
    (G17) STXBP1;
    (G18) GAP43;
    (G19) ATP1B1;
    (G20) MAOB;
    (G21) CHRM1;
    (G22) SLC7A14;
    (G23) NDUFS7;
    (G24) GPRIN1; or
    (G25) ATP8A1
    (B) Ribozyme nucleic acid for gene Gn transcript (c) Nucleic acid having RNAi activity for gene Gn transcript or precursor thereof
  3. 　タンパク質Pnの機能を抑制する物質が、以下の（ａ）～（ｃ）からなる群より選択される物質である、請求項１に記載の剤。
    （ａ）タンパク質Pnと結合する抗体
    （ｂ）タンパク質Pnと結合する低分子化合物
    （ｃ）タンパク質Pnとγ－セクレターゼとの結合活性を阻害する化合物     The agent according to claim 1, wherein the substance that suppresses the function of protein Pn is a substance selected from the group consisting of the following (a) to (c).
    (A) an antibody that binds to protein Pn (b) a low molecular compound that binds to protein Pn (c) a compound that inhibits the binding activity between protein Pn and γ-secretase
  4. 　Aβが、Aβ40またはAβ42である、請求項１～３のいずれかに記載の剤。 The agent according to any one of claims 1 to 3, wherein Aβ is Aβ40 or Aβ42.
  5. 　アルツハイマー病もしくは癌の治療または予防のための、請求項１～４のいずれかに記載の剤。 The agent according to any one of claims 1 to 4, for the treatment or prevention of Alzheimer's disease or cancer.
  6. 　哺乳動物において、以下のタンパク質：
    (P1) Probable phospholipid-transporting ATPase IIA；
    (P2) BDNF/NT-3 growth factors receptor precursor；
    (P3) ELAV-like protein 4；
    (P4) Coiled-coil domain-containing protein 136；
    (P5) Potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2；
    (P6) DnaJ homolog subfamily A member 2；
    (P7) Vesicle-associated membrane protein-associated protein A；
    (P8) Proton myo-inositol cotransporter；
    (P9) Leukocyte surface antigen CD47；
    (P10) Flotillin-1；
    (P11) Band 4.1 like 1；
    (P12) Regulator of G-protein signaling 7；
    (P13) Phospholipase D3；
    (P14) Ectoderm-neural cortex protein 1；
    (P15) synaptophysin；
    (P16) solute carrier family 2 (facilitated glucose transporter), member 3；
    (P17) syntaxin binding protein 1；
    (P18) growth associated protein 43；
    (P19) ATPase, Na+/K+ transporting, beta 1 polypeptide；
    (P20) Amine oxidase [flavin-containing] B；
    (P21) Muscarinic acetylcholine receptor M1；
    (P22) Probable cationic amino acid transporter；
    (P23) NADH dehydrogenase iron-sulfur protein 7；
    (P24) G-protein-regulated inducer of neurite outgrowth；および
    (P25) Probable phospholipid-transporting ATPase 1A
    から選ばれるタンパク質Pn（nは1～25のいずれかの整数）の発現または機能を抑制することを含む、該哺乳動物におけるAβ産生の抑制方法。     In mammals, the following proteins:
    (P1) Probable phospholipid-transporting ATPase IIA;
    (P2) BDNF / NT-3 growth factors receptor precursor;
    (P3) ELAV-like protein 4;
    (P4) Coiled-coil domain-containing protein 136;
    (P5) Potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 2;
    (P6) DnaJ homolog subfamily A member 2;
    (P7) Vesicle-associated membrane protein-associated protein A;
    (P8) Proton myo-inositol cotransporter;
    (P9) Leukocyte surface antigen CD47;
    (P10) Flotillin-1;
    (P11) Band 4.1 like 1;
    (P12) Regulator of G-protein signaling 7;
    (P13) Phospholipase D3;
    (P14) Ectoderm-neural cortex protein 1;
    (P15) synaptophysin;
    (P16) solute carrier family 2 (facilitated glucose transporter), member 3;
    (P17) syntaxin binding protein 1;
    (P18) growth associated protein 43;
    (P19) ATPase, Na + / K + transporting, beta 1 polypeptide;
    (P20) Amine oxidase [flavin-containing] B;
    (P21) Muscarinic acetylcholine receptor M1;
    (P22) Probable capable amino acid transporter;
    (P23) NADH dehydrogenase iron-sulfur protein 7;
    (P24) G-protein-regulated inducer of neurite outgrowth; and
    (P25) Probable phospholipid-transporting ATPase 1A
    A method for inhibiting Aβ production in a mammal, comprising inhibiting the expression or function of a protein Pn selected from (n is an integer of 1 to 25).
