WO2017026496A1 - Periostin gene expression-suppressing nucleic acid molecule, method for suppressing expression of periostin gene, and application therefor - Google Patents

Periostin gene expression-suppressing nucleic acid molecule, method for suppressing expression of periostin gene, and application therefor Download PDF

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WO2017026496A1
WO2017026496A1 PCT/JP2016/073500 JP2016073500W WO2017026496A1 WO 2017026496 A1 WO2017026496 A1 WO 2017026496A1 JP 2016073500 W JP2016073500 W JP 2016073500W WO 2017026496 A1 WO2017026496 A1 WO 2017026496A1
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expression
nucleotide
nucleic acid
acid molecule
seq
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Japanese (ja)
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寿徳 吉川
和正 高尾
雅文 吉野
マルクス ホスバッハ
モニカ クランペルト
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株式会社アクアセラピューティクス
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

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  • the present invention relates to a periostin gene expression-suppressing nucleic acid molecule, a periostin gene expression-suppressing method, and uses thereof.
  • Diabetic retinopathy is classified into simple diabetic retinopathy, preproliferative diabetic retinopathy and proliferative diabetic retinopathy (PDR) according to the stage. Among them, proliferative diabetic retinopathy may lead to blindness as well as decreased visual acuity.
  • Macular degeneration is a disease in which visual acuity decreases due to degeneration of the macular due to aging, stress, or the like. This condition can also lead to blindness in severe cases.
  • Non-patent Document 1 periostin gene expression is involved.
  • an object of the present invention is to provide a new molecule that suppresses the expression of the periostin gene, and to provide uses such as an expression suppression method using the same and a method for treating a periostin-related disease.
  • the periostin gene expression-suppressing nucleic acid molecule of the present invention is a periostin gene expression-suppressing nucleic acid molecule
  • the expression-suppressing nucleic acid molecule includes the following nucleotide (as4) as an expression-suppressing sequence for the periostin gene.
  • As4 a nucleotide comprising a base sequence that hybridizes under stringent conditions to a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 121 to 127, and having a function of suppressing expression of a periostin gene
  • the periostin gene expression-suppressing reagent of the present invention is characterized by comprising the expression-suppressing nucleic acid molecule of the present invention.
  • the periostin gene-related disease drug of the present invention is characterized by comprising the expression-suppressing nucleic acid molecule of the present invention.
  • the suppression method of the present invention is a method for suppressing the expression of a periostin gene, and is characterized by using the expression-suppressing nucleic acid molecule of the present invention.
  • the method for treating a periostin gene-related disease of the present invention includes a step of administering the expression-suppressing nucleic acid molecule of the present invention to a patient.
  • the expression-suppressing nucleic acid molecule of the present invention can suppress the expression of the periostin gene. For this reason, this invention is effective in the treatment of the disease caused by the expression of the periostin gene.
  • FIG. 1A is a graph showing the relative value of the expression level of periostin gene in vitro in Example 1 of the present invention.
  • FIG. 1B is a graph showing the relative value of the expression level of periostin gene in vitro in Example 1 of the present invention.
  • FIG. 1C is a graph showing the relative value of the expression level of periostin gene in vitro in Example 1 of the present invention.
  • FIG. 1D is a graph showing the relative value of the expression level of periostin gene in vitro in Example 1 of the present invention.
  • FIG. 2 is a graph showing the relative value of the expression level of the periostin gene in vitro in Example 2 of the present invention.
  • FIG. 3 is a graph showing the relative value of the expression level of the periostin gene in Example 4 of the present invention.
  • FIG. 4 is a graph showing the amount of protein in Example 4 of the present invention.
  • FIG. 5 is a graph showing the amount of hydroxyproline in Example 4 of the present invention.
  • FIG. 6 is a photograph showing the results of Masson trichrome staining in Example 4 of the present invention.
  • FIG. 7 is a graph showing the results of Ashcroft score in Example 4 of the present invention.
  • FIG. 8 is a graph showing the results of adhesion scores in Example 5 of the present invention.
  • FIG. 9 is a graph showing the measurement results of the expression level of the periostin gene in Example 5 of the present invention.
  • FIG. 10A is a photograph showing the results of Azan staining and periostin immunostaining of the tissue at the tip of the cecum in Example 5 of the present invention.
  • FIG. 10B is a photograph showing the results of Azan staining and periostin immunostaining of the cecal tip tissue in Example 5 of the present invention.
  • FIG. 10C is a photograph showing the results of Azan staining and periostin immunostaining of the tissue at the tip of the cecum in Example 5 of the present invention.
  • FIG. 10D is a photograph showing the results of Azan staining and periostin immunostaining of the tissue at the tip of the cecum in Example 5 of the present invention.
  • FIG. 10A is a photograph showing the results of Azan staining and periostin immunostaining of the tissue at the tip of the cecum in Example 5 of the present invention.
  • FIG. 10B is a photograph showing the results of Azan staining and periostin immunostaining of the
  • FIG. 10E is a photograph showing the results of Azan staining and periostin immunostaining of the cecal tip tissue in Example 5 of the present invention.
  • FIG. 10F is a photograph showing the results of Azan staining and periostin immunostaining of the tissue at the tip of the cecum in Example 5 of the present invention.
  • FIG. 11 is a graph showing the measurement results of the expression level of periostin gene in Example 6 of the present invention.
  • FIG. 12 is a graph showing the results of adhesion scores in Example 6 of the present invention.
  • FIG. 13A is a photograph showing the results of Azan staining and periostin immunostaining of the tissue at the tip of the cecum in Example 6 of the present invention.
  • FIG. 13B is a photograph showing the results of Azan staining and periostin immunostaining of the tissue at the tip of the cecum in Example 6 of the present invention.
  • FIG. 14 is a graph showing the relative value of the expression level of the periostin gene in vitro in Example 7 of the present invention.
  • FIG. 15 is a graph showing the relative value of the expression level of the periostin gene in vitro in Example 7 of the present invention.
  • the expression-suppressing nucleic acid molecule of the present invention (hereinafter also referred to as “nucleic acid molecule”) is, as described above, a periostin gene expression-suppressing nucleic acid molecule,
  • the expression-suppressing nucleic acid molecule includes the following nucleotide (as4) as an expression-suppressing sequence for the periostin gene.
  • As4 a nucleotide comprising a base sequence that hybridizes under stringent conditions to a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 121 to 127, and having a function of suppressing expression of a periostin gene
  • nucleic acid molecule of the present invention will be described.
  • nucleotide (as4) is also referred to as an as4 nucleotide.
  • the nucleic acid molecule of the present invention is a nucleic acid molecule for suppressing expression as an expression suppressing sequence of the periostin gene, and the nucleotide of the following (as1), (as2) or (as3) is used as an expression suppressing sequence of the periostin gene
  • nucleic acid molecules containing (As1) Nucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 1-120 and 217-232 (as2) In the nucleotide sequence of (as1), one or several bases are deleted, substituted and / or added.
  • a nucleotide having a function to suppress expression of a periostin gene (as3)
  • nucleic acid molecule containing the nucleotide (as1), (as2) or (as3) will be described, and then the as4 nucleotide will be described.
  • nucleotides (as1), (as2) or (as3) are referred to as as nucleotides, and are also referred to as as1, nucleotides, and as3 nucleotides, respectively.
  • the suppression of periostin gene expression is not particularly limited, and may be, for example, suppression of gene transcription itself or suppression of degradation of a gene transcription product. Further, the suppression of periostin gene expression may be, for example, suppression of periostin protein expression having an original function.
  • the protein When protein is expressed, the protein may be, for example, a protein in which the function is inhibited. It may be a protein lacking the function.
  • the expression suppression sequence may be, for example, a sequence consisting of the as4 nucleotide, a sequence containing the as4 nucleotide, or a partial sequence of the as4 nucleotide.
  • the expression suppression sequence may be, for example, a sequence consisting of the as1 nucleotide, the as2 nucleotide or the as3 nucleotide, or the as1 nucleotide , A sequence containing the as2 nucleotide or the as3 nucleotide, or a partial sequence of the as1 nucleotide, the as2 nucleotide or the as3 nucleotide.
  • the length of the expression suppression sequence is not particularly limited, and is, for example, 10 to 40 bases long, 11 to 30 bases long, or 12 to 20 bases long.
  • the numerical range of the number of bases discloses all positive integers belonging to the range.
  • the description “1 to 4 bases” includes “1, 2, 3, 4 bases”. "Means all disclosures (the same applies hereinafter).
  • the sequence of the as1 nucleotide is shown below.
  • the nucleotides consisting of the base sequences of SEQ ID NOs: 1-120 and 217-232, the expression suppressing sequence consisting of the nucleotides, the expression suppressing sequence containing the nucleotides, and the nucleic acid molecules containing the nucleotides are named as follows: (Name before the sequence number).
  • “1 or several” is not particularly limited. “One or several” is, for example, 1 to 7, 1 to 5, 1 to 4, 1, 2, or 3.
  • the as2 nucleotide only needs to have the same function as the as1 nucleotide, and more specifically, it only needs to have a function to suppress the expression of the periostin gene.
  • “And / or” means at least one of the meanings, and can also be expressed as “at least one selected from the group consisting of” (hereinafter the same).
  • the identity is, for example, 80% or more, 85% or more, 90% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more. It is.
  • the as3 nucleotide only needs to have the same function as the as1 nucleotide, and more specifically, it only needs to have a function to suppress the expression of the periostin gene.
  • the identity can be calculated with default parameters using analysis software such as BLAST and FASTA (hereinafter the same).
  • the total length of the nucleic acid molecule of the present invention is not particularly limited, and for example, description of the length of the expression suppressing sequence can be cited.
  • the base sequence of SEQ ID NO: 121 in the as4 nucleotide is as follows.
  • the base sequence of SEQ ID NO: 121 is the base sequence of mRNA variant 1 of the periostin gene.
  • the polynucleotide of SEQ ID NO: 121 can be obtained, for example, from Homo sapiens .
  • GeneBank has accession no.
  • the polynucleotide registered by NM_006475.2 is mentioned.
  • the base sequence of SEQ ID NO: 122 in the as4 nucleotide is as follows.
  • the base sequence of SEQ ID NO: 122 is the base sequence of mRNA variant 2 of the periostin gene.
  • the polynucleotide of SEQ ID NO: 122 can be obtained, for example, from Homo sapiens , for example, accession No. 1 to GeneBank. Examples thereof include polynucleotides registered under NM_001135934.1.
  • the base sequence of SEQ ID NO: 123 in the as4 nucleotide is as follows.
  • the base sequence of SEQ ID NO: 123 is the base sequence of mRNA variant 3 of the periostin gene.
  • the polynucleotide of SEQ ID NO: 123 can be obtained, for example, from Homo sapiens .
  • GeneBank has accession no.
  • the polynucleotide registered by NM_0011359355.1 is mentioned.
  • the base sequence of SEQ ID NO: 124 in the as4 nucleotide is as follows.
  • the base sequence of SEQ ID NO: 124 is the base sequence of mRNA variant 4 of the periostin gene.
  • the polynucleotide of SEQ ID NO: 124 can be obtained, for example, from Homo sapiens , for example, accession No.
  • the polynucleotide registered by NM_001135936.1 is mention
  • the base sequence of SEQ ID NO: 125 in the as4 nucleotide is as follows.
  • the base sequence of SEQ ID NO: 125 is the base sequence of mRNA variant 5 of the periostin gene.
  • the polynucleotide of SEQ ID NO: 125 can be obtained, for example, from Homo sapiens .
  • the polynucleotide registered by NM_0012866665.1 is mentioned.
  • the base sequence of SEQ ID NO: 126 in the as4 nucleotide is as follows.
  • the base sequence of SEQ ID NO: 126 is the base sequence of mRNA variant 6 of the periostin gene.
  • the polynucleotide of SEQ ID NO: 126 can be obtained, for example, from Homo sapiens .
  • GeneBank has accession no.
  • the base sequence of SEQ ID NO: 127 in the as4 nucleotide is as follows.
  • the base sequence of SEQ ID NO: 127 is the base sequence of mRNA variant 7 of the periostin gene.
  • the polynucleotide of SEQ ID NO: 127 can be obtained, for example, from Homo sapiens .
  • GeneBank has accession no.
  • the polynucleotide registered by NM_0012866677.1 is mentioned.
  • hybridization may be, for example, within a range in which the as4 nucleotide has a function of suppressing the expression of the periostin gene.
  • the “hybridizing base sequence” is, for example, complete with respect to a polynucleotide comprising any one of the base sequences of SEQ ID NOs: 121 to 127 (hereinafter also referred to as “periostin gene mRNA”). Or a partially complementary polynucleotide.
  • the hybridization can be detected by, for example, various hybridization assays.
  • the hybridization assay is not particularly limited, for example, Zanburuku (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) ,” [Cold Spring Harbor Laboratory Press (1989)] and the like can also be employed.
  • stringent conditions may be, for example, low stringency conditions, medium stringency conditions, or high stringency conditions.
  • Low stringent conditions are, for example, conditions of 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, and 32 ° C.
  • Medium stringent conditions are, for example, 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, 42 ° C.
  • High stringent conditions are, for example, conditions of 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C.
  • the degree of stringency can be set by those skilled in the art by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, time, and the like.
  • “Stringent conditions” are, for example, Zanburuku previously described (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) ,” [Cold Spring Harbor Laboratory Press ( 1989)] etc. can also be employed.
  • the as4 nucleotide is a polynucleotide partially complementary to the periostin gene mRNA
  • the as4 nucleotide is, for example, 80% or more, 85% or more, 90% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the bases are complementary to at least one periostin gene mRNA.
  • the position at which the as4 nucleotide hybridizes to the periostin gene mRNA is not particularly limited, and may be, for example, the 5 ′ untranslated region (5′UTR) of the periostin gene mRNA, or the coding region (CDS: coding'region), 3'untranslated region (3'UTR: Three'prime'untranslated'region), or any two or more regions.
  • the hybridizing position is preferably at least one of the CDS and the 3'UTR, and more preferably the 3'UTR, because the expression of the periostin gene can be further suppressed.
  • the 5′UTR, the CDS, and the 3′UTR in the base sequence of each periostin gene mRNA are, for example, the base sequences shown in Table 1 below, respectively.
  • Human periostin gene mRNA 5′UTR (SEQ ID NO: 128) 5'-AGACTCTCAGGTTGATGCAGTGTTCCCTCCCACAACTCTGACATGTATATAAATTCTGAGCTCTCCAAAGCCCACTGCCAGTTCTCTTCGGGGACTAACTGCAACGGAGAGACTCAAG-3 '
  • the hybridizing position is more preferably at least one of the CDS and the 3′UTR, and more preferably the following position because the expression of the periostin gene can be further suppressed. It is done.
  • CDS-3'UTR The 1229th to 3132rd base sequence in the base sequence of SEQ ID NO: 121 (the 1111st to 2511th base sequence in the base sequence of SEQ ID NO: 129 and the 1st to 503rd base sequence in the base sequence of SEQ ID NO: 130)
  • CDS 815 to 1127th nucleotide sequence in the nucleotide sequence of SEQ ID NO: 121 (697 to 1009th nucleotide sequence in the nucleotide sequence of SEQ ID NO: 129)
  • the 965th to 988th base sequences in the base sequence of SEQ ID NO: 121 (the 847th to 870th base sequences in the base sequence of SEQ ID NO: 129) 1105th to 1127
  • the position to hybridize is more preferably at least one of the CDS and the 3′UTR, more preferably the following position, since the expression of the periostin gene can be further suppressed.
  • This is a position corresponding to the illustration of the CDS and the 3′UTR at the position to be hybridized in SEQ ID NO: 121.
  • the corresponding position can be identified, for example, by comparing the base sequence of SEQ ID NO: 121 and the base sequence of SEQ ID NO: 122.
  • CDS-3'UTR The 1229 to 2961st base sequence in the base sequence of SEQ ID NO: 122 (the 1111 to 2340th base sequence in the base sequence of SEQ ID NO: 131 and the 1st to 503rd base sequence in the base sequence of SEQ ID NO: 130)
  • the hybridizing position is preferably at least one of the CDS and the 3′UTR, more preferably the following position, and more preferably, since the expression of the periostin gene can be further suppressed.
  • This is a position corresponding to the illustration of the CDS and the 3′UTR at the position to be hybridized in SEQ ID NO: 121.
  • the corresponding position can be identified by comparing the base sequence of SEQ ID NO: 121 and the base sequence of SEQ ID NO: 123, for example.
  • the hybridizing position is preferably at least one of the CDS and the 3′UTR, more preferably the following position, since the expression of the periostin gene can be further suppressed.
  • This is a position corresponding to the illustration of the CDS and the 3′UTR at the position to be hybridized in SEQ ID NO: 121.
  • the corresponding position can be specified, for example, by comparing the base sequence of SEQ ID NO: 121 and the base sequence of SEQ ID NO: 124.
  • CDS-3'UTR The 1229 to 2877th base sequence in the base sequence of SEQ ID NO: 124 (the 1111 to 2256th base sequence in the base sequence of SEQ ID NO: 133 and the 1st to 503rd base sequence in the base sequence of SEQ ID NO: 130)
  • the hybridizing position is preferably at least one of the CDS and the 3′UTR, more preferably the following position, and more preferably, since the expression of the periostin gene can be further suppressed.
  • This is a position corresponding to the illustration of the CDS and the 3′UTR at the position to be hybridized in SEQ ID NO: 121.
  • the corresponding position can be identified by comparing the base sequence of SEQ ID NO: 121 and the base sequence of SEQ ID NO: 125, for example.
  • CDS-3'UTR The 1229-3051st base sequence in the base sequence of SEQ ID NO: 125 (the 1111-2430th base sequence in the base sequence of SEQ ID NO: 134 and the 1st to 503rd base sequence in the base sequence of SEQ ID NO: 130)
  • the hybridizing position is preferably at least one of the CDS and the 3′UTR, more preferably the following position, since the expression of the periostin gene can be further suppressed.
  • This is a position corresponding to the illustration of the CDS and the 3′UTR at the position to be hybridized in SEQ ID NO: 121.
  • the corresponding position can be specified by comparing the base sequence of SEQ ID NO: 121 and the base sequence of SEQ ID NO: 126, for example.
  • CDS-3'UTR The 1229 to 2871st base sequence in the base sequence of SEQ ID NO: 126 (the 1111 to 2250th base sequence in the base sequence of SEQ ID NO: 135 and the 1st to 503rd base sequence in the base sequence of SEQ ID NO: 130)
  • the hybridizing position is preferably at least one of the CDS and the 3′UTR, more preferably the following position, since the expression of the periostin gene can be further suppressed.
  • This is a position corresponding to the illustration of the CDS and the 3′UTR at the position to be hybridized in SEQ ID NO: 121.
  • the corresponding position can be identified, for example, by comparing the base sequence of SEQ ID NO: 121 and the base sequence of SEQ ID NO: 127.
  • CDS-3'UTR The 1229 to 2787th base sequence in the base sequence of SEQ ID NO: 127 (the 1111 to 2166th base sequence in the base sequence of SEQ ID NO: 136 and the 1st to 503rd base sequence in the base sequence of SEQ ID NO: 130)
  • the length of the as4 nucleotide is not particularly limited, and for example, the description of the length of the expression suppressing sequence can be used.
  • the length of the as4 nucleotide excludes, for example, 19 bases.
  • as4 nucleotide examples include the as1 nucleotide, the as2 nucleotide, and the as3 nucleotide.
  • the expression of the periostin gene in human glioblastoma cells is, for example, 45% or more, 50% or more, 55% or more, 60% or more, 65 % Or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.
  • the suppression of the expression is based on, for example, the expression level of the periostin gene of the human glioblastoma cell when the nucleic acid molecule is 0 nmol / L, and the human glia when the reference and the nucleic acid molecule are present at 30 nmol / L. It can be calculated by comparing the expression level of periostin gene in blastoma cells.
  • the human glioblastoma cells include A172 cells (ATCC (registered trademark) CRL-1620 (trademark)) and the like.
  • the expression of the periostin gene in mouse fibroblasts is, for example, 45% or more, 50% or more, 55% or more, 60% or more, 65 % Or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.
  • the suppression of the expression is based on, for example, the expression level of the periostin gene of the mouse fibroblast when the nucleic acid molecule is 0 nmol / L, and the mouse when the reference and the nucleic acid molecule are present at 20 nmol / L. It can be calculated by comparing the expression level of the periostin gene in fibroblasts.
  • the mouse fibroblasts include NIH-3T3 cells (ATCC (registered trademark) CRL-1658 (trademark)) and the like.
  • the nucleic acid molecule of the present invention may further have an additional sequence.
  • the additional sequence may be added to at least one of the 3 ′ end and the 5 ′ end of the as nucleotide in the expression suppression sequence, for example. Is preferably added to the 5 ′ end.
  • the additional sequence is not particularly limited, and for example, the length and sequence are not particularly limited.
  • the additional sequence can be represented by (N) n , for example.
  • N is a base, and may be a natural base or an artificial base, for example. Examples of the natural base include A, C, G, U, and T.
  • the n is a positive integer and indicates the base length of the additional sequence.
  • the additional sequence (N) n has a length (n) of, for example, 1, 2, 3 bases, preferably 1 or 2 bases, and more preferably 2 bases. When the additional sequence (N) n has a length of 2 bases or more (n ⁇ 2), the consecutive bases (N) may be, for example, the same base or different bases. Examples of (N) n include UU, CU, UC, GA, AG, GC, UA, AA, CC, GU, UG, CG, AU, and TT from the 3 ′ side or the 5 ′ side.
  • examples of the expression suppression sequence include a sequence in which the as1 nucleotide and the additional sequence are linked.
  • the nucleic acid molecule is preferably a nucleic acid molecule having a protecting group, for example.
  • the protecting group is, for example, a modifying group that imparts resistance to a nuclease such as exonuclease with respect to the nucleic acid molecule.
  • Examples of the nucleic acid molecule having the protecting group include a nucleic acid molecule containing a modified nucleotide residue described later.
  • the structural unit of the nucleic acid molecule of the present invention is not particularly limited, and examples thereof include nucleotide residues.
  • the nucleotide residue include a ribonucleotide residue and a deoxyribonucleotide residue.
  • the nucleotide residue include an unmodified unmodified nucleotide residue and a modified modified nucleotide residue.
  • the nucleic acid molecule of the present invention may be, for example, a DNA molecule, an RNA molecule, or a molecule containing a DNA molecule and an RNA molecule.
  • the nucleic acid molecule of the present invention is a DNA molecule
  • the nucleic acid molecule may be, for example, a DNA molecule consisting of only deoxyribonucleotide residues, and in addition to deoxyribonucleotide residues, ribonucleotide residues and / or non-nucleotide residues It may be a DNA molecule containing
  • the nucleic acid molecule of the present invention is an RNA molecule
  • the nucleic acid molecule may be, for example, an RNA molecule consisting of only ribonucleotide residues, and in addition to ribonucleotide residues, deoxyribonucleotide residues and / or non-nucleotide residues RNA molecules containing
  • the nucleotide residue includes, for example, a sugar, a base and a phosphate as constituent elements.
  • examples of the nucleotide residue include a ribonucleotide residue and a deoxyribonucleotide residue as described above.
  • the ribonucleotide residue has, for example, a ribose residue as a sugar, and has adenine (A), guanine (G), cytosine (C) and uracil (U) as bases
  • the deoxyribonucleotide residue is For example, it has a deoxyribose residue as a sugar and has adenine (A), guanine (G), cytosine (C) and thymine (T) as bases.
  • the nucleotide residue includes an unmodified nucleotide residue and a modified nucleotide residue.
  • each of the constituent elements is, for example, the same or substantially the same as that existing in nature, preferably the same or substantially the same as that naturally occurring in the human body. .
  • the modified nucleotide residue is, for example, a nucleotide residue obtained by modifying the unmodified nucleotide residue.
  • the modified nucleotide residue for example, any of the constituent elements of the unmodified nucleotide residue may be modified.
  • “modification” refers to, for example, substitution, addition and / or deletion of the component, substitution, addition and / or deletion of atoms and / or functional groups in the component, and is referred to as “modification”. be able to.
  • modified nucleotide residue include naturally occurring nucleotide residues, artificially modified nucleotide residues, and the like. See, for example, Limbach et al.
  • modified nucleotide residue may be, for example, a residue of the nucleotide substitute.
  • Examples of the modification of the nucleotide residue include modification of a sugar-phosphate skeleton (hereinafter also referred to as “sugar phosphate skeleton”). Specifically, the modification of a sugar residue and the modification of a phosphate are possible. can give.
  • the deoxyribose residue and / or ribose residue may be modified.
  • the deoxyribose residue can modify the 2'-position carbon. Specifically, for example, hydrogen bonded to the 2'-position carbon can be substituted with a halogen such as fluoro.
  • the deoxyribose residue can be substituted with a ribose residue by, for example, substituting the hydrogen at the 2'-position with a hydroxyl group.
  • the ribose residue can be modified, for example, at the 2′-position carbon.
  • a hydroxyl group bonded to the 2′-position carbon can be replaced with hydrogen or a halogen such as fluoro.
  • the ribose residue may replace, for example, a hydroxyl group hydrogen bonded to the 2'-position carbon.
  • the ribose residue can be substituted with a deoxyribose residue by, for example, substituting the hydroxyl group at the 2'-position with hydrogen.
  • the ribose residue can be substituted with, for example, a stereoisomer, and can be substituted with, for example, an arabinose residue.
  • the modified nucleotide residue may be, for example, a bicyclic sugar residue obtained by crosslinking a cyclic structure of the sugar residue in the sugar residue constituting the nucleotide residue.
  • Specific examples of the modified nucleotide residue containing the bicyclic sugar residue are not particularly limited, and examples thereof include known bicyclic artificial nucleic acid monomer residues.
  • the bicyclic artificial nucleic acid monomer residues include, for example, cEt (constrained ethyl bicyclic acid, manufactured by Ionis Pharmaceuticals), LNA (TM), Locked Nucleic Acid, ENA (registered trademark, 2'-O, 4 '). -C-Ethylenebridged Nucleic Acid) and the like, preferably LNA.
  • the sugar phosphate skeleton may be substituted with a non-sugar phosphate skeleton having a non-deoxyribose residue, a non-ribose residue and / or a non-phosphate, for example.
  • the non-sugar phosphate skeleton include uncharged bodies of the sugar phosphate skeleton.
  • Examples of the substitute for the nucleotide substituted with the non-sugar phosphate skeleton include morpholino, cyclobutyl, pyrrolidine, PNA (peptide nucleic acid) and the like.
  • a phosphate group can be modified.
  • the phosphate group closest to the sugar residue is called an ⁇ -phosphate group.
  • the ⁇ -phosphate group is negatively charged, and the charge is evenly distributed over two oxygen atoms that are not bound to a sugar residue.
  • the four oxygen atoms in the ⁇ -phosphate group in the phosphodiester bond between nucleotide residues, the two oxygen atoms that are non-bonded to the sugar residue are hereinafter referred to as “non-linking oxygen”.
  • the two oxygen atoms bonded to the sugar residue are hereinafter referred to as “linking oxygen”.
  • the ⁇ -phosphate group is preferably subjected to, for example, a modification that makes it uncharged or a modification that makes the charge distribution in the unbound oxygen asymmetric.
  • the phosphate group may replace the non-bonded oxygen, for example.
  • the oxygen is, for example, one of S (sulfur), Se (selenium), B (boron), C (carbon), H (hydrogen), N (nitrogen), and OR (R is an alkyl group or an aryl group).
  • R is an alkyl group or an aryl group.
  • the non-bonded oxygen for example, both are preferably substituted, and more preferably, both are substituted with S.
  • the modified phosphate group include phosphorothioate, phosphorodithioate, phosphoroselenate, boranophosphate, boranophosphate ester, phosphonate hydrogen, phosphoramidate, alkyl or arylphosphonate, and phosphotriester. Among them, phosphorothioate and phosphorodithioate in which the two non-bonded oxygens are both substituted with S are preferable.
  • the phosphate group may substitute, for example, the bonded oxygen.
  • the oxygen can be substituted, for example, with any atom of S (sulfur), C (carbon) and N (nitrogen), and the modified phosphate group is, for example, a bridged phosphoramidate, S substituted with N Substituted bridged phosphorothioates, bridged methylene phosphonates substituted with C, and the like.
  • the binding oxygen substitution is preferably performed, for example, on at least one of the 5 ′ terminal nucleotide residue and the 3 ′ terminal nucleotide residue of the nucleic acid molecule of the present invention. For the 'side, substitution with N is preferred.
  • the phosphate group may be substituted with, for example, the phosphorus-free linker.
  • the linker include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioform acetal, form acetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethyl. Hydrazo, methyleneoxymethylimino and the like, preferably methylenecarbonylamino group and methylenemethylimino group.
  • nucleic acid molecule of the present invention for example, at least one nucleotide residue at the 3 'end and the 5' end may be modified.
  • the modification may be, for example, either the 3 'end or the 5' end, or both.
  • the modification is, for example, as described above, and is preferably performed on the terminal phosphate group.
  • the phosphate group may be modified entirely, or one or more atoms in the phosphate group may be modified. In the former case, for example, the entire phosphate group may be substituted or deleted.
  • Examples of the modification of the terminal nucleotide residue include addition of other molecules.
  • Examples of the other molecule include functional molecules such as a labeling substance and a protecting group as described later.
  • Examples of the protecting group include S (sulfur), Si (silicon), B (boron), ester-containing groups, and the like.
  • the functional molecule such as the labeling substance can be used for detecting the nucleic acid molecule of the present invention, for example.
  • the other molecule may be added to the phosphate group of the nucleotide residue, for example, or may be added to the phosphate group or the sugar residue via a spacer.
  • the terminal atom of the spacer can be added or substituted, for example, to the binding oxygen of the phosphate group or O, N, S or C of the sugar residue.
  • the binding site of the sugar residue is preferably, for example, C at the 3 'position or C at the 5' position, or an atom bonded thereto.
  • the spacer can be added or substituted at a terminal atom of a nucleotide substitute such as PNA.
  • the spacer is not particularly limited.
  • n is a positive integer
  • n 3 or 6 is preferable.
  • the molecule added to the terminal includes, for example, a dye, an intercalating agent (for example, acridine), a crosslinking agent (for example, psoralen, mitomycin C), an anticancer agent, a porphyrin (TPPC4, texaphyrin, suffirin).
  • an intercalating agent for example, acridine
  • a crosslinking agent for example, psoralen, mitomycin C
  • an anticancer agent for example, a porphyrin (TPPC4, texaphyrin, suffirin).
  • Polycyclic aromatic hydrocarbons eg phenazine, dihydrophenazine
  • artificial endonucleases eg EDTA
  • lipophilic carriers eg cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1 , 3-bis-O (hexadecyl) glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3 -(Oleoi ) Cholic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (eg, antennapedia peptide, Tat peptide, RGD peptide), alkylating agents, phosphate, amino,
  • the 5 ′ end may be modified with, for example, a phosphate group or a phosphate group analog.
  • the phosphate group is, for example, 5 ′ monophosphate ((HO) 2 (O) PO-5 ′), 5 ′ diphosphate ((HO) 2 (O) POP (HO) (O) —O— 5 '), 5' triphosphate ((HO) 2 (O) PO- (HO) (O) POP (HO) (O) -O-5 '), 5'-guanosine cap (7-methylated or Unmethylated, 7m-GO-5 '-(HO) (O) PO- (HO) (O) POP (HO) (O) -O-5'), 5'-adenosine cap (Appp), optional Modified or unmodified nucleotide cap structure (NO-5 '-(HO) (O) PO- (HO) (O) POP (HO) (O) -O-5'), 5 'mono
  • the base is not particularly limited.
  • the base may be, for example, a natural base or a non-natural base.
  • the base may be, for example, naturally derived or a synthetic product.
  • As the base for example, a general base or a modified analog thereof can be used.
  • Examples of the base include purine bases such as adenine and guanine, and pyrimidine bases such as cytosine, uracil and thymine.
  • Other examples of the base include inosine, xanthine, hypoxanthine, purine, isoguanine, isocytosine, and 7-deazaadenine.
  • the base examples include alkyl derivatives such as 2-aminoadenine and 6-methylated purine; alkyl derivatives such as 2-propylated purine; 5-halouracil and 5-halocytosine; 5-propynyluracil and 5-propynylcytosine; -Azouracil, 6-azocytosine and 6-azothymine; 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5- (2-aminopropyl) uracil, 5-aminoallyluracil; 8-halogenated, aminated, Thiolated, thioalkylated, hydroxylated and other 8-substituted purines; 5-trifluoromethylated and other 5-substituted pyrimidines; 7-methylguanine; 5-substituted pyrimidines; 6-azapyrimidines; N-2, N -6 and O-6 substituted purines (2-aminopropyladenyl 5-
  • the modified nucleotide residue may include, for example, a residue lacking a base, that is, an abasic sugar phosphate skeleton.
  • the modified nucleotide residues are, for example, US Provisional Application No. 60 / 465,665 (filing date: April 25, 2003) and International Application No. PCT / US04 / 07070 (filing date: 2004/3). The residues described on the 8th of May) can be used, and the present invention can incorporate these documents.
  • the nucleic acid molecule of the present invention may contain, for example, a labeling substance and be labeled with the labeling substance.
  • the labeling substance is not particularly limited, and examples thereof include fluorescent substances, dyes, isotopes and the like.
  • the labeling substance include fluorophores such as pyrene, TAMRA, fluorescein, Cy (registered trademark) 3 dye, and Cy (registered trademark) 5 dye, and examples of the dye include Alexa dyes such as Alexa488. It is done.
  • the isotope include a stable isotope and a radioactive isotope, and preferably a stable isotope.
  • the stable isotope has a low risk of exposure and does not require a dedicated facility, so that it is easy to handle and the cost can be reduced.
  • the stable isotope does not change the physical properties of the labeled compound, for example, and is excellent in properties as a tracer.
  • the stable isotope is not particularly limited, and examples thereof include 2 H, 13 C, 15 N, 17 O, 18 O, 33 S, 34 S, and 36 S.
  • the nucleic acid molecule of the present invention is, for example, a single-stranded nucleic acid molecule, and may have a 5 'side region, an internal region, and a 3' side region in the above order from the 5 'side to the 3' side.
  • the expression suppression sequence may be arranged in any one of the 5 ′ region, the internal region, and the 3 ′ region, or may be arranged over two or more regions, or all It may be arranged over the area.
  • the nucleic acid molecule of the present invention contains an as1 nucleotide
  • the 5 ′ lower-case base (a, u, g, c, t) is the 5 ′ region
  • upper-case bases (A) sandwiched between the lower-case base on the 5′-side and the lower-case base on the 3′-side , T, G, C) may be the internal region.
  • the nucleic acid molecule of the present invention contains as2 nucleotides or as3 nucleotides, for example, it is sandwiched between the 5 ′ lower case base, the 5 ′ lower case base and the 3 ′ lower case base in the as1 nucleotide.
  • the upper case base and the base corresponding to the 3 'side lower case base may be the 5' side region, the internal region, and the 3 'side region, respectively.
  • the nucleotide residues constituting the 5 'region, the internal region, and the 3' region are not particularly limited, and may be, for example, ribonucleotide residues or deoxyribonucleotide residues.
  • the internal region is preferably a deoxyribonucleotide residue, more preferably the modified deoxyribonucleotide residue, and more preferably the phosphate group because expression suppression can be induced by degradation of mRNA of the periostin gene via RNaseH. More preferred are modified deoxyribonucleotide residues that have been modified.
  • nucleotide residues constituting the 5′-side region and the 3′-side region are preferably ribonucleotide residues, the modified ribonucleotide residue in which the sugar residue is modified, or the phosphate group Is more preferably a modified ribonucleotide residue, more preferably a ribonucleotide residue in which the sugar residue and the phosphate group are modified.
  • nucleotide residues constituting the 5 ′ region and the 3 ′ region are ribonucleotide residues
  • nucleotide residues constituting the internal region are deoxyribonucleotide residues. It is preferably a group.
  • the nucleotide residues constituting the 5'-side region and the 3'-side region may be, for example, the unmodified nucleotide residue or the modified nucleotide residue. Since the 5′-side region and the 3′-side region can improve, for example, the nuclease resistance of the nucleic acid molecule, the nucleotide residues constituting at least one of the 5′-side region and the 3′-side region are modified as described above. Since it is preferably a nucleotide residue, and the nuclease resistance of the nucleic acid molecule can be further improved, the nucleotide residues constituting the 5 ′ side region and the 3 ′ side region are more preferably the modified nucleotide residues. preferable.
  • the 5 ′ region may be, for example, all of the nucleotide residues constituting the 5 ′ region may be the modified nucleotide residue, A part thereof may be the modified nucleotide residue. In the latter case, in the 5 ′ region, a continuous nucleotide residue may be the modified nucleotide residue, or a non-continuous nucleotide residue may be the modified nucleotide residue. .
  • the continuous nucleotide residue is the modified nucleotide residue, it is preferable that the nucleotide residue continuous from the 5 'terminal nucleotide residue in the 5' side region is the modified nucleotide residue.
  • the modified nucleotide residue is, for example, as described above, preferably a modified ribonucleotide residue in which the sugar residue is modified, or a modified ribonucleotide residue in which the phosphate group is modified, and more
  • the sugar residue and the phosphate group are modified ribonucleotide residues.
  • the 3 ′ side region when the 3 ′ side region includes a modified nucleotide residue, the 3 ′ side region, for example, all of the nucleotide residues constituting the 3 ′ side region may be the modified nucleotide residue, A part thereof may be the modified nucleotide residue. In the latter case, in the 3 ′ region, a continuous nucleotide residue may be the modified nucleotide residue, or a non-continuous nucleotide residue may be the modified nucleotide residue. . When the continuous nucleotide residue is the modified nucleotide residue, it is preferable that the nucleotide residue continuous from the 3 'terminal nucleotide residue in the 3' side region is the modified nucleotide residue.
  • the modified nucleotide residue is, for example, as described above, preferably a modified ribonucleotide residue in which the sugar residue is modified, or a modified ribonucleotide residue in which the phosphate group is modified, and more
  • the sugar residue and the phosphate group are modified ribonucleotide residues.
  • the internal region may be, for example, an unmodified nucleotide residue or a modified nucleotide residue.
  • the internal region includes a modified nucleotide residue, for example, all of the nucleotide residues constituting the internal region may be the modified nucleotide residue, or part of the internal region may be the modified nucleotide residue. It may be a nucleotide residue.
  • the internal region is preferably a modified deoxyribonucleotide residue in which the phosphate group is modified.
  • the nucleotide residues constituting the 5 ′ region and the 3 ′ region are modified ribonucleotide residues
  • the nucleotide residues constituting the internal region are: It is preferably a deoxyribonucleotide residue
  • the nucleotide residues constituting the 5 ′ region and the 3 ′ region are the modified ribonucleotide residues
  • the nucleotide residues constituting the internal region are modified More preferably, it is a deoxyribonucleotide residue
  • the nucleotide residues constituting the 5′-side region and the 3′-side region contain 2′-O-methylribose and have a phosphate group modified.
  • At least one of a nucleotide residue and LNA, and the nucleotide residue constituting the internal region is modified with a phosphate group modified More preferably a deoxyribonucleotide residues.
  • the modified nucleotide residue is preferably a modified ribonucleotide residue having a phosphate group modified.
  • the number of bases in each region is not particularly limited.
  • the number of bases in the 5′-side region is, for example, 1 to 14, 1 to 12, 2 to 11, 3 to 8, 3 to 6, 3 to 5, 2 to 5, 2 to 4 It is a piece.
  • the number of bases in the internal region is, for example, 5 to 16, 6 to 14, 8 to 14, 6 to 12, or 7 to 12.
  • the number of bases in the 3 ′ side region is, for example, 1 to 14, 1 to 12, 2 to 11, 3 to 8, 3 to 6, 3 to 5, 2 to 5, 2 to 4 It is a piece.
  • the number of bases in the 5 'side region and the number of bases in the 3' side region may be the same or different, for example, but the former is preferred.
  • the combination of the total length of the nucleic acid molecule, the number of bases in the 5 ′ side region, the number of bases in the internal region, and the number of bases in the 3 ′ side region is not particularly limited.
  • the nucleotide residues constituting the 5 ′ side region and the 3 ′ side region are modified ribonucleotide residues
  • the nucleotide residues constituting the internal region are deoxyribonucleotide residues.
  • it is at least one of a group and a modified deoxyribonucleotide residue for example, the following combinations can be mentioned.
  • the modified ribonucleotide residue is preferably a modified ribonucleotide residue containing 2′-O-methyl ribose and having a phosphate group modified.
  • Total length of nucleic acid molecule 12 to 40 base length, 15 to 30 base length, 18 to 20 base length 5 ′ base number: 1 to 14, 2 to 8, 3 to 6 internal region base number: 5-16, 7-15, 8-14, 3 ′ base number: 1-14, 2-8, 3-6
  • the nucleotide residues constituting the 5 ′ side region and the 3 ′ side region are bicyclic artificial nucleic acid monomer residues, and the nucleotides constituting the internal region
  • examples thereof include the following combinations.
  • Total length of nucleic acid molecule 12 to 40 base length, 13 to 40 base length, 14 to 30 base length, 16 to 20 base length 5 ′ base number of bases: 1 to 14, 2 to 8, 2 to 5 Number of bases in internal region: 5 to 16, 7 to 15, 8 to 14 Number of bases in 3 'side region: 1 to 14, 2 to 8, 2 to 5
  • the nucleic acid molecule of the present invention can suppress the expression of the periostin gene. For this reason, the nucleic acid molecule of the present invention can be used, for example, as a therapeutic agent for a disease caused by expression of the periostin gene (hereinafter also referred to as “periostin gene-related disease”).
  • treatment includes, for example, the meaning of prevention of the disease, improvement of the disease, and improvement of the prognosis of the disease.
  • the periostin gene-related disease is not particularly limited, for example, eye disease, skin disease, respiratory disease, kidney disease, liver disease, gastrointestinal disease, otolaryngology disease, cardiovascular disease, blood disease, bone joint disease, Examples include cancer, inflammatory diseases, and fibrotic diseases.
  • the eye disease is not particularly limited, and includes retinopathy, macular degeneration, pterygium, conjunctivitis, intraocular neovascularization, fiber scar after eye surgery, etc.
  • the retinopathy is, for example, proliferative diabetic retinopathy And proliferative retinopathy such as proliferative vitreoretinopathy.
  • the skin disease is not particularly limited, and examples thereof include atopic dermatitis, wound healing, hypertrophic scar, keloid, systemic scleroderma and the like.
  • the respiratory disease is not particularly limited, and examples thereof include bronchial asthma, airway inflammation, and pulmonary fibrosis.
  • the renal disease is not particularly limited, and examples thereof include chronic kidney disease and polycystic kidney disease.
  • the liver disease is not particularly limited, and examples thereof include nonalcoholic steatohepatitis and nonalcoholic fatty liver disease.
  • the digestive system disease is not particularly limited, and examples thereof include capsular peritoneal sclerosis.
  • the otolaryngology disease is not particularly limited, and examples thereof include eosinophil otitis media, allergic rhinitis, chronic sinusitis, IgG4-related sclerosing salivary glanditis, and nasal polyp.
  • the cardiovascular disease is not particularly limited, and examples thereof include acute myocardial infarction, atherosclerosis, abdominal aortic aneurysm, rheumatic valvular disease and the like.
  • the blood disease is not particularly limited, and examples thereof include myelofibrosis.
  • the bone joint disease is not particularly limited, and examples thereof include knee arthropathy.
  • the cancer is not particularly limited.
  • the inflammatory disease is not particularly limited, and examples thereof include atopic dermatitis, bronchial asthma, allergic rhinitis, chronic sinusitis, eosinophilic otitis media, interstitial pneumonia, and systemic lupus erythematosus.
  • the fibrotic disease is not particularly limited, and examples thereof include idiopathic pulmonary fibrosis, chronic kidney disease, cirrhosis, encapsulating peritoneal sclerosis, myelofibrosis, scleroderma, and Dupuytren's contracture.
  • the method for using the nucleic acid molecule of the present invention is not particularly limited, and for example, the nucleic acid molecule may be administered to the administration subject.
  • Examples of the administration subject include cells, tissues, and organs. Examples of the administration subject include humans and non-human animals other than humans. Examples of the non-human animals include non-human mammals such as mice, rats, rabbits, sheep, cows, horses, dogs, pigs, monkeys, and the like. The administration may be, for example, in vivo or in vitro .
  • the cells are not particularly limited, for example, various cultured cells such as human and mouse retinal pigment epithelial cells such as ARPE-19, fibroblasts such as NIH3T3, glioblastoma cells such as A172 cells, ES cells And stem cells such as hematopoietic stem cells, cells isolated from living organisms such as primary cultured cells, and the like.
  • the cells exclude, for example, human fertilized eggs and cells in human embryos and human individuals.
  • composition of the present invention refers to the description of the composition of the present invention, pharmaceuticals, periostin gene expression suppression method, periostin gene-related disease treatment method and the like described later can be referred to.
  • the nucleic acid molecule of the present invention is useful as, for example, a pharmaceutical because it can suppress the expression of the periostin gene as described above.
  • the nucleic acid molecule of the present invention can be synthesized by, for example, a genetic engineering technique or an organic synthetic technique, and can also be referred to as a synthetic DNA molecule, a synthetic RNA molecule, or a synthetic DNA / RNA molecule.
  • composition of the present invention comprises the expression-inhibiting nucleic acid molecule of the present invention.
  • the composition of the present invention is characterized by including the expression-suppressing nucleic acid molecule of the present invention, and other configurations are not limited at all.
  • the composition of the present invention can also be referred to as, for example, an expression suppression reagent.
  • administration of a periostin gene can be suppressed by administering to a subject in which the periostin gene is present, particularly a subject having a relatively high periostin gene expression, or a subject that is predicted to be relatively high.
  • the administration target is, for example, as described above.
  • the composition of the present invention is a pharmaceutical composition for a periostin gene-related disease, a therapeutic agent for a periostin gene-related disease. It can also be said to be a drug for periostin gene-related diseases.
  • periostin gene-related disease for example, by administering to a patient with a periostin gene-related disease, expression of the periostin gene can be suppressed and the disease can be treated.
  • the periostin gene-related disease is, for example, as described above.
  • treatment includes, for example, the meaning of prevention of the disease, improvement of the disease, and improvement of the prognosis of the disease.
  • the administration method is not particularly limited, and can be appropriately determined according to the administration subject, for example.
  • the administration target is a cell or the like separated from a living body
  • examples thereof include a method using a transfection reagent, an electroporation method, and a nanobubble method.
  • parenteral administration examples include parenteral administration and oral administration.
  • parenteral administration include local administration, subcutaneous administration, and intravenous administration.
  • the administration site for the eye disease include eyes and blood vessels.
  • the administration method is not particularly limited, and examples thereof include instillation, instillation, intravitreal injection, subconjunctival injection, subtenon injection, and intraanterior administration.
  • the administration conditions of the composition of the present invention for example, the number of administrations, the dosage and the like.
  • composition of the present invention is not particularly limited, and examples thereof include injections, intravenous infusions, eye drops, eye ointments, skin ointments, patches, inhalants, liquids, aerosols, pump sprays, oral Agents.
  • the amount of the expression-suppressing nucleic acid molecule is not particularly limited.
  • the administration conditions for the expression-suppressing nucleic acid molecule are not particularly limited.
  • the dose (total) per administration is, for example, 5 to 5000 mg, 50 to 500 mg, and the number of administrations is For example, once every 2 to 8 weeks.
  • the dose (total) per dose for one eyeball of a human adult male is, for example, 0.01 to 10 mg, preferably 0.1 to 1 mg.
  • the number of times is, for example, once every 2 to 8 weeks.
  • the compounding amount of the nucleic acid molecule is preferably contained at a concentration that can realize the exemplified administration conditions.
  • composition of the present invention may contain, for example, only the expression-suppressing nucleic acid molecule of the present invention, or may further contain other additives.
  • the amount of the additive is not particularly limited as long as it does not interfere with the function of the expression suppressing nucleic acid molecule.
  • the additive is not particularly limited, and for example, a pharmaceutically acceptable additive is preferable.
  • the type of the additive is not particularly limited, and can be appropriately selected depending on, for example, the type of administration target.
  • the additive may form, for example, a complex with the expression-suppressing nucleic acid molecule.
  • the additive can be said to be a complexing agent, for example.
  • the expression-suppressing nucleic acid molecule can be efficiently delivered, for example, by making the expression-suppressing nucleic acid molecule a complex.
  • the binding between the expression-suppressing nucleic acid molecule and the complexing agent is not particularly limited, and examples thereof include non-covalent binding. Examples of the complex include an inclusion complex.
  • the complexing agent is not particularly limited, and examples thereof include a polymer, cyclodextrin, adamantine and the like.
  • examples of the cyclodextrin include a linear cyclodextrin copolymer and a linear oxidized cyclodextrin copolymer.
  • the additive examples include a carrier, a binding substance to a target cell, a condensing agent, a fusing agent, an excipient, a base, a stabilizer, a preservative, and the like.
  • the periostin gene expression-suppressing reagent of the present invention comprises the expression-suppressing nucleic acid molecule of the present invention.
  • the expression-suppressing reagent of the present invention is characterized by including the expression-suppressing nucleic acid molecule of the present invention, and other configurations and conditions are not limited at all.
  • the expression-suppressing nucleic acid molecule of the present invention can be used as the expression-suppressing reagent of the present invention.
  • the expression suppression reagent of the present invention may further include, for example, components that can be used in the expression suppression method of the present invention described later.
  • the component include a buffer solution.
  • each component may be stored in one container, or each component may be stored separately in a plurality of containers.
  • the expression suppression reagent of the present invention is also referred to as an expression suppression kit, for example.
  • the expression suppression kit of the present invention may further include instructions for use, for example.
  • the medicinal product for periostin gene-related disease of the present invention comprises the nucleic acid molecule for suppressing expression of the present invention.
  • the medicinal product for periostin gene-related disease of the present invention is characterized by including the expression-suppressing nucleic acid molecule of the present invention, and other configurations and conditions are not limited at all.
  • the expression-suppressing nucleic acid molecule of the present invention can be used.
  • the diseases targeted by the present invention are, for example, as described above.
  • the suppression method of the present invention is a method of suppressing expression of a periostin gene, and uses the expression-suppressing nucleic acid molecule of the present invention, the composition of the present invention, or the drug for a periostin gene-related disease. It is characterized by that.
  • the suppression method of the present invention is characterized by using the expression-suppressing nucleic acid molecule of the present invention, and other steps and conditions are not limited at all.
  • the suppression method of the present invention includes, for example, a step of administering the expression-suppressing nucleic acid molecule to a subject in which a periostin gene is present, particularly to a subject in which periostin gene expression is relatively high or predicted to be relatively high. Including.
  • the administration step for example, the expression-suppressing nucleic acid molecule is brought into contact with the administration subject.
  • the administration target include cells, tissues or organs as described above.
  • Examples of the administration subject include humans and non-human animals as described above.
  • the administration may be, for example, in vivo or in vitro .
  • the expression-suppressing nucleic acid molecule may be administered alone, or the composition of the present invention containing the expression-suppressing nucleic acid molecule may be administered.
  • the administration method is not particularly limited, and can be appropriately selected depending on, for example, the type of administration target, and the above description can be used.