  7. 　有効量の、タンパク質Pnの発現または機能を抑制する1以上の物質を哺乳動物に投与することを含む、請求項６に記載の方法。 7. The method of claim 6, comprising administering to the mammal an effective amount of one or more substances that suppress the expression or function of protein Pn.
  8. 　タンパク質Pnの発現を抑制する物質が、以下の（ａ）～（ｃ）からなる群より選択される物質である、請求項７に記載の方法。
    （ａ）タンパク質Pnをコードする遺伝子Gn：
    (G1) ATP9A；
    (G2) NTRK2；
    (G3) ELAVL4；
    (G4) NAG6；
    (G5) HCN2；
    (G6) DNAJA2；
    (G7) VAPA；
    (G8) SLC2A13；
    (G9) CD47;
    (G10) FLOT1；
    (G11) EPB41L1；
    (G12) RGS7；
    (G13) PLD3；
    (G14) ENC1；
    (G15) SYP；
    (G16) SLC2A3；
    (G17) STXBP1；
    (G18) GAP43；
    (G19) ATP1B1；
    (G20) MAOB；
    (G21) CHRM1；
    (G22) SLC7A14；
    (G23) NDUFS7；
    (G24) GPRIN1；または
    (G25) ATP8A1
    の転写産物に対するアンチセンス核酸
    （ｂ）遺伝子Gnの転写産物に対するリボザイム核酸
    （ｃ）遺伝子Gnの転写産物に対してRNAi活性を有する核酸もしくはその前駆体     The method according to claim 7, wherein the substance that suppresses the expression of protein Pn is a substance selected from the group consisting of the following (a) to (c).
    (A) Gene Gn encoding protein Pn:
    (G1) ATP9A;
    (G2) NTRK2;
    (G3) ELAVL4;
    (G4) NAG6;
    (G5) HCN2;
    (G6) DNAJA2;
    (G7) VAPA;
    (G8) SLC2A13;
    (G9) CD47;
    (G10) FLOT1;
    (G11) EPB41L1;
    (G12) RGS7;
    (G13) PLD3;
    (G14) ENC1;
    (G15) SYP;
    (G16) SLC2A3;
    (G17) STXBP1;
    (G18) GAP43;
    (G19) ATP1B1;
    (G20) MAOB;
    (G21) CHRM1;
    (G22) SLC7A14;
    (G23) NDUFS7;
    (G24) GPRIN1; or
    (G25) ATP8A1
    (B) Ribozyme nucleic acid for gene Gn transcript (c) Nucleic acid having RNAi activity for gene Gn transcript or precursor thereof
  9. 　タンパク質Pnの機能を抑制する物質が、以下の（ａ）～（ｃ）からなる群より選択される物質である、請求項７に記載の方法。
    （ａ）タンパク質Pnと結合する抗体
    （ｂ）タンパク質Pnと結合する低分子化合物
    （ｃ）タンパク質Pnとγ－セクレターゼとの結合活性を阻害する化合物     The method according to claim 7, wherein the substance that suppresses the function of protein Pn is a substance selected from the group consisting of the following (a) to (c).
    (A) an antibody that binds to protein Pn (b) a low molecular compound that binds to protein Pn (c) a compound that inhibits the binding activity between protein Pn and γ-secretase
  10. 　Aβが、Aβ40またはAβ42である、請求項６～９のいずれかに記載の方法。 The method according to any one of claims 6 to 9, wherein Aβ is Aβ40 or Aβ42.
  11. 　アルツハイマー病もしくは癌の治療または予防のための、請求項６～１０のいずれかに記載の方法。 The method according to any one of claims 6 to 10, for the treatment or prevention of Alzheimer's disease or cancer.
  12. 　前記(G1）～(G25)から選ばれる少なくとも１つの遺伝子の発現量もしくは前記(P1)～(P25)から選ばれる少なくとも１つのタンパク質の活性を低下させる物質を選択することを特徴とする、Ａβ産生抑制物質のスクリーニング方法。 Aβ, wherein the expression level of at least one gene selected from (G1) to (G25) or a substance that decreases the activity of at least one protein selected from (P1) to (P25) is selected. Screening method for production inhibitory substance.