  • the method for treating a periostin gene-related disease of the present invention includes a step of administering the expression-suppressing nucleic acid molecule of the present invention to a patient.
  • the treatment method of the present invention is characterized by using the expression-suppressing nucleic acid molecule of the present invention for the treatment of a periostin gene-related disease, and other steps and conditions are not limited at all.
  • the diseases targeted by the present invention are, for example, as described above.
  • the suppression method of the present invention can be used.
  • the administration method is not particularly limited, and for example, as described above, either parenteral administration or oral administration may be used.
  • the expression suppressing nucleic acid molecule of the present invention is a nucleic acid molecule for suppressing periostin gene expression or periostin protein function, or a nucleic acid molecule for treating periostin gene-related diseases.
  • the expression-suppressing nucleic acid molecule of the present invention is a nucleic acid molecule for producing a periostin gene expression inhibitor or a periostin gene-related disease drug.
  • Example 1 The nucleic acid molecule of the present invention was synthesized, and the suppression of human periostin gene expression in vitro was confirmed.
  • nucleic acid molecules As the nucleic acid molecules of Examples, the nucleic acid molecules X13707, X13708, X13710 to X13712, X13714, X13715, X13717 to X13727, X13730, X13734 to X13743, X13745 to X13757, X13759 to X13763, Table 2A and B below X13765 to X13776, X13778, X13781, X13783 to X13788, X13791, X13792, X13795 to X13800, and X13802 to X13827 were obtained.
  • nucleic acid synthesizer (ABI3900, manufactured by Applied Biosystems)
  • the nucleic acid molecule was obtained by synthesizing a synthetic product containing the nucleic acid molecule and then purifying the resulting synthetic product by HPLC. . Purification of the nucleic acid molecule was confirmed by absorbance at 260 nm and electrospray ionization mass spectrometry.
  • “a, u, g, c” in the nucleotide sequences of the nucleic acid molecules in the following Tables 2A and B are modified ribonucleotide residues containing 2′-O-methylribose and phosphorothioate.
  • “A, T, G, C” were modified deoxyribonucleotide residues containing phosphorothioate.
  • “5 ′ end position” indicates the position of the base on the 5 ′ end side where each nucleic acid molecule hybridizes in the base sequence of SEQ ID NO: 121
  • “3 ′ end position” Indicates the position of the base on the 3 ′ end side where each nucleic acid molecule hybridizes in the base sequence of SEQ ID NO: 121.
  • the cells were cultured in the medium, and the culture was seeded in a 96-well plate at 1.5 ⁇ 10 4 cells / well. Further, each of the nucleic acid molecules was transfected using a transfection reagent Lipofectamine (registered trademark) 2000 (Invitrogen) according to the attached protocol. Specifically, transfection was performed so that the final concentration of the nucleic acid molecule was 3 or 30 nmol / L per well.
  • the cells in the wells were cultured for 24 hours. After culturing, the cells in each well were collected and attached except that the cells were lysed at 53 ° C. using an mRNA quantification reagent (QuantiGene 2.0, Affymetrix / Panomics (Axolabs customized product)).
  • the expression level of periostin gene was measured according to the protocol. Specifically, a cell lysate was prepared using the mRNA quantification reagent. Then, 50 ⁇ L of the cell lysate is reacted with the probe set for human periostin gene, and human periostin is calculated by calculating RLU (relative light unit) using an optical measuring instrument (Victor2-Light, manufactured by Perkin® Elmer).
  • RLU relative light unit
  • the expression level of the gene was measured.
  • the expression level of the human GAPDH gene is the same as that described above except that 10 ⁇ L of the cell lysate and the probe set for human GAPDH gene were reacted using Quantigene Explore Kit (Panomics) as the mRNA quantification reagent. ,It was measured.
  • the expression level of the human periostin gene was corrected by the expression level of the human GAPDH gene.
  • the expression level was calculated as the relative value of the expression level, assuming that the transfection reagent was added and the non-added cell group to which the nucleic acid molecule was not added was 1.
  • the control was measured in the same manner except that the nucleic acid molecule and the transfection reagent were not added.
  • each probe was a probe containing a polynucleotide having the base sequence shown in Table 3A below as a polynucleotide hybridizing to the mRNA of the human periostin gene.
  • each probe was a probe containing a polynucleotide having the nucleotide sequence shown in Table 3B below as a polynucleotide hybridizing to mRNA of the human GAPDH gene.
  • FIGS. 1A to 1D are graphs showing the relative value of the expression level, the vertical axis is the relative value of the expression level, the white bar in the figure indicates the result of 3 nmol / L, and the black bar is 30 nmol / L The result of L is shown.
  • the nucleic acid molecules of the above examples showed lower values than the control, so that it was confirmed that all of them had expression suppressing activity.
  • Example 2 The nucleic acid molecule of the present invention was synthesized, and the suppression of periostin gene expression in vitro was confirmed.
  • nucleic acid molecules The nucleic acid molecules X13464 to X13467, X13469 to X13471, X13473, X13474, and X13476 to X13480 shown in Table 4 below were obtained as the nucleic acid molecules of Examples in the same manner as in Example 1 (1).
  • “a, u, g, c” in the base sequence of each nucleic acid molecule shown in Table 4 below is a modified ribonucleotide residue containing 2′-O-methylribose and phosphorothioate
  • A, T, G, C were modified deoxyribonucleotide residues including phosphorothioate.
  • 5 ′ end position indicates the position of the base on the 5 ′ end side where each nucleic acid molecule hybridizes in the base sequence of SEQ ID NO: 121
  • 3 ′ end position In the base sequence of SEQ ID NO: 121, the position of the 3 ′ end base to which each nucleic acid molecule hybridizes is shown.
  • mice fibroblast NIH-3T3 cells (ATCC) were used as a medium, 10% FBS, 2 mmol / L L-glutamine, 100 U / mL penicillin, and Using DMEM containing 100 mg / mL streptomycin, the final concentration of the nucleic acid molecule is 20 nmol / L, the culture time after the transfection is 48 hours, and the probe set for the human periostin gene and the probe set for the human GAPDH gene are used. Instead, the relative value of the expression level was measured in the same manner as in Example 1 (2) except that the mouse periostin gene probe set and the mouse periostin gene probe set were used.
  • each probe was a probe containing a polynucleotide consisting of the nucleotide sequence shown in Table 5A below as a polynucleotide hybridizing to the mRNA of the mouse periostin gene.
  • each probe was a probe containing a polynucleotide having the nucleotide sequence shown in Table 5B below as a polynucleotide hybridizing to the mRNA of the mouse GAPDH gene.
  • FIG. 2 is a graph showing the relative value of the expression level, and the vertical axis represents the relative value of the expression level.
  • the nucleic acid molecule of the said Example showed a value lower than a non-added cell group, it has confirmed that all have expression suppression activity.
  • X13470, X13478, X13466, and X13479 showed extremely strong expression suppressing activity. From these results, it was found that the nucleic acid molecule of the present invention has an excellent expression suppressing activity.
  • Example 3 The half-inhibitory concentration of the nucleic acid molecule of the present invention was confirmed.
  • nucleic acid molecule instead of the nucleic acid molecule, X13470, X13478, X13466, and X13479 are used, and the final concentration of the nucleic acid molecule is 0.0102, 0.0305, 0.0914, 0.274, 0.823, 2.. Except for 47, 7.41, 22.2, 66.7, or 200 nmol / L, the relative value of the expression level was measured in the same manner as in Example 2 (2).
  • the half-inhibitory concentration of each nucleic acid molecule was calculated.
  • the results are shown in Table 6.
  • Table 6 it was confirmed that all the nucleic acid molecules had sufficiently low half-inhibitory concentrations and had excellent expression suppression activity.
  • the half-inhibitory concentrations of X13798, X13802, X13803, and X13810 were less than 1 nmol / L, and it was confirmed that they had extremely excellent expression suppressing activity. From these results, it was found that the nucleic acid molecule of the present invention has an excellent expression suppressing activity.
  • Example 4 Using a bleomycin-induced pulmonary fibrosis model mouse, it was confirmed that fibrosis was suppressed by administration of periostin expression-suppressing nucleic acid molecule.
  • nucleic acid molecule The nucleic acid molecule of SEQ ID NO: 217 (X17752) was used as a periostin expression-suppressing nucleic acid molecule to prepare a nucleic acid solution.
  • the nucleic acid molecule was prepared by the method of Example 1.
  • “a, u, g, c” is a modified ribonucleotide residue containing 2′-O-methylribose and phosphorothioate
  • A, T, G, C is a modified deoxyribonucleotide containing phosphorothioate.
  • Nucleotide residues. “C” and “C” were 5-methylcytosine.
  • X17752 SEQ ID NO: 217) 5'-caccaCTGTTCGTAAuuugg-3 '
  • an ALZET osmotic minipump (model 2001, manufactured by DURECT Corporation) into which 200 ⁇ L of bleomycin hydrochloride aqueous solution (10 mg / mL) had been previously injected was implanted subcutaneously in the back of the mouse.
  • a predetermined concentration (0.4 mg / mL or 2 mg / mL (Examples 4 (1) and (2), respectively)).
  • 50 ⁇ L of the nucleic acid solution was administered nasally to the mice.
  • the negative control was similarly used except that physiological saline was used in place of the bleomycin hydrochloride aqueous solution and the nucleic acid solution.
  • the control 1 was except that physiological saline was used in place of the nucleic acid solution.
  • Control 2 was treated in the same manner except that a control nucleic acid solution containing 2 mg / mL of the following control nucleic acid molecule was used instead of the nucleic acid solution.
  • a, u, g, c is a modified ribonucleotide residue containing 2′-O-methylribose and phosphorothioate
  • A, T, G, C is phosphorothioate.
  • the modified deoxyribonucleotide residues were included.
  • C and “C” were 5-methylcytosine.
  • Control nucleic acid molecule (SEQ ID NO: 233) 5'-cgacaTCGTGCGTCGuauau-3 '
  • the right lung (a part of the lower lobe) was used for measurement of the expression level of the mouse periostin gene in the lung tissue described later. Further, among the extracted lungs, the right lung (upper lobe, middle lobe, and accessory lobe) was fixed in formalin and used for histopathological analysis described later. Furthermore, among the extracted lungs, the left lung was used for measurement of the amount of hydroxyproline described later.
  • ReverTra Ace registered trademark
  • oligo oligo
  • Primer set for periostin gene amplification (SEQ ID NO: 234) 5'-CACGGCATGGTTATTCCTTCA-3 ' (SEQ ID NO: 235) 5'-TCAGGACACGGTCAATGACAT-3 ' GAPDH gene amplification primer set (SEQ ID NO: 236) 5'-TGGCCTTCCGTGTTCCTAC-3 ' (SEQ ID NO: 237) 5'-GAGTTGCTGTTGAAGTCGCA-3 '
  • the measurement result of the expression level of periostin gene is shown in FIG.
  • the horizontal axis indicates the type of sample, and the vertical axis indicates the expression level of the periostin gene.
  • the expression level of periostin gene was increased in controls 1 and 2 in which pulmonary fibrosis was induced by bleomycin and the expression-suppressing nucleic acid molecule of the present invention was not administered as compared with the negative control. .
  • the expression-suppressing nucleic acid molecule of the present invention when the expression-suppressing nucleic acid molecule of the present invention was administered (Examples 4 (1) and (2)), lung fibers caused by bleomycin were compared with the control nucleic acid molecule (Control 2).
  • the expression increase of the periostin gene at the time of induction of the disease was significantly suppressed, and the expression levels were 69.5% and 65.1%, respectively, compared with the control 2.
  • the amount of collagen protein was determined as a rabbit polyclonal anti-type I collagen antibody (manufactured by Rockland Immunochemicals, catalog number: 600-401-103) as a capture antibody, and as a detection antibody, a biotin-labeled rabbit polyclonal anti-type I collagen antibody ( Using Rockland Immunochemicals, catalog number 600-406-103), the measurement was performed by sandwich ELISA.
  • FIG. 4 is a graph showing the amount of protein in BALF, where (A) shows the amount of periostin protein and (B) shows the amount of type I collagen protein. 4 (A) and 4 (B), the horizontal axis indicates the type of sample, and the vertical axis indicates the amount of each protein.
  • the amount of periostin protein is increased in controls 1 and 2 in which pulmonary fibrosis is induced by bleomycin and the expression-suppressing nucleic acid molecule of the present invention is not administered as compared with the negative control. did.
  • FIG. 4 shows the amount of periostin protein in BALF, where (A) shows the amount of periostin protein and (B) shows the amount of type I collagen protein. 4 (A) and 4 (B), the horizontal axis indicates the type of sample, and the vertical axis indicates the amount of each protein.
  • the amount of periostin protein is increased in controls 1 and 2 in which pulmonary fibrosis is induced by bleomycin and the expression-s
  • the expression-suppressing nucleic acid molecule of the present invention when the expression-suppressing nucleic acid molecule of the present invention is administered, the amount of type I collagen protein when pulmonary fibrosis is induced by bleomycin, compared with the control nucleic acid molecule (control 2) Increase was significantly suppressed, with 82.0% and 52.9% protein content compared to Control 2, respectively. From these results, it was found that in the bleomycin-induced pulmonary fibrosis model mouse, the expression-inhibited nucleic acid molecule of the present invention can suppress the expression level of periostin gene and the amount of periostin protein, thereby suppressing the production of type I collagen. .
  • FIG. 5 is a graph showing the amount of hydroxyproline.
  • the horizontal axis indicates the type of sample, and the vertical axis indicates the amount of hydroxyproline.
  • pulmonary fibrosis was induced by bleomycin and the amount of hydroxyproline was increased in Controls 1 and 2 to which the expression suppressing nucleic acid molecule of the present invention was not administered.
  • FIG. 5 when the expression-suppressing nucleic acid molecule of the present invention was administered (Examples (1) and (2)), lung fibers caused by bleomycin were compared with the control nucleic acid molecule (Control 2).
  • FIG. 6 shows the results of Masson trichrome staining.
  • FIG. 6 is a photograph of a tissue section after Masson trichrome staining.
  • (A) is the result of negative control
  • (B) is the result of control 1
  • (C) is the result of control 2
  • (D) is the result of Example 4 (1)
  • (E ) Shows the result of Example 4 (2).
  • the expression-suppressing nucleic acid molecule of the present invention was administered (Examples 4 (1) and (2))
  • alveolar omission was suppressed as compared to the control nucleic acid molecule (Control 2).
  • FIG. 7 is a graph showing the Ashcroft score.
  • the horizontal axis indicates the sample type, and the vertical axis indicates the Ashcroft score.
  • the ashcroft score increased in controls 1 and 2 in which pulmonary fibrosis was induced by bleomycin and the expression-suppressing nucleic acid molecule of the present invention was not administered.
  • the expression-suppressing nucleic acid molecule of the present invention was administered (Examples 4 (1) and (2))
  • the Ashcroft score was significantly reduced as compared with the control nucleic acid molecule (Control 2).
  • the nucleic acid molecule of this invention was administered, the Ashcroft score became 4 or less, and it turned out that the fibrosis of the lung is suppressed greatly.
  • the expression-suppressing nucleic acid molecule of the present invention can suppress fibrosis in pulmonary fibrosis.
  • FIG. 8 The result of adhesion score is shown in FIG. In FIG. 8, the horizontal axis indicates the number of days after surgery, and the vertical axis indicates the adhesion score. As shown in FIG. 8, film-like weak adhesion formation with a score of about 1 was observed from 1 day after surgery. Furthermore, the adhesion score increased over time, and on the 14th, adhesion formation with an average score of 3 or more was observed.
  • periostin gene expression level After measurement of adhesion score, the cecal tip was collected, RNA was extracted by a conventional method, and periostin gene expression level and 18S rRNA expression level in the RNA were quantified by RT-PCR. did. The expression level of the periostin gene was a relative value corrected with 18S rRNA.
  • Primer set for periostin gene amplification (SEQ ID NO: 238) 5'-ATCAGGGGTCGGGATCAG-3 ' (SEQ ID NO: 239) 5'-GGAGCTGAAGTATTTCTTTTTGGT-3 ' 18s rRNA amplification primer set (SEQ ID NO: 240) 5'-GCAATTATTCCCCATGAACG-3 ' (SEQ ID NO: 241) 5'-GGGACTTAATCAACGCAAGC-3 '
  • the measurement result of the expression level of periostin gene is shown in FIG.
  • the horizontal axis indicates the number of days after surgery, and the vertical axis indicates the expression level of the periostin gene.
  • the expression of periostin tended to increase over time from day 1 to day 14 after surgery.
  • a tissue section was prepared from the collected cecal tip, and using a Mallory azocarmine G solution (Muto Kagaku Co.) and Mallory aniline blue orange G solution (Muto Kagaku Co.) Tissue staining was performed by Azan staining. Further, periostin was immunostained using an anti-periostin antibody (sc-49480, manufactured by Santa Cruz Biotechnology, Inc.) as a primary antibody and an LSAB staining kit (produced by DAKO). Then, it image
  • FIG. 10 shows the results of Azan staining and periostin immunostaining of the cecal tip tissue
  • FIGS. 10A to 10F show the control staining results (Normal) and 1, 3, 5, 7, 14 after the operation, respectively.
  • the dyeing result in day is shown.
  • 10A to F the upper photograph shows the staining result of Azan staining
  • the lower photograph shows the staining result of periostin immunostaining.
  • the left column photo shows the result of 20 times magnification
  • the right column photo shows the result of enclosing the rectangle in the left column photo with the magnification of 200 times in the region indicated by the arrow X.
  • a portion indicated by an arrow Y stained with Azan staining indicates a fibrosis site.
  • fibrosis formation at the adhesion site was confirmed from 1 day to 14 days after the operation.
  • FIG. 10A as a result of periostin immunostaining, expression of periostin was observed in the cecum tissue (the area surrounded by the solid line indicated by the arrow Z) in the cecal tissue that was not subjected to scratching.
  • FIGS. 10B to 10F in the tissue subjected to the abrasion treatment, strong expression of periostin was confirmed not only at the base but also at the adhesion site (the region surrounded by the solid line indicated by the arrow P). .
  • periostin gene expression increased in correlation with an increase in adhesion score, and that periostin was strongly expressed at the adhesion site. From these results, it was found that the formation of adhesions was correlated with the expression of periostin.
  • the nucleic acid molecule of SEQ ID NO: 217 (X17752) was used as the periostin expression-suppressing nucleic acid molecule.
  • Example 5 Using a 10-week-old male mouse (C57BL / 6J, obtained from Japan SLC), the cecum was scraped in the same manner as in Example 5. Thereafter, 0.2 mL of the nucleic acid solution was administered intraperitoneally, and the abdomen was closed. One week after the operation, 0.2 mL of the nucleic acid solution was again administered intraperitoneally. The dose of the nucleic acid molecule for each mouse was 40 mg / kg / dose.
  • Example 6 14 days after the operation, measurement of the expression level of the periostin gene, measurement of adhesion score, and histopathological analysis were performed on the mice of each group in the same manner as in Example 5 (Example 6).
  • Control 1 was carried out in the same manner except that the control nucleic acid molecule (SEQ ID NO: 233) was used in place of the periostin antisense nucleic acid.
  • Control 2 was carried out in the same manner except that PBS was used instead of the nucleic acid solution.
  • the measurement result of the expression level of the periostin gene is shown in FIG. In FIG. 11, the horizontal axis represents the sample name, and the vertical axis represents the expression level of the periostin gene. As shown in FIG. 11, in Example 6, the expression level of the periostin gene was significantly reduced as compared with Control 1 and Control 2. From these results, it was found that periostin gene expression can be suppressed by administration of periostin expression-suppressing nucleic acid molecules in postoperative adhesion model mice.
  • the horizontal axis indicates the sample name
  • the vertical axis indicates the adhesion score.
  • the adhesion score was significantly reduced as compared with Control 1 and Control 2. From these results, it was shown that the formation of adhesions is suppressed by administration of periostin expression-inhibiting nucleic acid molecules in postoperative adhesion model mice.
  • FIGS. 13A and 13B show the results of histopathological analysis.
  • FIG. 13A shows the staining result of Control 2
  • FIG. 13B shows the staining result of Example 6.
  • 13A and 13B the left column shows the staining result of Azan staining, and the right column shows the staining result of periostin immunostaining.
  • the upper part shows the result of 20 times magnification
  • the lower part shows the result of enlarging the area shown by the arrow X by 200 times with the square of the upper photograph.
  • FIG. 13A shows the staining result of Control 2
  • FIG. 13B shows the staining result of Example 6.
  • 13A and 13B the left column shows the staining result of Azan staining
  • the right column shows the staining result of periostin immunostaining.
  • the upper part shows the result of 20 times magnification
  • the lower part shows the result of enlarging the area shown by the arrow X by 200 times with the
  • FIG. 13A fibrosis formation was observed in control 2 as indicated by arrow Y, and strong expression of periostin was confirmed at the adhesion site (region surrounded by a solid line indicated by arrow P).
  • FIG. 13B administration of periostin antisense nucleic acid showed a decrease in fibrosis formation at the adhesion site as indicated by arrow Y in Example 6, and the adhesion site (enclosed by a solid line indicated by arrow P). In the region), it was confirmed that the expression of periostin was suppressed.
  • Example 7 Periostin expression-suppressing nucleic acid molecules having different lengths and periostin expression-suppressing nucleic acid molecules containing different modified nucleotide residues were synthesized, and human periostin gene expression suppression was confirmed in vitro .
  • nucleic acid molecules X22816, X16256, X22817, X22818, X22822, and X22823 shown in Table 7 below were obtained as the nucleic acid molecules of Examples. Specifically, it was obtained in the same manner as in Example 1 (1), and purification was confirmed.
  • nucleic acid molecules of the examples in the base sequences of the nucleic acid molecules in Table 7 below, “a, u, g, c” is a modified ribonucleotide residue containing 2′-O-methylribose and phosphorothioate, “A, T, G, C” were modified deoxyribonucleotide residues including phosphorothioate.
  • each nucleic acid molecule is a nucleic acid molecule in which only the modified nucleotide residue in X13792 is changed, or a nucleic acid molecule in which the modified nucleotide residue and the length are shortened.
  • WGW represents the lengths of the 5 ′ region, the internal region, and the 3 ′ region in each nucleic acid molecule.
  • nucleic acid molecules of Examples using LNA as modified nucleotide residues, nucleic acid molecules L-5105, L-4124, L-3143, L-4104, L-3123, L-2142, L in Table 8 below are used. -484, L-3103, and L-373 were obtained.
  • nucleic acid molecule of an Example WHEREIN In the base sequence of each nucleic acid molecule of following Table 8, "a, t, g, c" are the oxygen atom of 2'-position of a ribose ring, and the carbon atom of 4'-position.
  • a modified ribonucleotide residue cross-linked via methylene and containing phosphorothioate, ie LNA containing phosphorothioate, and “A, T, G, C” were modified deoxyribonucleotide residues containing phosphorothioate.
  • “c” was 5-methylcytosine.
  • Each nucleic acid molecule is a nucleic acid molecule in which only the modified nucleotide residue in X13792 is changed, or a nucleic acid molecule in which the modified nucleotide residue and the length are shortened.
  • WGW represents the lengths of the 5 'region, the internal region, and the 3' region in each nucleic acid molecule, respectively.
  • FIGS. FIG. 14 shows the results for the nucleic acid molecules in Table 7
  • FIG. 15 shows the results for the nucleic acid molecules in Table 8.
  • 14 and 15 are graphs showing the relative value of the expression level, the vertical axis is the relative value of the expression level, the black bar in the figure shows the result of 25 nmol / L, and the white bar is 75 nmol / L The result of L is shown.
  • the nucleic acid molecules of the above examples were confirmed to have expression suppression activity since the relative value of the expression level was lower than 1.
  • periostin expression-suppressing nucleic acid molecules having different lengths and periostin expression-suppressing nucleic acid molecules containing different modified nucleotide residues also have excellent expression-suppressing activity.
  • the expression of periostin gene or the function of periostin protein can be suppressed. Therefore, the present invention is effective for treatment of diseases caused by periostin gene expression or periostin protein.

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Abstract

Provided is a novel molecule for suppressing expression of the periostin gene. A nucleic acid molecule for suppressing expression of the periostin gene, wherein the expression-suppressing nucleic acid molecule is characterized in containing the following (as4) nucleotide as a periostin-gene-expression-suppressing sequence. (as4) A nucleotide having the ability to suppress expression of the periostin gene, said nucleotide comprising a base sequence that hybridizes under stringent conditions with a polynucleotide comprising any base sequence of SEQ ID NOS: 121-127. Since this expression-suppressing nucleic acid molecule can suppress expression of the periostin gene, said molecule can be used, for example, in the treatment of diseases resulting from periostin gene expression.

Description

ペリオスチン遺伝子の発現抑制核酸分子、ペリオスチン遺伝子の発現抑制方法、およびその用途Periostin gene expression suppressing nucleic acid molecule, periostin gene expression suppressing method, and use thereof
　本発明は、ペリオスチン遺伝子の発現抑制核酸分子、ペリオスチン遺伝子の発現抑制方法、およびその用途に関する。 The present invention relates to a periostin gene expression-suppressing nucleic acid molecule, a periostin gene expression-suppressing method, and uses thereof.
　近年、糖尿病患者の増加に伴い、合併症の一つである糖尿病網膜症（Diabetic　Retinopathy：DR）患者が増加している。糖尿病網膜症は、病期によって、単純糖尿病網膜症、前増殖糖尿病網膜症および増殖糖尿病網膜症（Proliferative　Diabetic　Retinopathy：PDR）に分類されている。中でも、増殖糖尿病網膜症は、視力の低下だけでなく、失明に至るおそれがある。 In recent years, with the increase of diabetic patients, the number of diabetic retinopathy (DR) patients, which is one of complications, is increasing. Diabetic retinopathy is classified into simple diabetic retinopathy, preproliferative diabetic retinopathy and proliferative diabetic retinopathy (PDR) according to the stage. Among them, proliferative diabetic retinopathy may lead to blindness as well as decreased visual acuity.
　また、眼疾患としては、同様に、加齢黄斑変性症（Age-related　Macular　Degeneration：AMD）等の黄斑変性症も問題となっている。黄斑変性症は、加齢やストレス等が原因となり、黄斑が変性して視力が低下する疾患である。この疾患も、重篤な場合は失明に至るおそれがある。 As eye diseases, macular degeneration such as age-related Macular Degeneration (AMD) is also a problem. Macular degeneration is a disease in which visual acuity decreases due to degeneration of the macular due to aging, stress, or the like. This condition can also lead to blindness in severe cases.
　これらの眼疾患は、網脈絡膜が酸欠状態等に陥り、酸素を補うために、網膜上下に新生血管が形成されることが発端となる。新生血管は、脆弱なため、破れて出血し易く、出血した血液が光路を遮ること等によって、視力が低下する。また、前記新生血管は、増殖組織といわれる線維性膜へと進展し、これが原因となって牽引性網膜剥離を生じることもある。そして、このような状態が維持され、繰り返されることによって、重篤化すると、失明に至るおそれがある（特許文献１）。このため、これらの眼疾患に関しては、治療薬となりうる新たな候補物質の提供が求められている。 These eye diseases start from the formation of new blood vessels above and below the retina in order to make the retina choroid deficient and supplement oxygen. Since the new blood vessel is fragile, it is easily broken and bleeds, and the visual acuity is reduced by the blood that has bleed interrupts the optical path. In addition, the new blood vessel develops into a fibrous membrane called a proliferating tissue, which may cause tractional retinal detachment. And if such a state is maintained and repeated and it becomes serious, there exists a possibility of leading to blindness (patent document 1). Therefore, for these eye diseases, provision of new candidate substances that can serve as therapeutic agents is demanded.
　これらの眼疾患については、ペリオスチン遺伝子の発現が関与していることが報告されている（非特許文献１）。 For these eye diseases, it has been reported that periostin gene expression is involved (Non-patent Document 1).
　また、眼疾患以外の疾患においても、ペリオスチン遺伝子の発現が関与していることが報告されている疾患もある。 In addition, some diseases other than eye diseases have been reported to involve periostin gene expression.
特開２０１１－２２０９６９号公報JP 2011-220969 A
　そこで、本発明は、ペリオスチン遺伝子の発現を抑制する新たな分子の提供、ならびに、それを用いた発現抑制方法およびペリオスチン関連疾患の治療方法等の用途の提供を目的とする。 Therefore, an object of the present invention is to provide a new molecule that suppresses the expression of the periostin gene, and to provide uses such as an expression suppression method using the same and a method for treating a periostin-related disease.
　前記目的を達成するために、本発明のペリオスチン遺伝子の発現抑制核酸分子は、ペリオスチン遺伝子の発現抑制核酸分子であり、
前記発現抑制核酸分子が、前記ペリオスチン遺伝子の発現抑制配列として、下記（ａｓ４）のヌクレオチドを含むことを特徴とする。
（ａｓ４）配列番号１２１～１２７のいずれか１つの塩基配列からなるポリヌクレオチドに対して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からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチド In order to achieve the above object, the periostin gene expression-suppressing nucleic acid molecule of the present invention is a periostin gene expression-suppressing nucleic acid molecule,
The expression-suppressing nucleic acid molecule includes the following nucleotide (as4) as an expression-suppressing sequence for the periostin gene.
(As4) a nucleotide comprising a base sequence that hybridizes under stringent conditions to a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 121 to 127, and having a function of suppressing expression of a periostin gene
　本発明のペリオスチン遺伝子の発現抑制試薬は、前記本発明の発現抑制核酸分子を含むことを特徴とする。 The periostin gene expression-suppressing reagent of the present invention is characterized by comprising the expression-suppressing nucleic acid molecule of the present invention.
　本発明のペリオスチン遺伝子関連疾患用医薬品は、本発明の前記発現抑制核酸分子を含むことを特徴とする。 The periostin gene-related disease drug of the present invention is characterized by comprising the expression-suppressing nucleic acid molecule of the present invention.
　本発明の抑制方法は、ペリオスチン遺伝子の発現抑制方法であって、本発明の前記発現抑制核酸分子を使用することを特徴とする。 The suppression method of the present invention is a method for suppressing the expression of a periostin gene, and is characterized by using the expression-suppressing nucleic acid molecule of the present invention.
　本発明のペリオスチン遺伝子関連疾患の治療方法は、本発明の前記発現抑制核酸分子を、患者に投与する工程を含むことを特徴とする。 The method for treating a periostin gene-related disease of the present invention includes a step of administering the expression-suppressing nucleic acid molecule of the present invention to a patient.
　本発明の発現抑制核酸分子によれば、ペリオスチン遺伝子の発現抑制が可能である。このため、本発明は、ペリオスチン遺伝子の発現が原因となる疾患の治療に有効である。 The expression-suppressing nucleic acid molecule of the present invention can suppress the expression of the periostin gene. For this reason, this invention is effective in the treatment of the disease caused by the expression of the periostin gene.
図１Ａは、本発明の実施例１におけるｉｎ　ｖｉｔｒｏでのペリオスチン遺伝子の発現量の相対値を示すグラフである。FIG. 1A is a graph showing the relative value of the expression level of periostin gene in vitro in Example 1 of the present invention. 図１Ｂは、本発明の実施例１におけるｉｎ　ｖｉｔｒｏでのペリオスチン遺伝子の発現量の相対値を示すグラフである。FIG. 1B is a graph showing the relative value of the expression level of periostin gene in vitro in Example 1 of the present invention. 図１Ｃは、本発明の実施例１におけるｉｎ　ｖｉｔｒｏでのペリオスチン遺伝子の発現量の相対値を示すグラフである。FIG. 1C is a graph showing the relative value of the expression level of periostin gene in vitro in Example 1 of the present invention. 図１Ｄは、本発明の実施例１におけるｉｎ　ｖｉｔｒｏでのペリオスチン遺伝子の発現量の相対値を示すグラフである。FIG. 1D is a graph showing the relative value of the expression level of periostin gene in vitro in Example 1 of the present invention. 図２は、本発明の実施例２におけるｉｎ　ｖｉｔｒｏでのペリオスチン遺伝子の発現量の相対値を示すグラフである。FIG. 2 is a graph showing the relative value of the expression level of the periostin gene in vitro in Example 2 of the present invention. 図３は、本発明の実施例４におけるペリオスチン遺伝子の発現量の相対値を示すグラフである。FIG. 3 is a graph showing the relative value of the expression level of the periostin gene in Example 4 of the present invention. 図４は、本発明の実施例４におけるタンパク質量を示すグラフである。FIG. 4 is a graph showing the amount of protein in Example 4 of the present invention. 図５は、本発明の実施例４におけるヒドロキシプロリン量を示すグラフである。FIG. 5 is a graph showing the amount of hydroxyproline in Example 4 of the present invention. 図６は、本発明の実施例４におけるマッソントリクローム染色の結果を示す写真であるFIG. 6 is a photograph showing the results of Masson trichrome staining in Example 4 of the present invention. 図７は、本発明の実施例４におけるアシュクロフトスコアの結果を示すグラフである。FIG. 7 is a graph showing the results of Ashcroft score in Example 4 of the present invention. 図８は、本発明の実施例５における癒着スコアの結果を示すグラフである。FIG. 8 is a graph showing the results of adhesion scores in Example 5 of the present invention. 図９は、本発明の実施例５におけるペリオスチン遺伝子の発現量の測定結果を示すグラフである。FIG. 9 is a graph showing the measurement results of the expression level of the periostin gene in Example 5 of the present invention. 図１０Ａは、本発明の実施例５における盲腸先端部の組織のアザン染色およびペリオスチン免疫染色の結果を示す写真である。FIG. 10A is a photograph showing the results of Azan staining and periostin immunostaining of the tissue at the tip of the cecum in Example 5 of the present invention. 図１０Ｂは、本発明の実施例５における盲腸先端部の組織のアザン染色およびペリオスチン免疫染色の結果を示す写真である。FIG. 10B is a photograph showing the results of Azan staining and periostin immunostaining of the cecal tip tissue in Example 5 of the present invention. 図１０Ｃは、本発明の実施例５における盲腸先端部の組織のアザン染色およびペリオスチン免疫染色の結果を示す写真である。FIG. 10C is a photograph showing the results of Azan staining and periostin immunostaining of the tissue at the tip of the cecum in Example 5 of the present invention. 図１０Ｄは、本発明の実施例５における盲腸先端部の組織のアザン染色およびペリオスチン免疫染色の結果を示す写真である。FIG. 10D is a photograph showing the results of Azan staining and periostin immunostaining of the tissue at the tip of the cecum in Example 5 of the present invention. 図１０Ｅは、本発明の実施例５における盲腸先端部の組織のアザン染色およびペリオスチン免疫染色の結果を示す写真である。FIG. 10E is a photograph showing the results of Azan staining and periostin immunostaining of the cecal tip tissue in Example 5 of the present invention. 図１０Ｆは、本発明の実施例５における盲腸先端部の組織のアザン染色およびペリオスチン免疫染色の結果を示す写真である。FIG. 10F is a photograph showing the results of Azan staining and periostin immunostaining of the tissue at the tip of the cecum in Example 5 of the present invention. 図１１は、本発明の実施例６におけるペリオスチン遺伝子の発現量の測定結果を示すグラフである。FIG. 11 is a graph showing the measurement results of the expression level of periostin gene in Example 6 of the present invention. 図１２は、本発明の実施例６における癒着スコアの結果を示すグラフである。FIG. 12 is a graph showing the results of adhesion scores in Example 6 of the present invention. 図１３Ａは、本発明の実施例６における盲腸先端部の組織のアザン染色およびペリオスチン免疫染色の結果を示す写真である。FIG. 13A is a photograph showing the results of Azan staining and periostin immunostaining of the tissue at the tip of the cecum in Example 6 of the present invention. 図１３Ｂは、本発明の実施例６における盲腸先端部の組織のアザン染色およびペリオスチン免疫染色の結果を示す写真である。FIG. 13B is a photograph showing the results of Azan staining and periostin immunostaining of the tissue at the tip of the cecum in Example 6 of the present invention. 図１４は、本発明の実施例７におけるｉｎ　ｖｉｔｒｏでのペリオスチン遺伝子の発現量の相対値を示すグラフである。FIG. 14 is a graph showing the relative value of the expression level of the periostin gene in vitro in Example 7 of the present invention. 図１５は、本発明の実施例７におけるｉｎ　ｖｉｔｒｏでのペリオスチン遺伝子の発現量の相対値を示すグラフである。FIG. 15 is a graph showing the relative value of the expression level of the periostin gene in vitro in Example 7 of the present invention.
　本明細書で使用する用語は、特に言及しない限り、当該技術分野で通常用いられる意味で用いることができる。 Unless otherwise stated, terms used in this specification can be used in the meaning normally used in the technical field.
＜発現抑制核酸分子＞
　本発明の発現抑制核酸分子（以下、「核酸分子」ともいう。）は、前述のように、ペリオスチン遺伝子の発現抑制核酸分子であり、
前記発現抑制核酸分子が、前記ペリオスチン遺伝子の発現抑制配列として、下記（ａｓ４）のヌクレオチドを含むことを特徴とする。
（ａｓ４）配列番号１２１～１２７のいずれか１つの塩基配列からなるポリヌクレオチドに対して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からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチド <Expression-suppressing nucleic acid molecule>
The expression-suppressing nucleic acid molecule of the present invention (hereinafter also referred to as “nucleic acid molecule”) is, as described above, a periostin gene expression-suppressing nucleic acid molecule,
The expression-suppressing nucleic acid molecule includes the following nucleotide (as4) as an expression-suppressing sequence for the periostin gene.
(As4) a nucleotide comprising a base sequence that hybridizes under stringent conditions to a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 121 to 127, and having a function of suppressing expression of a periostin gene
　以下、本発明の核酸分子について説明する。以下の説明において、前記（ａｓ４）のヌクレオチドを、ａｓ４ヌクレオチドともいう。 Hereinafter, the nucleic acid molecule of the present invention will be described. In the following description, the nucleotide (as4) is also referred to as an as4 nucleotide.
　本発明の核酸分子は、具体例として、前記ペリオスチン遺伝子の発現抑制配列として発現抑制用の核酸分子であり、ペリオスチン遺伝子の発現抑制配列として、下記（ａｓ１）、（ａｓ２）または（ａｓ３）のヌクレオチドを含む核酸分子があげられる。
（ａｓ１）配列番号１～１２０および２１７～２３２のいずれか一つの塩基配列からなるヌクレオチド
（ａｓ２）前記（ａｓ１）の塩基配列において、１もしくは数個の塩基が欠失、置換および／または付加された塩基配列からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチド
（ａｓ３）前記（ａｓ１）の塩基配列と８０％以上の同一性を有する塩基配列からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチド As a specific example, the nucleic acid molecule of the present invention is a nucleic acid molecule for suppressing expression as an expression suppressing sequence of the periostin gene, and the nucleotide of the following (as1), (as2) or (as3) is used as an expression suppressing sequence of the periostin gene And nucleic acid molecules containing
(As1) Nucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 1-120 and 217-232 (as2) In the nucleotide sequence of (as1), one or several bases are deleted, substituted and / or added. A nucleotide having a function to suppress expression of a periostin gene (as3) A nucleotide having a base sequence having 80% or more identity with the base sequence of (as1) and having a function to suppress expression of a periostin gene
　以下、前記（ａｓ１）、（ａｓ２）または（ａｓ３）のヌクレオチドを含む核酸分子について説明した上で、前記ａｓ４ヌクレオチドについて説明する。以下の説明において、前記（ａｓ１）、（ａｓ２）または（ａｓ３）のヌクレオチドを、ａｓヌクレオチドといい、それぞれ、ａｓ１ヌクレオチド、ａｓ２ヌクレオチド、ａｓ３ヌクレオチドともいう。 Hereinafter, the nucleic acid molecule containing the nucleotide (as1), (as2) or (as3) will be described, and then the as4 nucleotide will be described. In the following description, the nucleotides (as1), (as2) or (as3) are referred to as as nucleotides, and are also referred to as as1, nucleotides, and as3 nucleotides, respectively.
　本発明において、ペリオスチン遺伝子の発現の抑制は、特に制限されず、例えば、遺伝子の転写自体の抑制でも、遺伝子の転写産物を分解することによる抑制でもよい。また、ペリオスチン遺伝子の発現の抑制は、例えば、本来の機能を有するペリオスチンタンパク質の発現の抑制であってもよく、タンパク質が発現される場合、前記タンパク質は、例えば、前記機能が阻害されたタンパク質でもよいし、前記機能が欠失したタンパク質でもよい。 In the present invention, the suppression of periostin gene expression is not particularly limited, and may be, for example, suppression of gene transcription itself or suppression of degradation of a gene transcription product. Further, the suppression of periostin gene expression may be, for example, suppression of periostin protein expression having an original function. When protein is expressed, the protein may be, for example, a protein in which the function is inhibited. It may be a protein lacking the function.
　前記発現抑制配列は、例えば、前記ａｓ４ヌクレオチドからなる配列でもよいし、前記ａｓ４ヌクレオチドを含む配列でもよいし、前記ａｓ４ヌクレオチドの部分配列でもよい。また、前記ａｓ４ヌクレオチドが、前記ａｓ１ヌクレオチド、前記ａｓ２ヌクレオチドまたは前記ａｓ３ヌクレオチドの場合、前記発現抑制配列は、例えば、前記ａｓ１ヌクレオチド、前記ａｓ２ヌクレオチドまたは前記ａｓ３ヌクレオチドからなる配列でもよいし、前記ａｓ１ヌクレオチド、前記ａｓ２ヌクレオチドまたは前記ａｓ３ヌクレオチドを含む配列でもよいし、前記ａｓ１ヌクレオチド、前記ａｓ２ヌクレオチドまたは前記ａｓ３ヌクレオチドの部分配列でもよい。 The expression suppression sequence may be, for example, a sequence consisting of the as4 nucleotide, a sequence containing the as4 nucleotide, or a partial sequence of the as4 nucleotide. When the as4 nucleotide is the as1 nucleotide, the as2 nucleotide or the as3 nucleotide, the expression suppression sequence may be, for example, a sequence consisting of the as1 nucleotide, the as2 nucleotide or the as3 nucleotide, or the as1 nucleotide , A sequence containing the as2 nucleotide or the as3 nucleotide, or a partial sequence of the as1 nucleotide, the as2 nucleotide or the as3 nucleotide.
　前記発現抑制配列の長さは、特に制限されず、例えば、１０～４０塩基長、１１～３０塩基長、１２～２０塩基長である。本発明において、例えば、塩基数の数値範囲は、その範囲に属する正の整数を全て開示するものであり、例えば、「１～４塩基」との記載は、「１、２、３、４塩基」の全ての開示を意味する（以下、同様）。 The length of the expression suppression sequence is not particularly limited, and is, for example, 10 to 40 bases long, 11 to 30 bases long, or 12 to 20 bases long. In the present invention, for example, the numerical range of the number of bases discloses all positive integers belonging to the range. For example, the description “1 to 4 bases” includes “1, 2, 3, 4 bases”. "Means all disclosures (the same applies hereinafter).
　前記ａｓ１ヌクレオチドの配列を以下に示す。なお、前記配列番号１～１２０および２１７～２３２の塩基配列からなるヌクレオチド、前記ヌクレオチドからなる発現抑制配列、前記ヌクレオチドを含む発現抑制配列、および前記ヌクレオチドを含む核酸分子は、それぞれ、以下に示す名称（配列番号の前の名称）で表わすこともある。
Ｘ１３７０７（配列番号１）　5'-agggaATCATCTTGAgucuc-3'
Ｘ１３７０８（配列番号２）　5'-aagggAATCATCTTGagucu-3'
Ｘ１３７１０（配列番号３）　5'-cagcuGTACGATATAcgaag-3'
Ｘ１３７１１（配列番号４）　5'-cgaccCCTGATACGAcuaug-3'
Ｘ１３７１２（配列番号５）　5'-gacagCTGTACGATAuacga-3'
Ｘ１３７１４（配列番号６）　5'-agcugTACGATATACgaaga-3'
Ｘ１３７１５（配列番号７）　5'-agacaGCTGTACGATauacg-3'
Ｘ１３７１７（配列番号８）　5'-gacccCTGATACGACuauga-3'
Ｘ１３７１８（配列番号９）　5'-aauugGGCCACAAGAuccgu-3'
Ｘ１３７１９（配列番号１０）　5'-auuggGCCACAAGATccgug-3'
Ｘ１３７２０（配列番号１１）　5'-uaauuGGGCCACAAGauccg-3'
Ｘ１３７２１（配列番号１２）　5'-cucuaCGGATATCAGaaucc-3'
Ｘ１３７２２（配列番号１３）　5'-gcuacCACGAACAAAcuuaa-3'
Ｘ１３７２３（配列番号１４）　5'-gaaggTGCTACCACGaacaa-3'
Ｘ１３７２４（配列番号１５）　5'-cuaccACGAACAAACuuaau-3'
Ｘ１３７２５（配列番号１６）　5'-caaacCTCTACGGATaucag-3'
Ｘ１３７２６（配列番号１７）　5'-gcagaCAGCTGTACGauaua-3'
Ｘ１３７２７（配列番号１８）　5'-gaucuCGCGGAATATgugaa-3'
Ｘ１３７３０（配列番号１９）　5'-cagacAGCTGTACGAuauac-3'
Ｘ１３７３２（配列番号２０）　5'-ccuaaTTGGGCCACAagauc-3'
Ｘ１３７３４（配列番号２１）　5'-gagcaTTTTTGTCCCguauc-3'
Ｘ１３７３５（配列番号２２）　5'-ugaagGTGCTACCACgaaca-3'
Ｘ１３７３６（配列番号２３）　5'-uacggATATCAGAATccaag-3'
Ｘ１３７３７（配列番号２４）　5'-uugaaGGTGCTACCAcgaac-3'
Ｘ１３７３８（配列番号２５）　5'-auaccAGTTCTTACAagugc-3'
Ｘ１３７３９（配列番号２６）　5'-auccuTTCTAGGACAccucg-3'
Ｘ１３７４０（配列番号２７）　5'-accucTACGGATATCagaau-3'
Ｘ１３７４１（配列番号２８）　5'-ccucuACGGATATCAgaauc-3'
Ｘ１３７４２（配列番号２９）　5'-guaauTCAACATTCAcguug-3'
Ｘ１３７４３（配列番号３０）　5'-aggugCTACCACGAAcaaac-3'
Ｘ１３７４５（配列番号３１）　5'-ggauaGAGGAGTTTAucuac-3'
Ｘ１３７４６（配列番号３２）　5'-aggaaTTAGGACCTGaucaa-3'
Ｘ１３７４７（配列番号３３）　5'-ucaccGTCACATCCTaucuc-3'
Ｘ１３７４８（配列番号３４）　5'-caccgTCACATCCTAucuca-3'
Ｘ１３７４９（配列番号３５）　5'-uccuuTCTAGGACACcucgu-3'
Ｘ１３７５０（配列番号３６）　5'-ucuugAATTGAGGTAccaau-3'
Ｘ１３７５１（配列番号３７）　5'-guuguCCCAAGCCTCauuac-3'
Ｘ１３７５２（配列番号３８）　5'-ucaauGGGATAACAGuucac-3'
Ｘ１３７５３（配列番号３９）　5'-cauuuTTGTCCCGTAucaga-3'
Ｘ１３７５４（配列番号４０）　5'-uaggaCCTGATCAATcaaau-3'
Ｘ１３７５５（配列番号４１）　5'-agaauCAGGAATTAGgaccu-3'
Ｘ１３７５６（配列番号４２）　5'-augggATAACAGTTCacaac-3'
Ｘ１３７５７（配列番号４３）　5'-gaggaGTTTATCTACaacau-3'
Ｘ１３７５８（配列番号４４）　5'-gucauTCCCTTAAAAgcauc-3'
Ｘ１３７５９（配列番号４５）　5'-agaguATCATCAGAAaaugc-3'
Ｘ１３７６０（配列番号４６）　5'-ucuacAACATGAATTacacc-3'
Ｘ１３７６１（配列番号４７）　5'-gcuguACGATATACGaagac-3'
Ｘ１３７６２（配列番号４８）　5'-guacgATATACGAAGacucu-3'
Ｘ１３７６３（配列番号４９）　5'-aucucGCGGAATATGugaau-3'
Ｘ１３７６５（配列番号５０）　5'-gccuaATTGGGCCACaagau-3'
Ｘ１３７６６（配列番号５１）　5'-uacgaTATACGAAGAcucug-3'
Ｘ１３７６７（配列番号５２）　5'-augccAAGCCTAATTgggcc-3'
Ｘ１３７６８（配列番号５３）　5'-cuguaCGATATACGAagacu-3'
Ｘ１３７６９（配列番号５４）　5'-ccaaaCCTCTACGGAuauca-3'
Ｘ１３７７０（配列番号５５）　5'-uccaaACCTCTACGGauauc-3'
Ｘ１３７７１（配列番号５６）　5'-uguacGATATACGAAgacuc-3'
Ｘ１３７７２（配列番号５７）　5'-gugcuACCACGAACAaacuu-3'
Ｘ１３７７３（配列番号５８）　5'-cgauaTACGAAGACTcugag-3'
Ｘ１３７７４（配列番号５９）　5'-ccaagCCTCATTACTcggug-3'
Ｘ１３７７５（配列番号６０）　5'-ugcuaCCACGAACAAacuua-3'
Ｘ１３７７６（配列番号６１）　5'-gcucuCCAAACCTCTacgga-3'
Ｘ１３７７７（配列番号６２）　5'-ggugcTACCACGAACaaacu-3'
Ｘ１３７７８（配列番号６３）　5'-gauauACGAAGACTCugagc-3'
Ｘ１３７８０（配列番号６４）　5'-gucacCGTCACATCCuaucu-3'
Ｘ１３７８１（配列番号６５）　5'-cuaauTGGGCCACAAgaucc-3'
Ｘ１３７８３（配列番号６６）　5'-cugucACCGTCACATccuau-3'
Ｘ１３７８４（配列番号６７）　5'-cccaaGCCTCATTACucggu-3'
Ｘ１３７８５（配列番号６８）　5'-uaauuCAACATTCACguugc-3'
Ｘ１３７８６（配列番号６９）　5'-uaacaTACATACAAGgcucg-3'
Ｘ１３７８７（配列番号７０）　5'-aguaaACCCACTCATauaga-3'
Ｘ１３７８８（配列番号７１）　5'-accguCACATCCTATcucaa-3'
Ｘ１３７９１（配列番号７２）　5'-uuugaAGGTGCTACCacgaa-3'
Ｘ１３７９２（配列番号７３）　5'-gcucgGTCTTTTCAAuggga-3'
Ｘ１３７９３（配列番号７４）　5'-guaaaCCCACTCATAuagaa-3'
Ｘ１３７９５（配列番号７５）　5'-agcgcTTATCTTGTTuuaac-3'
Ｘ１３７９６（配列番号７６）　5'-ccugaTCAATCAAATggauc-3'
Ｘ１３７９７（配列番号７７）　5'-cuuucTAGGACACCTcgugg-3'
Ｘ１３７９８（配列番号７８）　5'-guacaCCAAGCACCTauuuu-3'
Ｘ１３７９９（配列番号７９）　5'-ugggcCACAAGATCCgugaa-3'
Ｘ１３８００（配列番号８０）　5'-gcauuTTTGTCCCGTaucag-3'
Ｘ１３８０２（配列番号８１）　5'-uauaaCTTAGCTTCCcaugg-3'
Ｘ１３８０３（配列番号８２）　5'-ggauaACAGTTCACAacucu-3'
Ｘ１３８０４（配列番号８３）　5'-gaaguCTTGAATTGAgguac-3'
Ｘ１３８０５（配列番号８４）　5'-caguaATTCAACATTcacgu-3'
Ｘ１３８０６（配列番号８５）　5'-ccaguAAACCCACTCauaua-3'
Ｘ１３８０７（配列番号８６）　5'-gaauuGAGGTACCAAuuugu-3'
Ｘ１３８０８（配列番号８７）　5'-uuguaATGAATCTGGugaca-3'
Ｘ１３８０９（配列番号８８）　5'-gaucaCACCATTATTuguca-3'
Ｘ１３８１０（配列番号８９）　5'-uguaaTGAATCTGGTgacaa-3'
Ｘ１３８１１（配列番号９０）　5'-gcacaAATAATGTCCagucu-3'
Ｘ１３８１２（配列番号９１）　5'-acacgGTCAATGACAuggac-3'
Ｘ１３８１３（配列番号９２）　5'-uggauAGAGGAGTTTaucua-3'
Ｘ１３８１４（配列番号９３）　5'-guaauGAATCTGGTGacaag-3'
Ｘ１３８１５（配列番号９４）　5'-gucccGTATCAGAATuucuu-3'
Ｘ１３８１６（配列番号９５）　5'-ccaagCCTAATTGGGccaca-3'
Ｘ１３８１７（配列番号９６）　5'-gcucaTTAAGGCCAAcuuuu-3'
Ｘ１３８１８（配列番号９７）　5'-gauccTTTCTAGGACaccuc-3'
Ｘ１３８１９（配列番号９８）　5'-acgaaCAAACTTAATuugga-3'
Ｘ１３８２０（配列番号９９）　5'-aauucAACATTCACGuugcu-3'
Ｘ１３８２１（配列番号１００）　5'-ggcacAAATAATGTCcaguc-3'
Ｘ１３８２２（配列番号１０１）　5'-auucaACATTCACGTugcuc-3'
Ｘ１３８２３（配列番号１０２）　5'-ucccgACCCCTGATAcgacu-3'
Ｘ１３８２４（配列番号１０３）　5'-ugucaCCGTCACATCcuauc-3'
Ｘ１３８２５（配列番号１０４）　5'-acgauATACGAAGACucuga-3'
Ｘ１３８２６（配列番号１０５）　5'-ucuccAAACCTCTACggaua-3'
Ｘ１３８２７（配列番号１０６）　5'-ugccaAGCCTAATTGggcca-3'
Ｘ１３４６４（配列番号１０７）　5'-uuuccAGCCAGCTCAauaac-3'
Ｘ１３４６５（配列番号１０８）　5'-gccagCTCAATAACTuguuu-3'
Ｘ１３４６６（配列番号１０９）　5'-agccaGCTCAATAACuuguu-3'
Ｘ１３４６７（配列番号１１０）　5'-gucauGATGTCAGATucuuu-3'
Ｘ１３４６９（配列番号１１１）　5'-uuccaGCCAGCTCAAuaacu-3'
Ｘ１３４７０（配列番号１１２）　5'-guuuuCCAGCCAGCTcaaua-3'
Ｘ１３４７１（配列番号１１３）　5'-uuuucCAGCCAGCTCaauaa-3'
Ｘ１３４７３（配列番号１１４）　5'-agccaCTTTGTCTCCcauga-3'
Ｘ１３４７４（配列番号１１５）　5'-gugccATAAACATGGucaau-3'
Ｘ１３４７６（配列番号１１６）　5'-uccagCCAGCTCAATaacuu-3'
Ｘ１３４７７（配列番号１１７）　5'-uguuuTCCAGCCAGCucaau-3'
Ｘ１３４７８（配列番号１１８）　5'-cagccAGCTCAATAAcuugu-3'
Ｘ１３４７９（配列番号１１９）　5'-ccagcCAGCTCAATAacuug-3'
Ｘ１３４８０（配列番号１２０）　5'-gggauTTCTTTGAAGgugcu-3'
Ｘ１７７５２（配列番号２１７）　5'-caccaCTGTTCGTAAuuugg-3'
Ｘ２２８１６（配列番号２１８）　5'-gcucggTCTTTTCAauggga-3'
Ｘ１６２５６（配列番号２１９）　5'-gcucgGTCTTTTCAAuggga-3'
Ｘ２２８１７（配列番号２２０）　5'-gcucGGTCTTTTCAATggga-3'
Ｘ２２８１８（配列番号２２１）　5'-gcuCGGTCTTTTCAATGgga-3'
Ｘ２２８２２（配列番号２２２）　5'-cucgGTCTTTTCAAuggg-3'
Ｘ２２８２３（配列番号２２３）　5'-cucGGTCTTTTCAATggg-3'
Ｌ－５１０５（配列番号２２４）　5'-gctcgGTCTTTTCAAtggga-3'
Ｌ－４１２４（配列番号２２５）　5'-gctcGGTCTTTTCAATggga-3'
Ｌ－３１４３（配列番号２２６）　5'-gctCGGTCTTTTCAATGgga-3'
Ｌ－４１０４（配列番号２２７）　5'-ctcgGTCTTTTCAAtggg-3'
Ｌ－３１２３（配列番号２２８）　5'-ctcGGTCTTTTCAATggg-3'
Ｌ－２１４２（配列番号２２９）　5'-ctCGGTCTTTTCAATGgg-3'
Ｌ－４８４　（配列番号２３０）　5'-tcggTCTTTTCAatgg-3'
Ｌ－３１０３（配列番号２３１）　5'-tcgGTCTTTTCAAtgg-3'
Ｌ－３７３　（配列番号２３２）　5'-ggtCTTTTCAatg-3' The sequence of the as1 nucleotide is shown below. The nucleotides consisting of the base sequences of SEQ ID NOs: 1-120 and 217-232, the expression suppressing sequence consisting of the nucleotides, the expression suppressing sequence containing the nucleotides, and the nucleic acid molecules containing the nucleotides are named as follows: (Name before the sequence number).