  13. 　以下の（ａ）～（ｃ）の工程を含む、Aβ産生抑制物質のスクリーニング方法。
    （ａ）(a1)遺伝子Gn（nは1～25のいずれかの整数）もしくは(a2) 遺伝子Gn（nは1～25のいずれかの整数）の転写調節領域の制御下にあるレポーター遺伝子を発現する細胞に、被検物質を接触させる工程
    （ｂ）前記細胞における(b1)遺伝子Gnもしくは(b2)レポーター遺伝子の発現量を測定する工程
    （ｃ）被検物質の非存在下において測定した場合と比較して、発現量を低下させる物質をAβ産生抑制物質の候補として選択する工程     A screening method for an Aβ production inhibitor comprising the following steps (a) to (c):
    A reporter gene under the control of the transcriptional regulatory region of (a) (a1) gene Gn (n is any integer from 1 to 25) or (a2) gene Gn (n is any integer from 1 to 25) A step of contacting a test substance with a cell to be expressed (b) a step of measuring the expression level of (b1) gene Gn or (b2) reporter gene in the cell (c) a measurement in the absence of the test substance Selecting a substance that reduces the expression level as a candidate for an Aβ production inhibitor compared to
  14. 　以下の（ａ）～（ｃ）の工程を含む、Aβ産生抑制物質のスクリーニング方法。
    （ａ）アミロイド前駆タンパク質を発現する細胞に、前記(P1)～(P25)から選ばれる少なくとも１つのタンパク質および被検物質を接触させる工程
    （ｂ）前記タンパク質の活性を測定する工程
    （ｃ）被検物質の非存在下において測定した場合と比較して、前記タンパク質の活性を低下させる物質を選択する工程     A screening method for an Aβ production inhibitor comprising the following steps (a) to (c):
    (A) contacting a cell expressing amyloid precursor protein with at least one protein selected from (P1) to (P25) and a test substance (b) measuring the activity of the protein (c) A step of selecting a substance that decreases the activity of the protein as compared to the case where it is measured in the absence of the test substance.
  15. 　前記タンパク質が前記細胞自体により提供される、請求項１４に記載の方法。 15. The method of claim 14, wherein the protein is provided by the cell itself.
  16. 　前記タンパク質の活性をAβの産生量を指標として測定することを特徴とする、請求項１４または１５に記載の方法。 The method according to claim 14 or 15, wherein the activity of the protein is measured using the production amount of Aβ as an index.
  17. 　以下の（ａ）～（ｃ）の工程を含む、Aβ産生抑制物質のスクリーニング方法。
    （ａ）γ－セクレターゼを発現する細胞もしくはその細胞膜画分に、前記(P1)～(P25)から選ばれる少なくとも１つのタンパク質および被検物質を接触させる工程
    （ｂ）γ－セクレターゼ活性を測定する工程
    （ｃ）被検物質の非存在下において測定した場合と比較して、γ－セクレターゼ活性を低下またはモジュレートさせる物質を選択する工程     A screening method for an Aβ production inhibitor comprising the following steps (a) to (c):
    (A) contacting at least one protein selected from (P1) to (P25) and a test substance with a cell expressing γ-secretase or a cell membrane fraction thereof; and (b) measuring γ-secretase activity. Step (c) A step of selecting a substance that reduces or modulates γ-secretase activity as compared with the case where it is measured in the absence of the test substance.
  18. 　前記タンパク質が前記細胞自体により提供される、請求項１７に記載の方法。 18. The method of claim 17, wherein the protein is provided by the cell itself.
  19. 　タンパク質Pn（nは1～25のいずれかの整数）とγ－セクレターゼとの結合活性を阻害する物質を選択することを特徴とする、Aβ産生抑制物質のスクリーニング方法。 A screening method for an Aβ production inhibitor, which comprises selecting a substance that inhibits the binding activity between protein Pn (n is an integer from 1 to 25) and γ-secretase.