X13707 (SEQ ID NO: 1) 5'-agggaATCATCTTGAgucuc-3 '
X13708 (SEQ ID NO: 2) 5'-aagggAATCATCTTGagucu-3 '
X13710 (SEQ ID NO: 3) 5'-cagcuGTACGATATAcgaag-3 '
X13711 (SEQ ID NO: 4) 5'-cgaccCCTGATACGAcuaug-3 '
X13712 (SEQ ID NO: 5) 5'-gacagCTGTACGATAuacga-3 '
X13714 (SEQ ID NO: 6) 5'-agcugTACGATATACgaaga-3 '
X13715 (SEQ ID NO: 7) 5'-agacaGCTGTACGATauacg-3 '
X13717 (sequence number 8) 5'-gacccCTGATACGACuauga-3 '
X13718 (SEQ ID NO: 9) 5'-aauugGGCCACAAGAuccgu-3 '
X13719 (SEQ ID NO: 10) 5'-auuggGCCACAAGATccgug-3 '
X13720 (SEQ ID NO: 11) 5'-uaauuGGGCCACAAGauccg-3 '
X13721 (SEQ ID NO: 12) 5'-cucuaCGGATATCAGaaucc-3 '
X13722 (SEQ ID NO: 13) 5'-gcuacCACGAACAAAcuuaa-3 '
X13723 (SEQ ID NO: 14) 5'-gaaggTGCTACCACGaacaa-3 '
X13724 (SEQ ID NO: 15) 5'-cuaccACGAACAAACuuaau-3 '
X13725 (SEQ ID NO: 16) 5'-caaacCTCTACGGATaucag-3 '
X13726 (SEQ ID NO: 17) 5'-gcagaCAGCTGTACGauaua-3 '
X13727 (SEQ ID NO: 18) 5'-gaucuCGCGGAATATgugaa-3 '
X13730 (SEQ ID NO: 19) 5'-cagacAGCTGTACGAuauac-3 '
X13732 (SEQ ID NO: 20) 5'-ccuaaTTGGGCCACAagauc-3 '
X13734 (SEQ ID NO: 21) 5'-gagcaTTTTTGTCCCguauc-3 '
X13735 (SEQ ID NO: 22) 5'-ugaagGTGCTACCACgaaca-3 '
X13736 (SEQ ID NO: 23) 5'-uacggATATCAGAATccaag-3 '
X13737 (SEQ ID NO: 24) 5'-uugaaGGTGCTACCAcgaac-3 '
X13738 (SEQ ID NO: 25) 5′-auaccAGTTCTTACAagugc-3 ′
X13739 (SEQ ID NO: 26) 5'-auccuTTCTAGGACAccucg-3 '
X13740 (SEQ ID NO: 27) 5'-accucTACGGATATCagaau-3 '
X13741 (SEQ ID NO: 28) 5'-ccucuACGGATATCAgaauc-3 '
X13742 (SEQ ID NO: 29) 5'-guaauTCAACATTCAcguug-3 '
X13743 (SEQ ID NO: 30) 5'-aggugCTACCACGAAcaaac-3 '
X13745 (SEQ ID NO: 31) 5'-ggauaGAGGAGTTTAucuac-3 '
X13746 (SEQ ID NO: 32) 5'-aggaaTTAGGACCTGaucaa-3 '
X13747 (SEQ ID NO: 33) 5'-ucaccGTCACATCCTaucuc-3 '
X13748 (SEQ ID NO: 34) 5'-caccgTCACATCCTAucuca-3 '
X13749 (SEQ ID NO: 35) 5'-uccuuTCTAGGACACcucgu-3 '
X13750 (SEQ ID NO: 36) 5'-ucuugAATTGAGGTAccaau-3 '
X13751 (SEQ ID NO: 37) 5'-guuguCCCAAGCCTCauuac-3 '
X13752 (SEQ ID NO: 38) 5'-ucaauGGGATAACAGuucac-3 '
X13753 (SEQ ID NO: 39) 5'-cauuuTTGTCCCGTAucaga-3 '
X13754 (SEQ ID NO: 40) 5'-uaggaCCTGATCAATcaaau-3 '
X13755 (SEQ ID NO: 41) 5'-agaauCAGGAATTAGgaccu-3 '
X13756 (SEQ ID NO: 42) 5'-augggATAACAGTTCacaac-3 '
X13757 (SEQ ID NO: 43) 5'-gaggaGTTTATCTACaacau-3 '
X13758 (SEQ ID NO: 44) 5'-gucauTCCCTTAAAAgcauc-3 '
X13759 (SEQ ID NO: 45) 5'-agaguATCATCAGAAaaugc-3 '
X13760 (SEQ ID NO: 46) 5'-ucuacAACATGAATTacacc-3 '
X13761 (SEQ ID NO: 47) 5'-gcuguACGATATACGaagac-3 '
X13762 (SEQ ID NO: 48) 5'-guacgATATACGAAGacucu-3 '
X13763 (SEQ ID NO: 49) 5'-aucucGCGGAATATGugaau-3 '
X13765 (SEQ ID NO: 50) 5'-gccuaATTGGGCCACaagau-3 '
X13766 (SEQ ID NO: 51) 5'-uacgaTATACGAAGAcucug-3 '
X13767 (SEQ ID NO: 52) 5'-augccAAGCCTAATTgggcc-3 '
X13768 (SEQ ID NO: 53) 5'-cuguaCGATATACGAagacu-3 '
X13769 (SEQ ID NO: 54) 5'-ccaaaCCTCTACGGAuauca-3 '
X13770 (SEQ ID NO: 55) 5'-uccaaACCTCTACGGauauc-3 '
X13771 (SEQ ID NO: 56) 5'-uguacGATATACGAAgacuc-3 '
X13772 (SEQ ID NO: 57) 5'-gugcuACCACGAACAaacuu-3 '
X13773 (SEQ ID NO: 58) 5'-cgauaTACGAAGACTcugag-3 '
X13774 (SEQ ID NO: 59) 5'-ccaagCCTCATTACTcggug-3 '
X13775 (SEQ ID NO: 60) 5'-ugcuaCCACGAACAAacuua-3 '
X13776 (SEQ ID NO: 61) 5'-gcucuCCAAACCTCTacgga-3 '
X13777 (SEQ ID NO: 62) 5'-ggugcTACCACGAACaaacu-3 '
X13778 (SEQ ID NO: 63) 5′-gauauACGAAGACTCugagc-3 ′
X13780 (SEQ ID NO: 64) 5'-gucacCGTCACATCCuaucu-3 '
X13781 (SEQ ID NO: 65) 5'-cuaauTGGGCCACAAgaucc-3 '
X13783 (SEQ ID NO: 66) 5'-cugucACCGTCACATccuau-3 '
X13784 (SEQ ID NO: 67) 5'-cccaaGCCTCATTACucggu-3 '
X13785 (SEQ ID NO: 68) 5'-uaauuCAACATTCACguugc-3 '
X13786 (SEQ ID NO: 69) 5'-uaacaTACATACAAGgcucg-3 '
X13787 (SEQ ID NO: 70) 5'-aguaaACCCACTCATauaga-3 '
X13788 (SEQ ID NO: 71) 5'-accguCACATCCTATcucaa-3 '
X13791 (SEQ ID NO: 72) 5'-uuugaAGGTGCTACCacgaa-3 '
X13792 (SEQ ID NO: 73) 5'-gcucgGTCTTTTCAAuggga-3 '
X13793 (SEQ ID NO: 74) 5'-guaaaCCCACTCATAuagaa-3 '
X13795 (SEQ ID NO: 75) 5'-agcgcTTATCTTGTTuuaac-3 '
X13796 (SEQ ID NO: 76) 5'-ccugaTCAATCAAATggauc-3 '
X13797 (SEQ ID NO: 77) 5'-cuuucTAGGACACCTcgugg-3 '
X13798 (SEQ ID NO: 78) 5'-guacaCCAAGCACCTauuuu-3 '
X13799 (SEQ ID NO: 79) 5'-ugggcCACAAGATCCgugaa-3 '
X13800 (SEQ ID NO: 80) 5'-gcauuTTTGTCCCGTaucag-3 '
X13802 (SEQ ID NO: 81) 5'-uauaaCTTAGCTTCCcaugg-3 '
X13803 (SEQ ID NO: 82) 5'-ggauaACAGTTCACAacucu-3 '
X13804 (SEQ ID NO: 83) 5'-gaaguCTTGAATTGAgguac-3 '
X13805 (SEQ ID NO: 84) 5'-caguaATTCAACATTcacgu-3 '
X13806 (SEQ ID NO: 85) 5'-ccaguAAACCCACTCauaua-3 '
X13807 (SEQ ID NO: 86) 5'-gaauuGAGGTACCAAuuugu-3 '
X13808 (SEQ ID NO: 87) 5'-uuguaATGAATCTGGugaca-3 '
X13809 (SEQ ID NO: 88) 5'-gaucaCACCATTATTuguca-3 '
X13810 (SEQ ID NO: 89) 5'-uguaaTGAATCTGGTgacaa-3 '
X13811 (SEQ ID NO: 90) 5'-gcacaAATAATGTCCagucu-3 '
X13812 (SEQ ID NO: 91) 5'-acacgGTCAATGACAuggac-3 '
X13813 (SEQ ID NO: 92) 5'-uggauAGAGGAGTTTaucua-3 '
X13814 (SEQ ID NO: 93) 5'-guaauGAATCTGGTGacaag-3 '
X13815 (SEQ ID NO: 94) 5'-gucccGTATCAGAATuucuu-3 '
X13816 (SEQ ID NO: 95) 5'-ccaagCCTAATTGGGccaca-3 '
X13817 (SEQ ID NO: 96) 5'-gcucaTTAAGGCCAAcuuuu-3 '
X13818 (SEQ ID NO: 97) 5'-gauccTTTCTAGGACaccuc-3 '
X13819 (SEQ ID NO: 98) 5'-acgaaCAAACTTAATuugga-3 '
X13820 (SEQ ID NO: 99) 5'-aauucAACATTCACGuugcu-3 '
X13821 (SEQ ID NO: 100) 5'-ggcacAAATAATGTCcaguc-3 '
X13822 (SEQ ID NO: 101) 5'-auucaACATTCACGTugcuc-3 '
X13823 (SEQ ID NO: 102) 5'-ucccgACCCCTGATAcgacu-3 '
X13824 (SEQ ID NO: 103) 5'-ugucaCCGTCACATCcuauc-3 '
X13825 (sequence number 104) 5'-acgauATACGAAGACucuga-3 '
X13826 (SEQ ID NO: 105) 5'-ucuccAAACCTCTACggaua-3 '
X13827 (SEQ ID NO: 106) 5'-ugccaAGCCTAATTGggcca-3 '
X13464 (SEQ ID NO: 107) 5'-uuuccAGCCAGCTCAauaac-3 '
X13465 (SEQ ID NO: 108) 5'-gccagCTCAATAACTuguuu-3 '
X13466 (sequence number 109) 5'-agccaGCTCAATAACuuguu-3 '
X13467 (SEQ ID NO: 110) 5'-gucauGATGTCAGATucuuu-3 '
X13469 (SEQ ID NO: 111) 5'-uuccaGCCAGCTCAAuaacu-3 '
X13470 (SEQ ID NO: 112) 5′-guuuuCCAGCCAGCTcaaua-3 ′
X13471 (SEQ ID NO: 113) 5'-uuuucCAGCCAGCTCaauaa-3 '
X13473 (sequence number 114) 5'-agccaCTTTGTCTCCcauga-3 '
X13474 (SEQ ID NO: 115) 5'-gugccATAAACATGGucaau-3 '
X13476 (SEQ ID NO: 116) 5'-uccagCCAGCTCAATaacuu-3 '
X13477 (SEQ ID NO: 117) 5'-uguuuTCCAGCCAGCucaau-3 '
X13478 (sequence number 118) 5'-cagccAGCTCAATAAcuugu-3 '
X13479 (SEQ ID NO: 119) 5'-ccagcCAGCTCAATAacuug-3 '
X13480 (SEQ ID NO: 120) 5'-gggauTTCTTTGAAGgugcu-3 '
X17752 (SEQ ID NO: 217) 5'-caccaCTGTTCGTAAuuugg-3 '
X22816 (SEQ ID NO: 218) 5'-gcucggTCTTTTCAauggga-3 '
X16256 (SEQ ID NO: 219) 5'-gcucgGTCTTTTCAAuggga-3 '
X22817 (SEQ ID NO: 220) 5'-gcucGGTCTTTTCAATggga-3 '
X22818 (SEQ ID NO: 221) 5'-gcuCGGTCTTTTCAATGgga-3 '
X22822 (SEQ ID NO: 222) 5'-cucgGTCTTTTCAAuggg-3 '
X22823 (SEQ ID NO: 223) 5'-cucGGTCTTTTCAATggg-3 '
L-5105 (SEQ ID NO: 224) 5'-gctcgGTCTTTTCAAtggga-3 '
L-4124 (SEQ ID NO: 225) 5'-gctcGGTCTTTTCAATggga-3 '
L-3143 (SEQ ID NO: 226) 5'-gctCGGTCTTTTCAATGgga-3 '
L-4104 (SEQ ID NO: 227) 5'-ctcgGTCTTTTCAAtggg-3 '
L-3123 (SEQ ID NO: 228) 5'-ctcGGTCTTTTCAATggg-3 '
L-2142 (SEQ ID NO: 229) 5'-ctCGGTCTTTTCAATGgg-3 '
L-484 (SEQ ID NO: 230) 5'-tcggTCTTTTCAatgg-3 '
L-3103 (SEQ ID NO: 231) 5'-tcgGTCTTTTCAAtgg-3 '
L-373 (SEQ ID NO: 232) 5'-ggtCTTTTCAatg-3 '
　前記ａｓ２ヌクレオチドにおいて、「１もしくは数個」は、特に制限されない。「１もしくは数個」は、例えば、１～７個、１～５個、１～４個、１個、２個または３個である。前記ａｓ２ヌクレオチドは、例えば、前記ａｓ１ヌクレオチドと同様の機能を有していればよく、より詳細には、ペリオスチン遺伝子の発現抑制機能を有していればよい。「および／または」とは、少なくともいずれか１つの意味であり、「からなる群から選択された少なくとも１つの」と表すこともできる（以下、同様）。 In the as2 nucleotide, “1 or several” is not particularly limited. “One or several” is, for example, 1 to 7, 1 to 5, 1 to 4, 1, 2, or 3. For example, the as2 nucleotide only needs to have the same function as the as1 nucleotide, and more specifically, it only needs to have a function to suppress the expression of the periostin gene. “And / or” means at least one of the meanings, and can also be expressed as “at least one selected from the group consisting of” (hereinafter the same).
　前記ａｓ３ヌクレオチドにおいて、同一性は、例えば、８０％以上、８５％以上、９０％以上、９３％以上、９４％以上、９５％以上、９６％以上、９７％以上、９８％以上、９９％以上である。前記ａｓ３ヌクレオチドは、例えば、前記ａｓ１ヌクレオチドと同様の機能を有していればよく、より詳細には、ペリオスチン遺伝子の発現抑制機能を有していればよい。前記同一性は、例えば、ＢＬＡＳＴ、ＦＡＳＴＡ等の解析ソフトウェアを用いて、デフォルトのパラメータにより算出できる（以下、同様）。 In the as3 nucleotide, the identity is, for example, 80% or more, 85% or more, 90% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more. It is. For example, the as3 nucleotide only needs to have the same function as the as1 nucleotide, and more specifically, it only needs to have a function to suppress the expression of the periostin gene. The identity can be calculated with default parameters using analysis software such as BLAST and FASTA (hereinafter the same).
　本発明の核酸分子の全長は、特に制限されず、例えば、前記発現抑制配列の長さの説明を援用できる。 The total length of the nucleic acid molecule of the present invention is not particularly limited, and for example, description of the length of the expression suppressing sequence can be cited.
　つぎに、前記ａｓ４ヌクレオチドについて説明する。 Next, the as4 nucleotide will be described.
　前記ａｓ４ヌクレオチドにおける配列番号１２１の塩基配列は、以下の通りである。配列番号１２１の塩基配列は、ペリオスチン遺伝子のｍＲＮＡ　ｖａｒｉａｎｔ　１の塩基配列である。配列番号１２１のポリヌクレオチドは、例えば、Ｈｏｍｏ　ｓａｐｉｅｎｓから得ることができ、例えば、ＧｅｎｅＢａｎｋにアクセッションＮｏ．ＮＭ＿００６４７５．２で登録されているポリヌクレオチドがあげられる。 The base sequence of SEQ ID NO: 121 in the as4 nucleotide is as follows. The base sequence of SEQ ID NO: 121 is the base sequence of mRNA variant 1 of the periostin gene. The polynucleotide of SEQ ID NO: 121 can be obtained, for example, from Homo sapiens . For example, GeneBank has accession no. The polynucleotide registered by NM_006475.2 is mentioned.
ヒトペリオスチン遺伝子ｍＲＮＡ　ｖａｒｉａｎｔ　１（配列番号１２１）
5'-AGACTCTCAGGTTGATGCAGTGTTCCCTCCCACAACTCTGACATGTATATAAATTCTGAGCTCTCCAAAGCCCACTGCCAGTTCTCTTCGGGGACTAACTGCAACGGAGAGACTCAAGATGATTCCCTTTTTACCCATGTTTTCTCTACTATTGCTGCTTATTGTTAACCCTATAAACGCCAACAATCATTATGACAAGATCTTGGCTCATAGTCGTATCAGGGGTCGGGACCAAGGCCCAAATGTCTGTGCCCTTCAACAGATTTTGGGCACCAAAAAGAAATACTTCAGCACTTGTAAGAACTGGTATAAAAAGTCCATCTGTGGACAGAAAACGACTGTGTTATATGAATGTTGCCCTGGTTATATGAGAATGGAAGGAATGAAAGGCTGCCCAGCAGTTTTGCCCATTGACCATGTTTATGGCACTCTGGGCATCGTGGGAGCCACCACAACGCAGCGCTATTCTGACGCCTCAAAACTGAGGGAGGAGATCGAGGGAAAGGGATCCTTCACTTACTTTGCACCGAGTAATGAGGCTTGGGACAACTTGGATTCTGATATCCGTAGAGGTTTGGAGAGCAACGTGAATGTTGAATTACTGAATGCTTTACATAGTCACATGATTAATAAGAGAATGTTGACCAAGGACTTAAAAAATGGCATGATTATTCCTTCAATGTATAACAATTTGGGGCTTTTCATTAACCATTATCCTAATGGGGTTGTCACTGTTAATTGTGCTCGAATCATCCATGGGAACCAGATTGCAACAAATGGTGTTGTCCATGTCATTGACCGTGTGCTTACACAAATTGGTACCTCAATTCAAGACTTCATTGAAGCAGAAGATGACCTTTCATCTTTTAGAGCAGCTGCCATCACATCGGACATATTGGAGGCCCTTGGAAGAGACGGTCACTTCACACTCTTTGCTCCCACCAATGAGGCTTTTGAGAAACTTCCACGAGGTGTCCTAGAAAGGATCATGGGAGACAAAGTGGCTTCCGAAGCTCTTATGAAGTACCACATCTTAAATACTCTCCAGTGTTCTGAGTCTATTATGGGAGGAGCAGTCTTTGAGACGCTGGAAGGAAATACAATTGAGATAGGATGTGACGGTGACAGTATAACAGTAAATGGAATCAAAATGGTGAACAAAAAGGATATTGTGACAAATAATGGTGTGATCCATTTGATTGATCAGGTCCTAATTCCTGATTCTGCCAAACAAGTTATTGAGCTGGCTGGAAAACAGCAAACCACCTTCACGGATCTTGTGGCCCAATTAGGCTTGGCATCTGCTCTGAGGCCAGATGGAGAATACACTTTGCTGGCACCTGTGAATAATGCATTTTCTGATGATACTCTCAGCATGGATCAGCGCCTCCTTAAATTAATTCTGCAGAATCACATATTGAAAGTAAAAGTTGGCCTTAATGAGCTTTACAACGGGCAAATACTGGAAACCATCGGAGGCAAACAGCTCAGAGTCTTCGTATATCGTACAGCTGTCTGCATTGAAAATTCATGCATGGAGAAAGGGAGTAAGCAAGGGAGAAACGGTGCGATTCACATATTCCGCGAGATCATCAAGCCAGCAGAGAAATCCCTCCATGAAAAGTTAAAACAAGATAAGCGCTTTAGCACCTTCCTCAGCCTACTTGAAGCTGCAGACTTGAAAGAGCTCCTGACACAACCTGGAGACTGGACATTATTTGTGCCAACCAATGATGCTTTTAAGGGAATGACTAGTGAAGAAAAAGAAATTCTGATACGGGACAAAAATGCTCTTCAAAACATCATTCTTTATCACCTGACACCAGGAGTTTTCATTGGAAAAGGATTTGAACCTGGTGTTACTAACATTTTAAAGACCACACAAGGAAGCAAAATCTTTCTGAAAGAAGTAAATGATACACTTCTGGTGAATGAATTGAAATCAAAAGAATCTGACATCATGACAACAAATGGTGTAATTCATGTTGTAGATAAACTCCTCTATCCAGCAGACACACCTGTTGGAAATGATCAACTGCTGGAAATACTTAATAAATTAATCAAATACATCCAAATTAAGTTTGTTCGTGGTAGCACCTTCAAAGAAATCCCCGTGACTGTCTATACAACTAAAATTATAACCAAAGTTGTGGAACCAAAAATTAAAGTGATTGAAGGCAGTCTTCAGCCTATTATCAAAACTGAAGGACCCACACTAACAAAAGTCAAAATTGAAGGTGAACCTGAATTCAGACTGATTAAAGAAGGTGAAACAATAACTGAAGTGATCCATGGAGAGCCAATTATTAAAAAATACACCAAAATCATTGATGGAGTGCCTGTGGAAATAACTGAAAAAGAGACACGAGAAGAACGAATCATTACAGGTCCTGAAATAAAATACACTAGGATTTCTACTGGAGGTGGAGAAACAGAAGAAACTCTGAAGAAATTGTTACAAGAAGAGGTCACCAAGGTCACCAAATTCATTGAAGGTGGTGATGGTCATTTATTTGAAGATGAAGAAATTAAAAGACTGCTTCAGGGAGACACACCCGTGAGGAAGTTGCAAGCCAACAAAAAAGTTCAAGGATCTAGAAGACGATTAAGGGAAGGTCGTTCTCAGTGAAAATCCAAAAACCAGAAAAAAATGTTTATACAACCCTAAGTCAATAACCTGACCTTAGAAAATTGTGAGAGCCAAGTTGACTTCAGGAACTGAAACATCAGCACAAAGAAGCAATCATCAAATAATTCTGAACACAAATTTAATATTTTTTTTTCTGAATGAGAAACATGAGGGAAATTGTGGAGTTAGCCTCCTGTGGTAAAGGAATTGAAGAAAATATAACACCTTACACCCTTTTTCATCTTGACATTAAAAGTTCTGGCTAACTTTGGAATCCATTAGAGAAAAATCCTTGTCACCAGATTCATTACAATTCAAATCGAAGAGTTGTGAACTGTTATCCCATTGAAAAGACCGAGCCTTGTATGTATGTTATGGATACATAAAATGCACGCAAGCCATTATCTCTCCATGGGAAGCTAAGTTATAAAAATAGGTGCTTGGTGTACAAAACTTTTTATATCAAAAGGCTTTGCACATTTCTATATGAGTGGGTTTACTGGTAAATTATGTTATTTTTTACAACTAATTTTGTACTCTCAGAATGTTTGTCATATGCTTCTTGCAATGCATATTTTTTAATCTCAAACGTTTCAATAAAACCATTTTTCAGATATAAAGAGAATTACTTCAAATTGAGTAATTCAGAAAAACTCAAGATTTAAGTTAAAAAGTGGTTTGGACTTGGGAACAGGACTTTATACCTCTTTTACTGTAACAAGTACTCATTAAAGGAAATTGAATGAAATTAAAAAAAAAAA-3' Human periostin gene mRNA variant 1 (SEQ ID NO: 121)
5 '-3'
　前記ａｓ４ヌクレオチドにおける配列番号１２２の塩基配列は、以下の通りである。配列番号１２２の塩基配列は、ペリオスチン遺伝子のｍＲＮＡ　ｖａｒｉａｎｔ　２の塩基配列である。配列番号１２２のポリヌクレオチドは、例えば、Ｈｏｍｏ　ｓａｐｉｅｎｓから得ることができ、例えば、ＧｅｎｅＢａｎｋにアクセッションＮｏ．ＮＭ＿００１１３５９３４．１で登録されているポリヌクレオチドがあげられる。 The base sequence of SEQ ID NO: 122 in the as4 nucleotide is as follows. The base sequence of SEQ ID NO: 122 is the base sequence of mRNA variant 2 of the periostin gene. The polynucleotide of SEQ ID NO: 122 can be obtained, for example, from Homo sapiens , for example, accession No. 1 to GeneBank. Examples thereof include polynucleotides registered under NM_001135934.1.
ヒトペリオスチン遺伝子ｍＲＮＡ　ｖａｒｉａｎｔ　２（配列番号１２２）
5'-AGACTCTCAGGTTGATGCAGTGTTCCCTCCCACAACTCTGACATGTATATAAATTCTGAGCTCTCCAAAGCCCACTGCCAGTTCTCTTCGGGGACTAACTGCAACGGAGAGACTCAAGATGATTCCCTTTTTACCCATGTTTTCTCTACTATTGCTGCTTATTGTTAACCCTATAAACGCCAACAATCATTATGACAAGATCTTGGCTCATAGTCGTATCAGGGGTCGGGACCAAGGCCCAAATGTCTGTGCCCTTCAACAGATTTTGGGCACCAAAAAGAAATACTTCAGCACTTGTAAGAACTGGTATAAAAAGTCCATCTGTGGACAGAAAACGACTGTGTTATATGAATGTTGCCCTGGTTATATGAGAATGGAAGGAATGAAAGGCTGCCCAGCAGTTTTGCCCATTGACCATGTTTATGGCACTCTGGGCATCGTGGGAGCCACCACAACGCAGCGCTATTCTGACGCCTCAAAACTGAGGGAGGAGATCGAGGGAAAGGGATCCTTCACTTACTTTGCACCGAGTAATGAGGCTTGGGACAACTTGGATTCTGATATCCGTAGAGGTTTGGAGAGCAACGTGAATGTTGAATTACTGAATGCTTTACATAGTCACATGATTAATAAGAGAATGTTGACCAAGGACTTAAAAAATGGCATGATTATTCCTTCAATGTATAACAATTTGGGGCTTTTCATTAACCATTATCCTAATGGGGTTGTCACTGTTAATTGTGCTCGAATCATCCATGGGAACCAGATTGCAACAAATGGTGTTGTCCATGTCATTGACCGTGTGCTTACACAAATTGGTACCTCAATTCAAGACTTCATTGAAGCAGAAGATGACCTTTCATCTTTTAGAGCAGCTGCCATCACATCGGACATATTGGAGGCCCTTGGAAGAGACGGTCACTTCACACTCTTTGCTCCCACCAATGAGGCTTTTGAGAAACTTCCACGAGGTGTCCTAGAAAGGATCATGGGAGACAAAGTGGCTTCCGAAGCTCTTATGAAGTACCACATCTTAAATACTCTCCAGTGTTCTGAGTCTATTATGGGAGGAGCAGTCTTTGAGACGCTGGAAGGAAATACAATTGAGATAGGATGTGACGGTGACAGTATAACAGTAAATGGAATCAAAATGGTGAACAAAAAGGATATTGTGACAAATAATGGTGTGATCCATTTGATTGATCAGGTCCTAATTCCTGATTCTGCCAAACAAGTTATTGAGCTGGCTGGAAAACAGCAAACCACCTTCACGGATCTTGTGGCCCAATTAGGCTTGGCATCTGCTCTGAGGCCAGATGGAGAATACACTTTGCTGGCACCTGTGAATAATGCATTTTCTGATGATACTCTCAGCATGGATCAGCGCCTCCTTAAATTAATTCTGCAGAATCACATATTGAAAGTAAAAGTTGGCCTTAATGAGCTTTACAACGGGCAAATACTGGAAACCATCGGAGGCAAACAGCTCAGAGTCTTCGTATATCGTACAGCTGTCTGCATTGAAAATTCATGCATGGAGAAAGGGAGTAAGCAAGGGAGAAACGGTGCGATTCACATATTCCGCGAGATCATCAAGCCAGCAGAGAAATCCCTCCATGAAAAGTTAAAACAAGATAAGCGCTTTAGCACCTTCCTCAGCCTACTTGAAGCTGCAGACTTGAAAGAGCTCCTGACACAACCTGGAGACTGGACATTATTTGTGCCAACCAATGATGCTTTTAAGGGAATGACTAGTGAAGAAAAAGAAATTCTGATACGGGACAAAAATGCTCTTCAAAACATCATTCTTTATCACCTGACACCAGGAGTTTTCATTGGAAAAGGATTTGAACCTGGTGTTACTAACATTTTAAAGACCACACAAGGAAGCAAAATCTTTCTGAAAGAAGTAAATGATACACTTCTGGTGAATGAATTGAAATCAAAAGAATCTGACATCATGACAACAAATGGTGTAATTCATGTTGTAGATAAACTCCTCTATCCAGCAGACACACCTGTTGGAAATGATCAACTGCTGGAAATACTTAATAAATTAATCAAATACATCCAAATTAAGTTTGTTCGTGGTAGCACCTTCAAAGAAATCCCCGTGACTGTCTATAAGCCAATTATTAAAAAATACACCAAAATCATTGATGGAGTGCCTGTGGAAATAACTGAAAAAGAGACACGAGAAGAACGAATCATTACAGGTCCTGAAATAAAATACACTAGGATTTCTACTGGAGGTGGAGAAACAGAAGAAACTCTGAAGAAATTGTTACAAGAAGAGGTCACCAAGGTCACCAAATTCATTGAAGGTGGTGATGGTCATTTATTTGAAGATGAAGAAATTAAAAGACTGCTTCAGGGAGACACACCCGTGAGGAAGTTGCAAGCCAACAAAAAAGTTCAAGGATCTAGAAGACGATTAAGGGAAGGTCGTTCTCAGTGAAAATCCAAAAACCAGAAAAAAATGTTTATACAACCCTAAGTCAATAACCTGACCTTAGAAAATTGTGAGAGCCAAGTTGACTTCAGGAACTGAAACATCAGCACAAAGAAGCAATCATCAAATAATTCTGAACACAAATTTAATATTTTTTTTTCTGAATGAGAAACATGAGGGAAATTGTGGAGTTAGCCTCCTGTGGTAAAGGAATTGAAGAAAATATAACACCTTACACCCTTTTTCATCTTGACATTAAAAGTTCTGGCTAACTTTGGAATCCATTAGAGAAAAATCCTTGTCACCAGATTCATTACAATTCAAATCGAAGAGTTGTGAACTGTTATCCCATTGAAAAGACCGAGCCTTGTATGTATGTTATGGATACATAAAATGCACGCAAGCCATTATCTCTCCATGGGAAGCTAAGTTATAAAAATAGGTGCTTGGTGTACAAAACTTTTTATATCAAAAGGCTTTGCACATTTCTATATGAGTGGGTTTACTGGTAAATTATGTTATTTTTTACAACTAATTTTGTACTCTCAGAATGTTTGTCATATGCTTCTTGCAATGCATATTTTTTAATCTCAAACGTTTCAATAAAACCATTTTTCAGATATAAAGAGAATTACTTCAAATTGAGTAATTCAGAAAAACTCAAGATTTAAGTTAAAAAGTGGTTTGGACTTGGGAACAGGACTTTATACCTCTTTTACTGTAACAAGTACTCATTAAAGGAAATTGAATGAAATTAAAAAAAAAAA-3' Human periostin gene mRNA variant 2 (SEQ ID NO: 122)
5 '-3'
　前記ａｓ４ヌクレオチドにおける配列番号１２３の塩基配列は、以下の通りである。配列番号１２３の塩基配列は、ペリオスチン遺伝子のｍＲＮＡ　ｖａｒｉａｎｔ３の塩基配列である。配列番号１２３のポリヌクレオチドは、例えば、Ｈｏｍｏ　ｓａｐｉｅｎｓから得ることができ、例えば、ＧｅｎｅＢａｎｋにアクセッションＮｏ．ＮＭ＿００１１３５９３５．１で登録されているポリヌクレオチドがあげられる。 The base sequence of SEQ ID NO: 123 in the as4 nucleotide is as follows. The base sequence of SEQ ID NO: 123 is the base sequence of mRNA variant 3 of the periostin gene. The polynucleotide of SEQ ID NO: 123 can be obtained, for example, from Homo sapiens . For example, GeneBank has accession no. The polynucleotide registered by NM_0011359355.1 is mentioned.
ヒトペリオスチン遺伝子ｍＲＮＡ　ｖａｒｉａｎｔ　３（配列番号１２３）
5'-AGACTCTCAGGTTGATGCAGTGTTCCCTCCCACAACTCTGACATGTATATAAATTCTGAGCTCTCCAAAGCCCACTGCCAGTTCTCTTCGGGGACTAACTGCAACGGAGAGACTCAAGATGATTCCCTTTTTACCCATGTTTTCTCTACTATTGCTGCTTATTGTTAACCCTATAAACGCCAACAATCATTATGACAAGATCTTGGCTCATAGTCGTATCAGGGGTCGGGACCAAGGCCCAAATGTCTGTGCCCTTCAACAGATTTTGGGCACCAAAAAGAAATACTTCAGCACTTGTAAGAACTGGTATAAAAAGTCCATCTGTGGACAGAAAACGACTGTGTTATATGAATGTTGCCCTGGTTATATGAGAATGGAAGGAATGAAAGGCTGCCCAGCAGTTTTGCCCATTGACCATGTTTATGGCACTCTGGGCATCGTGGGAGCCACCACAACGCAGCGCTATTCTGACGCCTCAAAACTGAGGGAGGAGATCGAGGGAAAGGGATCCTTCACTTACTTTGCACCGAGTAATGAGGCTTGGGACAACTTGGATTCTGATATCCGTAGAGGTTTGGAGAGCAACGTGAATGTTGAATTACTGAATGCTTTACATAGTCACATGATTAATAAGAGAATGTTGACCAAGGACTTAAAAAATGGCATGATTATTCCTTCAATGTATAACAATTTGGGGCTTTTCATTAACCATTATCCTAATGGGGTTGTCACTGTTAATTGTGCTCGAATCATCCATGGGAACCAGATTGCAACAAATGGTGTTGTCCATGTCATTGACCGTGTGCTTACACAAATTGGTACCTCAATTCAAGACTTCATTGAAGCAGAAGATGACCTTTCATCTTTTAGAGCAGCTGCCATCACATCGGACATATTGGAGGCCCTTGGAAGAGACGGTCACTTCACACTCTTTGCTCCCACCAATGAGGCTTTTGAGAAACTTCCACGAGGTGTCCTAGAAAGGATCATGGGAGACAAAGTGGCTTCCGAAGCTCTTATGAAGTACCACATCTTAAATACTCTCCAGTGTTCTGAGTCTATTATGGGAGGAGCAGTCTTTGAGACGCTGGAAGGAAATACAATTGAGATAGGATGTGACGGTGACAGTATAACAGTAAATGGAATCAAAATGGTGAACAAAAAGGATATTGTGACAAATAATGGTGTGATCCATTTGATTGATCAGGTCCTAATTCCTGATTCTGCCAAACAAGTTATTGAGCTGGCTGGAAAACAGCAAACCACCTTCACGGATCTTGTGGCCCAATTAGGCTTGGCATCTGCTCTGAGGCCAGATGGAGAATACACTTTGCTGGCACCTGTGAATAATGCATTTTCTGATGATACTCTCAGCATGGATCAGCGCCTCCTTAAATTAATTCTGCAGAATCACATATTGAAAGTAAAAGTTGGCCTTAATGAGCTTTACAACGGGCAAATACTGGAAACCATCGGAGGCAAACAGCTCAGAGTCTTCGTATATCGTACAGCTGTCTGCATTGAAAATTCATGCATGGAGAAAGGGAGTAAGCAAGGGAGAAACGGTGCGATTCACATATTCCGCGAGATCATCAAGCCAGCAGAGAAATCCCTCCATGAAAAGTTAAAACAAGATAAGCGCTTTAGCACCTTCCTCAGCCTACTTGAAGCTGCAGACTTGAAAGAGCTCCTGACACAACCTGGAGACTGGACATTATTTGTGCCAACCAATGATGCTTTTAAGGGAATGACTAGTGAAGAAAAAGAAATTCTGATACGGGACAAAAATGCTCTTCAAAACATCATTCTTTATCACCTGACACCAGGAGTTTTCATTGGAAAAGGATTTGAACCTGGTGTTACTAACATTTTAAAGACCACACAAGGAAGCAAAATCTTTCTGAAAGAAGTAAATGATACACTTCTGGTGAATGAATTGAAATCAAAAGAATCTGACATCATGACAACAAATGGTGTAATTCATGTTGTAGATAAACTCCTCTATCCAGCAGACACACCTGTTGGAAATGATCAACTGCTGGAAATACTTAATAAATTAATCAAATACATCCAAATTAAGTTTGTTCGTGGTAGCACCTTCAAAGAAATCCCCGTGACTGTCTATAGACCCACACTAACAAAAGTCAAAATTGAAGGTGAACCTGAATTCAGACTGATTAAAGAAGGTGAAACAATAACTGAAGTGATCCATGGAGAGCCAATTATTAAAAAATACACCAAAATCATTGATGGAGTGCCTGTGGAAATAACTGAAAAAGAGACACGAGAAGAACGAATCATTACAGGTCCTGAAATAAAATACACTAGGATTTCTACTGGAGGTGGAGAAACAGAAGAAACTCTGAAGAAATTGTTACAAGAAGACACACCCGTGAGGAAGTTGCAAGCCAACAAAAAAGTTCAAGGATCTAGAAGACGATTAAGGGAAGGTCGTTCTCAGTGAAAATCCAAAAACCAGAAAAAAATGTTTATACAACCCTAAGTCAATAACCTGACCTTAGAAAATTGTGAGAGCCAAGTTGACTTCAGGAACTGAAACATCAGCACAAAGAAGCAATCATCAAATAATTCTGAACACAAATTTAATATTTTTTTTTCTGAATGAGAAACATGAGGGAAATTGTGGAGTTAGCCTCCTGTGGTAAAGGAATTGAAGAAAATATAACACCTTACACCCTTTTTCATCTTGACATTAAAAGTTCTGGCTAACTTTGGAATCCATTAGAGAAAAATCCTTGTCACCAGATTCATTACAATTCAAATCGAAGAGTTGTGAACTGTTATCCCATTGAAAAGACCGAGCCTTGTATGTATGTTATGGATACATAAAATGCACGCAAGCCATTATCTCTCCATGGGAAGCTAAGTTATAAAAATAGGTGCTTGGTGTACAAAACTTTTTATATCAAAAGGCTTTGCACATTTCTATATGAGTGGGTTTACTGGTAAATTATGTTATTTTTTACAACTAATTTTGTACTCTCAGAATGTTTGTCATATGCTTCTTGCAATGCATATTTTTTAATCTCAAACGTTTCAATAAAACCATTTTTCAGATATAAAGAGAATTACTTCAAATTGAGTAATTCAGAAAAACTCAAGATTTAAGTTAAAAAGTGGTTTGGACTTGGGAACAGGACTTTATACCTCTTTTACTGTAACAAGTACTCATTAAAGGAAATTGAATGAAATTAAAAAAAAAAA-3' Human periostin gene mRNA variant 3 (SEQ ID NO: 123)
5 '-3'
　前記ａｓ４ヌクレオチドにおける配列番号１２４の塩基配列は、以下の通りである。配列番号１２４の塩基配列は、ペリオスチン遺伝子のｍＲＮＡ　ｖａｒｉａｎｔ　４の塩基配列である。配列番号１２４のポリヌクレオチドは、例えば、Ｈｏｍｏ　ｓａｐｉｅｎｓから得ることができ、例えば、ＧｅｎｅＢａｎｋにアクセッションＮｏ．ＮＭ＿００１１３５９３６．１で登録されているポリヌクレオチドがあげられる。 The base sequence of SEQ ID NO: 124 in the as4 nucleotide is as follows. The base sequence of SEQ ID NO: 124 is the base sequence of mRNA variant 4 of the periostin gene. The polynucleotide of SEQ ID NO: 124 can be obtained, for example, from Homo sapiens , for example, accession No. The polynucleotide registered by NM_001135936.1 is mention | raise | lifted.
ヒトペリオスチン遺伝子ｍＲＮＡ　ｖａｒｉａｎｔ　４（配列番号１２４）
5'-AGACTCTCAGGTTGATGCAGTGTTCCCTCCCACAACTCTGACATGTATATAAATTCTGAGCTCTCCAAAGCCCACTGCCAGTTCTCTTCGGGGACTAACTGCAACGGAGAGACTCAAGATGATTCCCTTTTTACCCATGTTTTCTCTACTATTGCTGCTTATTGTTAACCCTATAAACGCCAACAATCATTATGACAAGATCTTGGCTCATAGTCGTATCAGGGGTCGGGACCAAGGCCCAAATGTCTGTGCCCTTCAACAGATTTTGGGCACCAAAAAGAAATACTTCAGCACTTGTAAGAACTGGTATAAAAAGTCCATCTGTGGACAGAAAACGACTGTGTTATATGAATGTTGCCCTGGTTATATGAGAATGGAAGGAATGAAAGGCTGCCCAGCAGTTTTGCCCATTGACCATGTTTATGGCACTCTGGGCATCGTGGGAGCCACCACAACGCAGCGCTATTCTGACGCCTCAAAACTGAGGGAGGAGATCGAGGGAAAGGGATCCTTCACTTACTTTGCACCGAGTAATGAGGCTTGGGACAACTTGGATTCTGATATCCGTAGAGGTTTGGAGAGCAACGTGAATGTTGAATTACTGAATGCTTTACATAGTCACATGATTAATAAGAGAATGTTGACCAAGGACTTAAAAAATGGCATGATTATTCCTTCAATGTATAACAATTTGGGGCTTTTCATTAACCATTATCCTAATGGGGTTGTCACTGTTAATTGTGCTCGAATCATCCATGGGAACCAGATTGCAACAAATGGTGTTGTCCATGTCATTGACCGTGTGCTTACACAAATTGGTACCTCAATTCAAGACTTCATTGAAGCAGAAGATGACCTTTCATCTTTTAGAGCAGCTGCCATCACATCGGACATATTGGAGGCCCTTGGAAGAGACGGTCACTTCACACTCTTTGCTCCCACCAATGAGGCTTTTGAGAAACTTCCACGAGGTGTCCTAGAAAGGATCATGGGAGACAAAGTGGCTTCCGAAGCTCTTATGAAGTACCACATCTTAAATACTCTCCAGTGTTCTGAGTCTATTATGGGAGGAGCAGTCTTTGAGACGCTGGAAGGAAATACAATTGAGATAGGATGTGACGGTGACAGTATAACAGTAAATGGAATCAAAATGGTGAACAAAAAGGATATTGTGACAAATAATGGTGTGATCCATTTGATTGATCAGGTCCTAATTCCTGATTCTGCCAAACAAGTTATTGAGCTGGCTGGAAAACAGCAAACCACCTTCACGGATCTTGTGGCCCAATTAGGCTTGGCATCTGCTCTGAGGCCAGATGGAGAATACACTTTGCTGGCACCTGTGAATAATGCATTTTCTGATGATACTCTCAGCATGGATCAGCGCCTCCTTAAATTAATTCTGCAGAATCACATATTGAAAGTAAAAGTTGGCCTTAATGAGCTTTACAACGGGCAAATACTGGAAACCATCGGAGGCAAACAGCTCAGAGTCTTCGTATATCGTACAGCTGTCTGCATTGAAAATTCATGCATGGAGAAAGGGAGTAAGCAAGGGAGAAACGGTGCGATTCACATATTCCGCGAGATCATCAAGCCAGCAGAGAAATCCCTCCATGAAAAGTTAAAACAAGATAAGCGCTTTAGCACCTTCCTCAGCCTACTTGAAGCTGCAGACTTGAAAGAGCTCCTGACACAACCTGGAGACTGGACATTATTTGTGCCAACCAATGATGCTTTTAAGGGAATGACTAGTGAAGAAAAAGAAATTCTGATACGGGACAAAAATGCTCTTCAAAACATCATTCTTTATCACCTGACACCAGGAGTTTTCATTGGAAAAGGATTTGAACCTGGTGTTACTAACATTTTAAAGACCACACAAGGAAGCAAAATCTTTCTGAAAGAAGTAAATGATACACTTCTGGTGAATGAATTGAAATCAAAAGAATCTGACATCATGACAACAAATGGTGTAATTCATGTTGTAGATAAACTCCTCTATCCAGCAGACACACCTGTTGGAAATGATCAACTGCTGGAAATACTTAATAAATTAATCAAATACATCCAAATTAAGTTTGTTCGTGGTAGCACCTTCAAAGAAATCCCCGTGACTGTCTATAAGCCAATTATTAAAAAATACACCAAAATCATTGATGGAGTGCCTGTGGAAATAACTGAAAAAGAGACACGAGAAGAACGAATCATTACAGGTCCTGAAATAAAATACACTAGGATTTCTACTGGAGGTGGAGAAACAGAAGAAACTCTGAAGAAATTGTTACAAGAAGACACACCCGTGAGGAAGTTGCAAGCCAACAAAAAAGTTCAAGGATCTAGAAGACGATTAAGGGAAGGTCGTTCTCAGTGAAAATCCAAAAACCAGAAAAAAATGTTTATACAACCCTAAGTCAATAACCTGACCTTAGAAAATTGTGAGAGCCAAGTTGACTTCAGGAACTGAAACATCAGCACAAAGAAGCAATCATCAAATAATTCTGAACACAAATTTAATATTTTTTTTTCTGAATGAGAAACATGAGGGAAATTGTGGAGTTAGCCTCCTGTGGTAAAGGAATTGAAGAAAATATAACACCTTACACCCTTTTTCATCTTGACATTAAAAGTTCTGGCTAACTTTGGAATCCATTAGAGAAAAATCCTTGTCACCAGATTCATTACAATTCAAATCGAAGAGTTGTGAACTGTTATCCCATTGAAAAGACCGAGCCTTGTATGTATGTTATGGATACATAAAATGCACGCAAGCCATTATCTCTCCATGGGAAGCTAAGTTATAAAAATAGGTGCTTGGTGTACAAAACTTTTTATATCAAAAGGCTTTGCACATTTCTATATGAGTGGGTTTACTGGTAAATTATGTTATTTTTTACAACTAATTTTGTACTCTCAGAATGTTTGTCATATGCTTCTTGCAATGCATATTTTTTAATCTCAAACGTTTCAATAAAACCATTTTTCAGATATAAAGAGAATTACTTCAAATTGAGTAATTCAGAAAAACTCAAGATTTAAGTTAAAAAGTGGTTTGGACTTGGGAACAGGACTTTATACCTCTTTTACTGTAACAAGTACTCATTAAAGGAAATTGAATGAAATTAAAAAAAAAAA-3' Human periostin gene mRNA variant 4 (SEQ ID NO: 124)
5 '-3'
　前記ａｓ４ヌクレオチドにおける配列番号１２５の塩基配列は、以下の通りである。配列番号１２５の塩基配列は、ペリオスチン遺伝子のｍＲＮＡ　ｖａｒｉａｎｔ　５の塩基配列である。配列番号１２５のポリヌクレオチドは、例えば、Ｈｏｍｏ　ｓａｐｉｅｎｓから得ることができ、例えば、ＧｅｎｅＢａｎｋにアクセッションＮｏ．ＮＭ＿００１２８６６６５．１で登録されているポリヌクレオチドがあげられる。 The base sequence of SEQ ID NO: 125 in the as4 nucleotide is as follows. The base sequence of SEQ ID NO: 125 is the base sequence of mRNA variant 5 of the periostin gene. The polynucleotide of SEQ ID NO: 125 can be obtained, for example, from Homo sapiens . The polynucleotide registered by NM_0012866665.1 is mentioned.