  20. 　以下の（ａ）～（ｃ）の工程を含む、Aβ産生抑制物質のスクリーニング方法。
    （ａ）タンパク質Pn（nは1～25のいずれかの整数）およびγ－セクレターゼと、被検物質とを接触させる工程
    （ｂ）タンパク質Pnとγ－セクレターゼとの結合活性を測定する工程
    （ｃ）被検物質の非存在下において測定した場合と比較して、前記結合活性を低下させる物質を選択する工程     A screening method for an Aβ production inhibitor comprising the following steps (a) to (c):
    (A) contacting protein Pn (n is an integer of 1 to 25) and γ-secretase with a test substance (b) measuring the binding activity between protein Pn and γ-secretase (c ) A step of selecting a substance that reduces the binding activity as compared with the case where it is measured in the absence of the test substance.
  21. 　前記(G1）～(G25)から選ばれる少なくとも１つの遺伝子を導入した非ヒト動物において、アルツハイマー病の病態を反映する表現型を改善する物質を選択することを特徴とする、アルツハイマー病の治療または予防薬のスクリーニングまたは薬効評価方法。 A non-human animal into which at least one gene selected from (G1) to (G25) is introduced, wherein a substance that improves the phenotype reflecting the pathology of Alzheimer's disease is selected; Prophylactic drug screening or efficacy evaluation method.
  22. 　以下の（ａ）～（ｃ）の工程を含む、アルツハイマー病の治療または予防薬のスクリーニングまたは薬効評価方法。
    （ａ）前記(G1）～(G25)から選ばれる少なくとも１つの遺伝子を導入した非ヒト動物に被検物質を投与する工程
    （ｂ）該動物における、アルツハイマー病の病態を反映する少なくとも１つの表現型を評価する工程
    （ｃ）被検物質の非投与時において評価した場合と比較して、前記表現型を改善させる物質を選択する工程     A screening or drug efficacy evaluation method for a therapeutic or prophylactic agent for Alzheimer's disease comprising the following steps (a) to (c):
    (A) a step of administering a test substance to a non-human animal into which at least one gene selected from the above (G1) to (G25) has been introduced; (b) at least one expression reflecting the pathology of Alzheimer's disease in the animal A step of evaluating the type (c) a step of selecting a substance that improves the phenotype as compared with the case where the test substance is evaluated at the time of non-administration
  23. 　前記表現型が、脳組織、脳脊髄液、血液中などの組織中のAβ量、神経細胞死、中枢神経系の炎症反応、認知能力、アミロイドプラークの蓄積量、脳内血流量および脳内グルコース代謝量からなる群より選択される、請求項２２に記載の方法。 The phenotype is brain tissue, cerebrospinal fluid, Aβ amount in tissues such as blood, neuronal cell death, central nervous system inflammatory reaction, cognitive ability, amyloid plaque accumulation, cerebral blood flow and cerebral glucose 23. The method of claim 22, wherein the method is selected from the group consisting of metabolic amounts.
  24. 　アルツハイマー病または癌の発症または発症リスクの判定方法であって、以下の（ａ）～（ｃ）の工程を含む方法。
    （ａ）被験動物由来の試料を提供する工程
    （ｂ）該試料中の、前記(G1）～(G25)から選ばれる少なくとも１つの遺伝子の発現量もしくは前記(P1)～(P25)から選ばれる少なくとも１つのタンパク質の活性を測定する工程
    （ｃ）正常動物由来の試料において測定した場合と比較して、前記発現量もしくは活性が上昇している被験動物を、アルツハイマー病またはダウン症を発症しているか、将来発症するリスクが高いと判定する工程     A method for determining the onset or risk of developing Alzheimer's disease or cancer, comprising the following steps (a) to (c):
    (A) providing a sample derived from a test animal (b) the expression level of at least one gene selected from (G1) to (G25) in the sample or selected from (P1) to (P25) A step of measuring the activity of at least one protein (c) is the subject animal having an increased expression level or activity compared to the case of measuring in a sample derived from a normal animal developing Alzheimer's disease or Down's syndrome? The process of determining that the risk of developing in the future is high
PCT/JP2010/059568 2009-06-04 2010-06-04 Method for screening of inhibitor using factor capable of enhancing production of amyloid-beta peptide, and inhibitor obtained by same WO2010140694A1 (en)

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KR101883515B1 (en) 2010-12-28 2018-07-30 다이닛본 스미토모 세이야꾸 가부시끼가이샤 Diagnostic drug and diagnostic method for alzheimer's disease
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WO2018196782A1 (en) * 2017-04-27 2018-11-01 The University Of Hong Kong Use of hcn inhibitors for treatment of cancer
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