ヒトペリオスチン遺伝子ｍＲＮＡ　ｖａｒｉａｎｔ　５（配列番号１２５）
5'-AGACTCTCAGGTTGATGCAGTGTTCCCTCCCACAACTCTGACATGTATATAAATTCTGAGCTCTCCAAAGCCCACTGCCAGTTCTCTTCGGGGACTAACTGCAACGGAGAGACTCAAGATGATTCCCTTTTTACCCATGTTTTCTCTACTATTGCTGCTTATTGTTAACCCTATAAACGCCAACAATCATTATGACAAGATCTTGGCTCATAGTCGTATCAGGGGTCGGGACCAAGGCCCAAATGTCTGTGCCCTTCAACAGATTTTGGGCACCAAAAAGAAATACTTCAGCACTTGTAAGAACTGGTATAAAAAGTCCATCTGTGGACAGAAAACGACTGTGTTATATGAATGTTGCCCTGGTTATATGAGAATGGAAGGAATGAAAGGCTGCCCAGCAGTTTTGCCCATTGACCATGTTTATGGCACTCTGGGCATCGTGGGAGCCACCACAACGCAGCGCTATTCTGACGCCTCAAAACTGAGGGAGGAGATCGAGGGAAAGGGATCCTTCACTTACTTTGCACCGAGTAATGAGGCTTGGGACAACTTGGATTCTGATATCCGTAGAGGTTTGGAGAGCAACGTGAATGTTGAATTACTGAATGCTTTACATAGTCACATGATTAATAAGAGAATGTTGACCAAGGACTTAAAAAATGGCATGATTATTCCTTCAATGTATAACAATTTGGGGCTTTTCATTAACCATTATCCTAATGGGGTTGTCACTGTTAATTGTGCTCGAATCATCCATGGGAACCAGATTGCAACAAATGGTGTTGTCCATGTCATTGACCGTGTGCTTACACAAATTGGTACCTCAATTCAAGACTTCATTGAAGCAGAAGATGACCTTTCATCTTTTAGAGCAGCTGCCATCACATCGGACATATTGGAGGCCCTTGGAAGAGACGGTCACTTCACACTCTTTGCTCCCACCAATGAGGCTTTTGAGAAACTTCCACGAGGTGTCCTAGAAAGGATCATGGGAGACAAAGTGGCTTCCGAAGCTCTTATGAAGTACCACATCTTAAATACTCTCCAGTGTTCTGAGTCTATTATGGGAGGAGCAGTCTTTGAGACGCTGGAAGGAAATACAATTGAGATAGGATGTGACGGTGACAGTATAACAGTAAATGGAATCAAAATGGTGAACAAAAAGGATATTGTGACAAATAATGGTGTGATCCATTTGATTGATCAGGTCCTAATTCCTGATTCTGCCAAACAAGTTATTGAGCTGGCTGGAAAACAGCAAACCACCTTCACGGATCTTGTGGCCCAATTAGGCTTGGCATCTGCTCTGAGGCCAGATGGAGAATACACTTTGCTGGCACCTGTGAATAATGCATTTTCTGATGATACTCTCAGCATGGATCAGCGCCTCCTTAAATTAATTCTGCAGAATCACATATTGAAAGTAAAAGTTGGCCTTAATGAGCTTTACAACGGGCAAATACTGGAAACCATCGGAGGCAAACAGCTCAGAGTCTTCGTATATCGTACAGCTGTCTGCATTGAAAATTCATGCATGGAGAAAGGGAGTAAGCAAGGGAGAAACGGTGCGATTCACATATTCCGCGAGATCATCAAGCCAGCAGAGAAATCCCTCCATGAAAAGTTAAAACAAGATAAGCGCTTTAGCACCTTCCTCAGCCTACTTGAAGCTGCAGACTTGAAAGAGCTCCTGACACAACCTGGAGACTGGACATTATTTGTGCCAACCAATGATGCTTTTAAGGGAATGACTAGTGAAGAAAAAGAAATTCTGATACGGGACAAAAATGCTCTTCAAAACATCATTCTTTATCACCTGACACCAGGAGTTTTCATTGGAAAAGGATTTGAACCTGGTGTTACTAACATTTTAAAGACCACACAAGGAAGCAAAATCTTTCTGAAAGAAGTAAATGATACACTTCTGGTGAATGAATTGAAATCAAAAGAATCTGACATCATGACAACAAATGGTGTAATTCATGTTGTAGATAAACTCCTCTATCCAGCAGACACACCTGTTGGAAATGATCAACTGCTGGAAATACTTAATAAATTAATCAAATACATCCAAATTAAGTTTGTTCGTGGTAGCACCTTCAAAGAAATCCCCGTGACTGTCTATAGACCCACACTAACAAAAGTCAAAATTGAAGGTGAACCTGAATTCAGACTGATTAAAGAAGGTGAAACAATAACTGAAGTGATCCATGGAGAGCCAATTATTAAAAAATACACCAAAATCATTGATGGAGTGCCTGTGGAAATAACTGAAAAAGAGACACGAGAAGAACGAATCATTACAGGTCCTGAAATAAAATACACTAGGATTTCTACTGGAGGTGGAGAAACAGAAGAAACTCTGAAGAAATTGTTACAAGAAGAGGTCACCAAGGTCACCAAATTCATTGAAGGTGGTGATGGTCATTTATTTGAAGATGAAGAAATTAAAAGACTGCTTCAGGGAGACACACCCGTGAGGAAGTTGCAAGCCAACAAAAAAGTTCAAGGATCTAGAAGACGATTAAGGGAAGGTCGTTCTCAGTGAAAATCCAAAAACCAGAAAAAAATGTTTATACAACCCTAAGTCAATAACCTGACCTTAGAAAATTGTGAGAGCCAAGTTGACTTCAGGAACTGAAACATCAGCACAAAGAAGCAATCATCAAATAATTCTGAACACAAATTTAATATTTTTTTTTCTGAATGAGAAACATGAGGGAAATTGTGGAGTTAGCCTCCTGTGGTAAAGGAATTGAAGAAAATATAACACCTTACACCCTTTTTCATCTTGACATTAAAAGTTCTGGCTAACTTTGGAATCCATTAGAGAAAAATCCTTGTCACCAGATTCATTACAATTCAAATCGAAGAGTTGTGAACTGTTATCCCATTGAAAAGACCGAGCCTTGTATGTATGTTATGGATACATAAAATGCACGCAAGCCATTATCTCTCCATGGGAAGCTAAGTTATAAAAATAGGTGCTTGGTGTACAAAACTTTTTATATCAAAAGGCTTTGCACATTTCTATATGAGTGGGTTTACTGGTAAATTATGTTATTTTTTACAACTAATTTTGTACTCTCAGAATGTTTGTCATATGCTTCTTGCAATGCATATTTTTTAATCTCAAACGTTTCAATAAAACCATTTTTCAGATATAAAGAGAATTACTTCAAATTGAGTAATTCAGAAAAACTCAAGATTTAAGTTAAAAAGTGGTTTGGACTTGGGAACAGGACTTTATACCTCTTTTACTGTAACAAGTACTCATTAAAGGAAATTGAATGAAATTAAAAAAAAAAA-3' Human periostin gene mRNA variant 5 (SEQ ID NO: 125)
5 '-3'
　前記ａｓ４ヌクレオチドにおける配列番号１２６の塩基配列は、以下の通りである。配列番号１２６の塩基配列は、ペリオスチン遺伝子のｍＲＮＡ　ｖａｒｉａｎｔ　６の塩基配列である。配列番号１２６のポリヌクレオチドは、例えば、Ｈｏｍｏ　ｓａｐｉｅｎｓから得ることができ、例えば、ＧｅｎｅＢａｎｋにアクセッションＮｏ．ＮＭ＿００１２８６６６６．１で登録されているポリヌクレオチドがあげられる。 The base sequence of SEQ ID NO: 126 in the as4 nucleotide is as follows. The base sequence of SEQ ID NO: 126 is the base sequence of mRNA variant 6 of the periostin gene. The polynucleotide of SEQ ID NO: 126 can be obtained, for example, from Homo sapiens . For example, GeneBank has accession no. The polynucleotide registered by NM_0012866666.
ヒトペリオスチン遺伝子ｍＲＮＡ　ｖａｒｉａｎｔ　６（配列番号１２６）
5'-AGACTCTCAGGTTGATGCAGTGTTCCCTCCCACAACTCTGACATGTATATAAATTCTGAGCTCTCCAAAGCCCACTGCCAGTTCTCTTCGGGGACTAACTGCAACGGAGAGACTCAAGATGATTCCCTTTTTACCCATGTTTTCTCTACTATTGCTGCTTATTGTTAACCCTATAAACGCCAACAATCATTATGACAAGATCTTGGCTCATAGTCGTATCAGGGGTCGGGACCAAGGCCCAAATGTCTGTGCCCTTCAACAGATTTTGGGCACCAAAAAGAAATACTTCAGCACTTGTAAGAACTGGTATAAAAAGTCCATCTGTGGACAGAAAACGACTGTGTTATATGAATGTTGCCCTGGTTATATGAGAATGGAAGGAATGAAAGGCTGCCCAGCAGTTTTGCCCATTGACCATGTTTATGGCACTCTGGGCATCGTGGGAGCCACCACAACGCAGCGCTATTCTGACGCCTCAAAACTGAGGGAGGAGATCGAGGGAAAGGGATCCTTCACTTACTTTGCACCGAGTAATGAGGCTTGGGACAACTTGGATTCTGATATCCGTAGAGGTTTGGAGAGCAACGTGAATGTTGAATTACTGAATGCTTTACATAGTCACATGATTAATAAGAGAATGTTGACCAAGGACTTAAAAAATGGCATGATTATTCCTTCAATGTATAACAATTTGGGGCTTTTCATTAACCATTATCCTAATGGGGTTGTCACTGTTAATTGTGCTCGAATCATCCATGGGAACCAGATTGCAACAAATGGTGTTGTCCATGTCATTGACCGTGTGCTTACACAAATTGGTACCTCAATTCAAGACTTCATTGAAGCAGAAGATGACCTTTCATCTTTTAGAGCAGCTGCCATCACATCGGACATATTGGAGGCCCTTGGAAGAGACGGTCACTTCACACTCTTTGCTCCCACCAATGAGGCTTTTGAGAAACTTCCACGAGGTGTCCTAGAAAGGATCATGGGAGACAAAGTGGCTTCCGAAGCTCTTATGAAGTACCACATCTTAAATACTCTCCAGTGTTCTGAGTCTATTATGGGAGGAGCAGTCTTTGAGACGCTGGAAGGAAATACAATTGAGATAGGATGTGACGGTGACAGTATAACAGTAAATGGAATCAAAATGGTGAACAAAAAGGATATTGTGACAAATAATGGTGTGATCCATTTGATTGATCAGGTCCTAATTCCTGATTCTGCCAAACAAGTTATTGAGCTGGCTGGAAAACAGCAAACCACCTTCACGGATCTTGTGGCCCAATTAGGCTTGGCATCTGCTCTGAGGCCAGATGGAGAATACACTTTGCTGGCACCTGTGAATAATGCATTTTCTGATGATACTCTCAGCATGGATCAGCGCCTCCTTAAATTAATTCTGCAGAATCACATATTGAAAGTAAAAGTTGGCCTTAATGAGCTTTACAACGGGCAAATACTGGAAACCATCGGAGGCAAACAGCTCAGAGTCTTCGTATATCGTACAGCTGTCTGCATTGAAAATTCATGCATGGAGAAAGGGAGTAAGCAAGGGAGAAACGGTGCGATTCACATATTCCGCGAGATCATCAAGCCAGCAGAGAAATCCCTCCATGAAAAGTTAAAACAAGATAAGCGCTTTAGCACCTTCCTCAGCCTACTTGAAGCTGCAGACTTGAAAGAGCTCCTGACACAACCTGGAGACTGGACATTATTTGTGCCAACCAATGATGCTTTTAAGGGAATGACTAGTGAAGAAAAAGAAATTCTGATACGGGACAAAAATGCTCTTCAAAACATCATTCTTTATCACCTGACACCAGGAGTTTTCATTGGAAAAGGATTTGAACCTGGTGTTACTAACATTTTAAAGACCACACAAGGAAGCAAAATCTTTCTGAAAGAAGTAAATGATACACTTCTGGTGAATGAATTGAAATCAAAAGAATCTGACATCATGACAACAAATGGTGTAATTCATGTTGTAGATAAACTCCTCTATCCAGCAGACACACCTGTTGGAAATGATCAACTGCTGGAAATACTTAATAAATTAATCAAATACATCCAAATTAAGTTTGTTCGTGGTAGCACCTTCAAAGAAATCCCCGTGACTGTCTATAGTCCTGAAATAAAATACACTAGGATTTCTACTGGAGGTGGAGAAACAGAAGAAACTCTGAAGAAATTGTTACAAGAAGAGGTCACCAAGGTCACCAAATTCATTGAAGGTGGTGATGGTCATTTATTTGAAGATGAAGAAATTAAAAGACTGCTTCAGGGAGACACACCCGTGAGGAAGTTGCAAGCCAACAAAAAAGTTCAAGGATCTAGAAGACGATTAAGGGAAGGTCGTTCTCAGTGAAAATCCAAAAACCAGAAAAAAATGTTTATACAACCCTAAGTCAATAACCTGACCTTAGAAAATTGTGAGAGCCAAGTTGACTTCAGGAACTGAAACATCAGCACAAAGAAGCAATCATCAAATAATTCTGAACACAAATTTAATATTTTTTTTTCTGAATGAGAAACATGAGGGAAATTGTGGAGTTAGCCTCCTGTGGTAAAGGAATTGAAGAAAATATAACACCTTACACCCTTTTTCATCTTGACATTAAAAGTTCTGGCTAACTTTGGAATCCATTAGAGAAAAATCCTTGTCACCAGATTCATTACAATTCAAATCGAAGAGTTGTGAACTGTTATCCCATTGAAAAGACCGAGCCTTGTATGTATGTTATGGATACATAAAATGCACGCAAGCCATTATCTCTCCATGGGAAGCTAAGTTATAAAAATAGGTGCTTGGTGTACAAAACTTTTTATATCAAAAGGCTTTGCACATTTCTATATGAGTGGGTTTACTGGTAAATTATGTTATTTTTTACAACTAATTTTGTACTCTCAGAATGTTTGTCATATGCTTCTTGCAATGCATATTTTTTAATCTCAAACGTTTCAATAAAACCATTTTTCAGATATAAAGAGAATTACTTCAAATTGAGTAATTCAGAAAAACTCAAGATTTAAGTTAAAAAGTGGTTTGGACTTGGGAACAGGACTTTATACCTCTTTTACTGTAACAAGTACTCATTAAAGGAAATTGAATGAAATTAAAAAAAAAAA-3' Human periostin gene mRNA variant 6 (SEQ ID NO: 126)
5 '-3'
　前記ａｓ４ヌクレオチドにおける配列番号１２７の塩基配列は、以下の通りである。配列番号１２７の塩基配列は、ペリオスチン遺伝子のｍＲＮＡ　ｖａｒｉａｎｔ　７の塩基配列である。配列番号１２７のポリヌクレオチドは、例えば、Ｈｏｍｏ　ｓａｐｉｅｎｓから得ることができ、例えば、ＧｅｎｅＢａｎｋにアクセッションＮｏ．ＮＭ＿００１２８６６６７．１で登録されているポリヌクレオチドがあげられる。 The base sequence of SEQ ID NO: 127 in the as4 nucleotide is as follows. The base sequence of SEQ ID NO: 127 is the base sequence of mRNA variant 7 of the periostin gene. The polynucleotide of SEQ ID NO: 127 can be obtained, for example, from Homo sapiens . For example, GeneBank has accession no. The polynucleotide registered by NM_0012866677.1 is mentioned.
ヒトペリオスチン遺伝子ｍＲＮＡ　ｖａｒｉａｎｔ　７（配列番号１２７）
5'-AGACTCTCAGGTTGATGCAGTGTTCCCTCCCACAACTCTGACATGTATATAAATTCTGAGCTCTCCAAAGCCCACTGCCAGTTCTCTTCGGGGACTAACTGCAACGGAGAGACTCAAGATGATTCCCTTTTTACCCATGTTTTCTCTACTATTGCTGCTTATTGTTAACCCTATAAACGCCAACAATCATTATGACAAGATCTTGGCTCATAGTCGTATCAGGGGTCGGGACCAAGGCCCAAATGTCTGTGCCCTTCAACAGATTTTGGGCACCAAAAAGAAATACTTCAGCACTTGTAAGAACTGGTATAAAAAGTCCATCTGTGGACAGAAAACGACTGTGTTATATGAATGTTGCCCTGGTTATATGAGAATGGAAGGAATGAAAGGCTGCCCAGCAGTTTTGCCCATTGACCATGTTTATGGCACTCTGGGCATCGTGGGAGCCACCACAACGCAGCGCTATTCTGACGCCTCAAAACTGAGGGAGGAGATCGAGGGAAAGGGATCCTTCACTTACTTTGCACCGAGTAATGAGGCTTGGGACAACTTGGATTCTGATATCCGTAGAGGTTTGGAGAGCAACGTGAATGTTGAATTACTGAATGCTTTACATAGTCACATGATTAATAAGAGAATGTTGACCAAGGACTTAAAAAATGGCATGATTATTCCTTCAATGTATAACAATTTGGGGCTTTTCATTAACCATTATCCTAATGGGGTTGTCACTGTTAATTGTGCTCGAATCATCCATGGGAACCAGATTGCAACAAATGGTGTTGTCCATGTCATTGACCGTGTGCTTACACAAATTGGTACCTCAATTCAAGACTTCATTGAAGCAGAAGATGACCTTTCATCTTTTAGAGCAGCTGCCATCACATCGGACATATTGGAGGCCCTTGGAAGAGACGGTCACTTCACACTCTTTGCTCCCACCAATGAGGCTTTTGAGAAACTTCCACGAGGTGTCCTAGAAAGGATCATGGGAGACAAAGTGGCTTCCGAAGCTCTTATGAAGTACCACATCTTAAATACTCTCCAGTGTTCTGAGTCTATTATGGGAGGAGCAGTCTTTGAGACGCTGGAAGGAAATACAATTGAGATAGGATGTGACGGTGACAGTATAACAGTAAATGGAATCAAAATGGTGAACAAAAAGGATATTGTGACAAATAATGGTGTGATCCATTTGATTGATCAGGTCCTAATTCCTGATTCTGCCAAACAAGTTATTGAGCTGGCTGGAAAACAGCAAACCACCTTCACGGATCTTGTGGCCCAATTAGGCTTGGCATCTGCTCTGAGGCCAGATGGAGAATACACTTTGCTGGCACCTGTGAATAATGCATTTTCTGATGATACTCTCAGCATGGATCAGCGCCTCCTTAAATTAATTCTGCAGAATCACATATTGAAAGTAAAAGTTGGCCTTAATGAGCTTTACAACGGGCAAATACTGGAAACCATCGGAGGCAAACAGCTCAGAGTCTTCGTATATCGTACAGCTGTCTGCATTGAAAATTCATGCATGGAGAAAGGGAGTAAGCAAGGGAGAAACGGTGCGATTCACATATTCCGCGAGATCATCAAGCCAGCAGAGAAATCCCTCCATGAAAAGTTAAAACAAGATAAGCGCTTTAGCACCTTCCTCAGCCTACTTGAAGCTGCAGACTTGAAAGAGCTCCTGACACAACCTGGAGACTGGACATTATTTGTGCCAACCAATGATGCTTTTAAGGGAATGACTAGTGAAGAAAAAGAAATTCTGATACGGGACAAAAATGCTCTTCAAAACATCATTCTTTATCACCTGACACCAGGAGTTTTCATTGGAAAAGGATTTGAACCTGGTGTTACTAACATTTTAAAGACCACACAAGGAAGCAAAATCTTTCTGAAAGAAGTAAATGATACACTTCTGGTGAATGAATTGAAATCAAAAGAATCTGACATCATGACAACAAATGGTGTAATTCATGTTGTAGATAAACTCCTCTATCCAGCAGACACACCTGTTGGAAATGATCAACTGCTGGAAATACTTAATAAATTAATCAAATACATCCAAATTAAGTTTGTTCGTGGTAGCACCTTCAAAGAAATCCCCGTGACTGTCTATAGTCCTGAAATAAAATACACTAGGATTTCTACTGGAGGTGGAGAAACAGAAGAAACTCTGAAGAAATTGTTACAAGAAGACACACCCGTGAGGAAGTTGCAAGCCAACAAAAAAGTTCAAGGATCTAGAAGACGATTAAGGGAAGGTCGTTCTCAGTGAAAATCCAAAAACCAGAAAAAAATGTTTATACAACCCTAAGTCAATAACCTGACCTTAGAAAATTGTGAGAGCCAAGTTGACTTCAGGAACTGAAACATCAGCACAAAGAAGCAATCATCAAATAATTCTGAACACAAATTTAATATTTTTTTTTCTGAATGAGAAACATGAGGGAAATTGTGGAGTTAGCCTCCTGTGGTAAAGGAATTGAAGAAAATATAACACCTTACACCCTTTTTCATCTTGACATTAAAAGTTCTGGCTAACTTTGGAATCCATTAGAGAAAAATCCTTGTCACCAGATTCATTACAATTCAAATCGAAGAGTTGTGAACTGTTATCCCATTGAAAAGACCGAGCCTTGTATGTATGTTATGGATACATAAAATGCACGCAAGCCATTATCTCTCCATGGGAAGCTAAGTTATAAAAATAGGTGCTTGGTGTACAAAACTTTTTATATCAAAAGGCTTTGCACATTTCTATATGAGTGGGTTTACTGGTAAATTATGTTATTTTTTACAACTAATTTTGTACTCTCAGAATGTTTGTCATATGCTTCTTGCAATGCATATTTTTTAATCTCAAACGTTTCAATAAAACCATTTTTCAGATATAAAGAGAATTACTTCAAATTGAGTAATTCAGAAAAACTCAAGATTTAAGTTAAAAAGTGGTTTGGACTTGGGAACAGGACTTTATACCTCTTTTACTGTAACAAGTACTCATTAAAGGAAATTGAATGAAATTAAAAAAAAAAA-3' Human periostin gene mRNA variant 7 (SEQ ID NO: 127)
5 '-3'
　前記ａｓ４ヌクレオチドにおいて、「ハイブリダイズ」は、例えば、前記ａｓ４ヌクレオチドが、ペリオスチン遺伝子の発現抑制機能を有する範囲であればよい。前記ａｓ４ヌクレオチドにおいて、「ハイブリダイズする塩基配列」は、例えば、前記配列番号１２１～１２７のいずれか一つの塩基配列からなるポリヌクレオチド（以下、「ペリオスチン遺伝子ｍＲＮＡ」ともいう。）に対して、完全または部分的に相補的なポリヌクレオチドである。前記ハイブリダイズは、例えば、各種ハイブリダイゼーションアッセイにより検出できる。前記ハイブリダイゼーションアッセイは、特に制限されず、例えば、ザンブルーク（Ｓａｍｂｒｏｏｋ）ら編「モレキュラー・クローニング：ア・ラボラトリーマニュアル第２版（Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ：　Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ　２ｎｄ　Ｅｄ．）」〔Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ　（１９８９）〕等に記載されている方法を採用することもできる。 In the as4 nucleotide, “hybridization” may be, for example, within a range in which the as4 nucleotide has a function of suppressing the expression of the periostin gene. In the as4 nucleotide, the “hybridizing base sequence” is, for example, complete with respect to a polynucleotide comprising any one of the base sequences of SEQ ID NOs: 121 to 127 (hereinafter also referred to as “periostin gene mRNA”). Or a partially complementary polynucleotide. The hybridization can be detected by, for example, various hybridization assays. The hybridization assay is not particularly limited, for example, Zanburuku (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) ," [Cold Spring Harbor Laboratory Press (1989)] and the like can also be employed.
　前記ａｓ４ヌクレオチドにおいて、「ストリンジェントな条件」は、例えば、低ストリンジェントな条件、中ストリンジェントな条件、高ストリンジェントな条件のいずれでもよい。「低ストリンジェントな条件」は、例えば、５×ＳＳＣ、５×デンハルト溶液、０．５％ＳＤＳ、５０％ホルムアミド、３２℃の条件である。「中ストリンジェントな条件」は、例えば、５×ＳＳＣ、５×デンハルト溶液、０．５％ＳＤＳ、５０％ホルムアミド、４２℃の条件である。「高ストリンジェントな条件」は、例えば、５×ＳＳＣ、５×デンハルト溶液、０．５％ＳＤＳ、５０％ホルムアミド、５０℃の条件である。ストリンジェンシーの程度は、当業者であれば、例えば、温度、塩濃度、プローブの濃度および長さ、イオン強度、時間等の条件を適宜選択することで、設定可能である。「ストリンジェントな条件」は、例えば、前述したザンブルーク（Ｓａｍｂｒｏｏｋ）ら編「モレキュラー・クローニング：ア・ラボラトリーマニュアル第２版（Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ：　Ａ　Ｌａｂｏｒａｔｏｒｙ　Ｍａｎｕａｌ　２ｎｄ　Ｅｄ．）」〔Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ　（１９８９）〕等に記載の条件を採用することもできる。 In the as4 nucleotide, “stringent conditions” may be, for example, low stringency conditions, medium stringency conditions, or high stringency conditions. “Low stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, and 32 ° C. “Medium stringent conditions” are, for example, 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 42 ° C. “High stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C. The degree of stringency can be set by those skilled in the art by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, time, and the like. "Stringent conditions" are, for example, Zanburuku previously described (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) ," [Cold Spring Harbor Laboratory Press ( 1989)] etc. can also be employed.
　前記ａｓ４ヌクレオチドが、ペリオスチン遺伝子ｍＲＮＡに対して、部分的に相補的なポリヌクレオチドの場合、前記ａｓ４ヌクレオチドにおいて、例えば、８０％以上、８５％以上、９０％以上、９３％以上、９４％以上、９５％以上、９６％以上、９７％以上、９８％以上、９９％以上の塩基が、少なくとも一つのペリオスチン遺伝子ｍＲＮＡに対して、相補的である。 When the as4 nucleotide is a polynucleotide partially complementary to the periostin gene mRNA, the as4 nucleotide is, for example, 80% or more, 85% or more, 90% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of the bases are complementary to at least one periostin gene mRNA.
　前記ａｓ４ヌクレオチドがペリオスチン遺伝子ｍＲＮＡにハイブリダイズする位置は、特に制限されず、例えば、前記ペリオスチン遺伝子ｍＲＮＡの５’非翻訳領域（５’ＵＴＲ：Five prime untranslated region）でもよいし、コーディング領域（ＣＤＳ：coding region）でもよいし、３’非翻訳領域（３’ＵＴＲ：Three prime untranslated region）でもよいし、いずれか２つ以上の領域でもよい。前記ハイブリダイズする位置は、ペリオスチン遺伝子の発現をより抑制できることから、好ましくは、前記ＣＤＳおよび前記３’ＵＴＲの少なくとも一方であり、より好ましくは、前記３’ＵＴＲである。各ペリオスチン遺伝子ｍＲＮＡの塩基配列における前記５’ＵＴＲ、前記ＣＤＳおよび前記３’ＵＴＲは、例えば、それぞれ、下記表１の塩基配列である。 The position at which the as4 nucleotide hybridizes to the periostin gene mRNA is not particularly limited, and may be, for example, the 5 ′ untranslated region (5′UTR) of the periostin gene mRNA, or the coding region (CDS: coding'region), 3'untranslated region (3'UTR: Three'prime'untranslated'region), or any two or more regions. The hybridizing position is preferably at least one of the CDS and the 3'UTR, and more preferably the 3'UTR, because the expression of the periostin gene can be further suppressed. The 5′UTR, the CDS, and the 3′UTR in the base sequence of each periostin gene mRNA are, for example, the base sequences shown in Table 1 below, respectively.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
ヒトペリオスチン遺伝子ｍＲＮＡ　５’ＵＴＲ（配列番号１２８）
5'-AGACTCTCAGGTTGATGCAGTGTTCCCTCCCACAACTCTGACATGTATATAAATTCTGAGCTCTCCAAAGCCCACTGCCAGTTCTCTTCGGGGACTAACTGCAACGGAGAGACTCAAG-3' Human periostin gene mRNA 5′UTR (SEQ ID NO: 128)
5'-AGACTCTCAGGTTGATGCAGTGTTCCCTCCCACAACTCTGACATGTATATAAATTCTGAGCTCTCCAAAGCCCACTGCCAGTTCTCTTCGGGGACTAACTGCAACGGAGAGACTCAAG-3 '
ヒトペリオスチン遺伝子ｍＲＮＡ　ｖａｒｉａｎｔ　１　ＣＤＳ（配列番号１２９）
5'-ATGATTCCCTTTTTACCCATGTTTTCTCTACTATTGCTGCTTATTGTTAACCCTATAAACGCCAACAATCATTATGACAAGATCTTGGCTCATAGTCGTATCAGGGGTCGGGACCAAGGCCCAAATGTCTGTGCCCTTCAACAGATTTTGGGCACCAAAAAGAAATACTTCAGCACTTGTAAGAACTGGTATAAAAAGTCCATCTGTGGACAGAAAACGACTGTGTTATATGAATGTTGCCCTGGTTATATGAGAATGGAAGGAATGAAAGGCTGCCCAGCAGTTTTGCCCATTGACCATGTTTATGGCACTCTGGGCATCGTGGGAGCCACCACAACGCAGCGCTATTCTGACGCCTCAAAACTGAGGGAGGAGATCGAGGGAAAGGGATCCTTCACTTACTTTGCACCGAGTAATGAGGCTTGGGACAACTTGGATTCTGATATCCGTAGAGGTTTGGAGAGCAACGTGAATGTTGAATTACTGAATGCTTTACATAGTCACATGATTAATAAGAGAATGTTGACCAAGGACTTAAAAAATGGCATGATTATTCCTTCAATGTATAACAATTTGGGGCTTTTCATTAACCATTATCCTAATGGGGTTGTCACTGTTAATTGTGCTCGAATCATCCATGGGAACCAGATTGCAACAAATGGTGTTGTCCATGTCATTGACCGTGTGCTTACACAAATTGGTACCTCAATTCAAGACTTCATTGAAGCAGAAGATGACCTTTCATCTTTTAGAGCAGCTGCCATCACATCGGACATATTGGAGGCCCTTGGAAGAGACGGTCACTTCACACTCTTTGCTCCCACCAATGAGGCTTTTGAGAAACTTCCACGAGGTGTCCTAGAAAGGATCATGGGAGACAAAGTGGCTTCCGAAGCTCTTATGAAGTACCACATCTTAAATACTCTCCAGTGTTCTGAGTCTATTATGGGAGGAGCAGTCTTTGAGACGCTGGAAGGAAATACAATTGAGATAGGATGTGACGGTGACAGTATAACAGTAAATGGAATCAAAATGGTGAACAAAAAGGATATTGTGACAAATAATGGTGTGATCCATTTGATTGATCAGGTCCTAATTCCTGATTCTGCCAAACAAGTTATTGAGCTGGCTGGAAAACAGCAAACCACCTTCACGGATCTTGTGGCCCAATTAGGCTTGGCATCTGCTCTGAGGCCAGATGGAGAATACACTTTGCTGGCACCTGTGAATAATGCATTTTCTGATGATACTCTCAGCATGGATCAGCGCCTCCTTAAATTAATTCTGCAGAATCACATATTGAAAGTAAAAGTTGGCCTTAATGAGCTTTACAACGGGCAAATACTGGAAACCATCGGAGGCAAACAGCTCAGAGTCTTCGTATATCGTACAGCTGTCTGCATTGAAAATTCATGCATGGAGAAAGGGAGTAAGCAAGGGAGAAACGGTGCGATTCACATATTCCGCGAGATCATCAAGCCAGCAGAGAAATCCCTCCATGAAAAGTTAAAACAAGATAAGCGCTTTAGCACCTTCCTCAGCCTACTTGAAGCTGCAGACTTGAAAGAGCTCCTGACACAACCTGGAGACTGGACATTATTTGTGCCAACCAATGATGCTTTTAAGGGAATGACTAGTGAAGAAAAAGAAATTCTGATACGGGACAAAAATGCTCTTCAAAACATCATTCTTTATCACCTGACACCAGGAGTTTTCATTGGAAAAGGATTTGAACCTGGTGTTACTAACATTTTAAAGACCACACAAGGAAGCAAAATCTTTCTGAAAGAAGTAAATGATACACTTCTGGTGAATGAATTGAAATCAAAAGAATCTGACATCATGACAACAAATGGTGTAATTCATGTTGTAGATAAACTCCTCTATCCAGCAGACACACCTGTTGGAAATGATCAACTGCTGGAAATACTTAATAAATTAATCAAATACATCCAAATTAAGTTTGTTCGTGGTAGCACCTTCAAAGAAATCCCCGTGACTGTCTATACAACTAAAATTATAACCAAAGTTGTGGAACCAAAAATTAAAGTGATTGAAGGCAGTCTTCAGCCTATTATCAAAACTGAAGGACCCACACTAACAAAAGTCAAAATTGAAGGTGAACCTGAATTCAGACTGATTAAAGAAGGTGAAACAATAACTGAAGTGATCCATGGAGAGCCAATTATTAAAAAATACACCAAAATCATTGATGGAGTGCCTGTGGAAATAACTGAAAAAGAGACACGAGAAGAACGAATCATTACAGGTCCTGAAATAAAATACACTAGGATTTCTACTGGAGGTGGAGAAACAGAAGAAACTCTGAAGAAATTGTTACAAGAAGAGGTCACCAAGGTCACCAAATTCATTGAAGGTGGTGATGGTCATTTATTTGAAGATGAAGAAATTAAAAGACTGCTTCAGGGAGACACACCCGTGAGGAAGTTGCAAGCCAACAAAAAAGTTCAAGGATCTAGAAGACGATTAAGGGAAGGTCGTTCTCAGTGA-3' Human periostin gene mRNA variant 1 CDS (SEQ ID NO: 129)
5 '-3'
ヒトペリオスチン遺伝子ｍＲＮＡ　　３’ＵＴＲ（配列番号１３０）
5'-AAATCCAAAAACCAGAAAAAAATGTTTATACAACCCTAAGTCAATAACCTGACCTTAGAAAATTGTGAGAGCCAAGTTGACTTCAGGAACTGAAACATCAGCACAAAGAAGCAATCATCAAATAATTCTGAACACAAATTTAATATTTTTTTTTCTGAATGAGAAACATGAGGGAAATTGTGGAGTTAGCCTCCTGTGGTAAAGGAATTGAAGAAAATATAACACCTTACACCCTTTTTCATCTTGACATTAAAAGTTCTGGCTAACTTTGGAATCCATTAGAGAAAAATCCTTGTCACCAGATTCATTACAATTCAAATCGAAGAGTTGTGAACTGTTATCCCATTGAAAAGACCGAGCCTTGTATGTATGTTATGGATACATAAAATGCACGCAAGCCATTATCTCTCCATGGGAAGCTAAGTTATAAAAATAGGTGCTTGGTGTACAAAACTTTTTATATCAAAAGGCTTTGCACATTTCTATATGAGTGGGTTTACTGGTAAATTATGTTATTTTTTACAACTAATTTTGTACTCTCAGAATGTTTGTCATATGCTTCTTGCAATGCATATTTTTTAATCTCAAACGTTTCAATAAAACCATTTTTCAGATATAAAGAGAATTACTTCAAATTGAGTAATTCAGAAAAACTCAAGATTTAAGTTAAAAAGTGGTTTGGACTTGGGAACAGGACTTTATACCTCTTTTACTGTAACAAGTACTCATTAAAGGAAATTGAATGAAATTAAAAAAAAAAA-3' Human periostin gene mRNA 3′UTR (SEQ ID NO: 130)
5 '-3'
ヒトペリオスチン遺伝子ｍＲＮＡ　ｖａｒｉａｎｔ　２　ＣＤＳ（配列番号１３１）
5'-ATGATTCCCTTTTTACCCATGTTTTCTCTACTATTGCTGCTTATTGTTAACCCTATAAACGCCAACAATCATTATGACAAGATCTTGGCTCATAGTCGTATCAGGGGTCGGGACCAAGGCCCAAATGTCTGTGCCCTTCAACAGATTTTGGGCACCAAAAAGAAATACTTCAGCACTTGTAAGAACTGGTATAAAAAGTCCATCTGTGGACAGAAAACGACTGTGTTATATGAATGTTGCCCTGGTTATATGAGAATGGAAGGAATGAAAGGCTGCCCAGCAGTTTTGCCCATTGACCATGTTTATGGCACTCTGGGCATCGTGGGAGCCACCACAACGCAGCGCTATTCTGACGCCTCAAAACTGAGGGAGGAGATCGAGGGAAAGGGATCCTTCACTTACTTTGCACCGAGTAATGAGGCTTGGGACAACTTGGATTCTGATATCCGTAGAGGTTTGGAGAGCAACGTGAATGTTGAATTACTGAATGCTTTACATAGTCACATGATTAATAAGAGAATGTTGACCAAGGACTTAAAAAATGGCATGATTATTCCTTCAATGTATAACAATTTGGGGCTTTTCATTAACCATTATCCTAATGGGGTTGTCACTGTTAATTGTGCTCGAATCATCCATGGGAACCAGATTGCAACAAATGGTGTTGTCCATGTCATTGACCGTGTGCTTACACAAATTGGTACCTCAATTCAAGACTTCATTGAAGCAGAAGATGACCTTTCATCTTTTAGAGCAGCTGCCATCACATCGGACATATTGGAGGCCCTTGGAAGAGACGGTCACTTCACACTCTTTGCTCCCACCAATGAGGCTTTTGAGAAACTTCCACGAGGTGTCCTAGAAAGGATCATGGGAGACAAAGTGGCTTCCGAAGCTCTTATGAAGTACCACATCTTAAATACTCTCCAGTGTTCTGAGTCTATTATGGGAGGAGCAGTCTTTGAGACGCTGGAAGGAAATACAATTGAGATAGGATGTGACGGTGACAGTATAACAGTAAATGGAATCAAAATGGTGAACAAAAAGGATATTGTGACAAATAATGGTGTGATCCATTTGATTGATCAGGTCCTAATTCCTGATTCTGCCAAACAAGTTATTGAGCTGGCTGGAAAACAGCAAACCACCTTCACGGATCTTGTGGCCCAATTAGGCTTGGCATCTGCTCTGAGGCCAGATGGAGAATACACTTTGCTGGCACCTGTGAATAATGCATTTTCTGATGATACTCTCAGCATGGATCAGCGCCTCCTTAAATTAATTCTGCAGAATCACATATTGAAAGTAAAAGTTGGCCTTAATGAGCTTTACAACGGGCAAATACTGGAAACCATCGGAGGCAAACAGCTCAGAGTCTTCGTATATCGTACAGCTGTCTGCATTGAAAATTCATGCATGGAGAAAGGGAGTAAGCAAGGGAGAAACGGTGCGATTCACATATTCCGCGAGATCATCAAGCCAGCAGAGAAATCCCTCCATGAAAAGTTAAAACAAGATAAGCGCTTTAGCACCTTCCTCAGCCTACTTGAAGCTGCAGACTTGAAAGAGCTCCTGACACAACCTGGAGACTGGACATTATTTGTGCCAACCAATGATGCTTTTAAGGGAATGACTAGTGAAGAAAAAGAAATTCTGATACGGGACAAAAATGCTCTTCAAAACATCATTCTTTATCACCTGACACCAGGAGTTTTCATTGGAAAAGGATTTGAACCTGGTGTTACTAACATTTTAAAGACCACACAAGGAAGCAAAATCTTTCTGAAAGAAGTAAATGATACACTTCTGGTGAATGAATTGAAATCAAAAGAATCTGACATCATGACAACAAATGGTGTAATTCATGTTGTAGATAAACTCCTCTATCCAGCAGACACACCTGTTGGAAATGATCAACTGCTGGAAATACTTAATAAATTAATCAAATACATCCAAATTAAGTTTGTTCGTGGTAGCACCTTCAAAGAAATCCCCGTGACTGTCTATAAGCCAATTATTAAAAAATACACCAAAATCATTGATGGAGTGCCTGTGGAAATAACTGAAAAAGAGACACGAGAAGAACGAATCATTACAGGTCCTGAAATAAAATACACTAGGATTTCTACTGGAGGTGGAGAAACAGAAGAAACTCTGAAGAAATTGTTACAAGAAGAGGTCACCAAGGTCACCAAATTCATTGAAGGTGGTGATGGTCATTTATTTGAAGATGAAGAAATTAAAAGACTGCTTCAGGGAGACACACCCGTGAGGAAGTTGCAAGCCAACAAAAAAGTTCAAGGATCTAGAAGACGATTAAGGGAAGGTCGTTCTCAGTGA-3' Human periostin gene mRNA variant 2 CDS (SEQ ID NO: 131)
5 '-3'
ヒトペリオスチン遺伝子ｍＲＮＡ　ｖａｒｉａｎｔ　３　ＣＤＳ（配列番号１３２）
5'-ATGATTCCCTTTTTACCCATGTTTTCTCTACTATTGCTGCTTATTGTTAACCCTATAAACGCCAACAATCATTATGACAAGATCTTGGCTCATAGTCGTATCAGGGGTCGGGACCAAGGCCCAAATGTCTGTGCCCTTCAACAGATTTTGGGCACCAAAAAGAAATACTTCAGCACTTGTAAGAACTGGTATAAAAAGTCCATCTGTGGACAGAAAACGACTGTGTTATATGAATGTTGCCCTGGTTATATGAGAATGGAAGGAATGAAAGGCTGCCCAGCAGTTTTGCCCATTGACCATGTTTATGGCACTCTGGGCATCGTGGGAGCCACCACAACGCAGCGCTATTCTGACGCCTCAAAACTGAGGGAGGAGATCGAGGGAAAGGGATCCTTCACTTACTTTGCACCGAGTAATGAGGCTTGGGACAACTTGGATTCTGATATCCGTAGAGGTTTGGAGAGCAACGTGAATGTTGAATTACTGAATGCTTTACATAGTCACATGATTAATAAGAGAATGTTGACCAAGGACTTAAAAAATGGCATGATTATTCCTTCAATGTATAACAATTTGGGGCTTTTCATTAACCATTATCCTAATGGGGTTGTCACTGTTAATTGTGCTCGAATCATCCATGGGAACCAGATTGCAACAAATGGTGTTGTCCATGTCATTGACCGTGTGCTTACACAAATTGGTACCTCAATTCAAGACTTCATTGAAGCAGAAGATGACCTTTCATCTTTTAGAGCAGCTGCCATCACATCGGACATATTGGAGGCCCTTGGAAGAGACGGTCACTTCACACTCTTTGCTCCCACCAATGAGGCTTTTGAGAAACTTCCACGAGGTGTCCTAGAAAGGATCATGGGAGACAAAGTGGCTTCCGAAGCTCTTATGAAGTACCACATCTTAAATACTCTCCAGTGTTCTGAGTCTATTATGGGAGGAGCAGTCTTTGAGACGCTGGAAGGAAATACAATTGAGATAGGATGTGACGGTGACAGTATAACAGTAAATGGAATCAAAATGGTGAACAAAAAGGATATTGTGACAAATAATGGTGTGATCCATTTGATTGATCAGGTCCTAATTCCTGATTCTGCCAAACAAGTTATTGAGCTGGCTGGAAAACAGCAAACCACCTTCACGGATCTTGTGGCCCAATTAGGCTTGGCATCTGCTCTGAGGCCAGATGGAGAATACACTTTGCTGGCACCTGTGAATAATGCATTTTCTGATGATACTCTCAGCATGGATCAGCGCCTCCTTAAATTAATTCTGCAGAATCACATATTGAAAGTAAAAGTTGGCCTTAATGAGCTTTACAACGGGCAAATACTGGAAACCATCGGAGGCAAACAGCTCAGAGTCTTCGTATATCGTACAGCTGTCTGCATTGAAAATTCATGCATGGAGAAAGGGAGTAAGCAAGGGAGAAACGGTGCGATTCACATATTCCGCGAGATCATCAAGCCAGCAGAGAAATCCCTCCATGAAAAGTTAAAACAAGATAAGCGCTTTAGCACCTTCCTCAGCCTACTTGAAGCTGCAGACTTGAAAGAGCTCCTGACACAACCTGGAGACTGGACATTATTTGTGCCAACCAATGATGCTTTTAAGGGAATGACTAGTGAAGAAAAAGAAATTCTGATACGGGACAAAAATGCTCTTCAAAACATCATTCTTTATCACCTGACACCAGGAGTTTTCATTGGAAAAGGATTTGAACCTGGTGTTACTAACATTTTAAAGACCACACAAGGAAGCAAAATCTTTCTGAAAGAAGTAAATGATACACTTCTGGTGAATGAATTGAAATCAAAAGAATCTGACATCATGACAACAAATGGTGTAATTCATGTTGTAGATAAACTCCTCTATCCAGCAGACACACCTGTTGGAAATGATCAACTGCTGGAAATACTTAATAAATTAATCAAATACATCCAAATTAAGTTTGTTCGTGGTAGCACCTTCAAAGAAATCCCCGTGACTGTCTATAGACCCACACTAACAAAAGTCAAAATTGAAGGTGAACCTGAATTCAGACTGATTAAAGAAGGTGAAACAATAACTGAAGTGATCCATGGAGAGCCAATTATTAAAAAATACACCAAAATCATTGATGGAGTGCCTGTGGAAATAACTGAAAAAGAGACACGAGAAGAACGAATCATTACAGGTCCTGAAATAAAATACACTAGGATTTCTACTGGAGGTGGAGAAACAGAAGAAACTCTGAAGAAATTGTTACAAGAAGACACACCCGTGAGGAAGTTGCAAGCCAACAAAAAAGTTCAAGGATCTAGAAGACGATTAAGGGAAGGTCGTTCTCAGTGA-3' Human periostin gene mRNA variant 3 CDS (SEQ ID NO: 132)
5 '-3'
ヒトペリオスチン遺伝子ｍＲＮＡ　ｖａｒｉａｎｔ　４　ＣＤＳ（配列番号１３３）
5'-ATGATTCCCTTTTTACCCATGTTTTCTCTACTATTGCTGCTTATTGTTAACCCTATAAACGCCAACAATCATTATGACAAGATCTTGGCTCATAGTCGTATCAGGGGTCGGGACCAAGGCCCAAATGTCTGTGCCCTTCAACAGATTTTGGGCACCAAAAAGAAATACTTCAGCACTTGTAAGAACTGGTATAAAAAGTCCATCTGTGGACAGAAAACGACTGTGTTATATGAATGTTGCCCTGGTTATATGAGAATGGAAGGAATGAAAGGCTGCCCAGCAGTTTTGCCCATTGACCATGTTTATGGCACTCTGGGCATCGTGGGAGCCACCACAACGCAGCGCTATTCTGACGCCTCAAAACTGAGGGAGGAGATCGAGGGAAAGGGATCCTTCACTTACTTTGCACCGAGTAATGAGGCTTGGGACAACTTGGATTCTGATATCCGTAGAGGTTTGGAGAGCAACGTGAATGTTGAATTACTGAATGCTTTACATAGTCACATGATTAATAAGAGAATGTTGACCAAGGACTTAAAAAATGGCATGATTATTCCTTCAATGTATAACAATTTGGGGCTTTTCATTAACCATTATCCTAATGGGGTTGTCACTGTTAATTGTGCTCGAATCATCCATGGGAACCAGATTGCAACAAATGGTGTTGTCCATGTCATTGACCGTGTGCTTACACAAATTGGTACCTCAATTCAAGACTTCATTGAAGCAGAAGATGACCTTTCATCTTTTAGAGCAGCTGCCATCACATCGGACATATTGGAGGCCCTTGGAAGAGACGGTCACTTCACACTCTTTGCTCCCACCAATGAGGCTTTTGAGAAACTTCCACGAGGTGTCCTAGAAAGGATCATGGGAGACAAAGTGGCTTCCGAAGCTCTTATGAAGTACCACATCTTAAATACTCTCCAGTGTTCTGAGTCTATTATGGGAGGAGCAGTCTTTGAGACGCTGGAAGGAAATACAATTGAGATAGGATGTGACGGTGACAGTATAACAGTAAATGGAATCAAAATGGTGAACAAAAAGGATATTGTGACAAATAATGGTGTGATCCATTTGATTGATCAGGTCCTAATTCCTGATTCTGCCAAACAAGTTATTGAGCTGGCTGGAAAACAGCAAACCACCTTCACGGATCTTGTGGCCCAATTAGGCTTGGCATCTGCTCTGAGGCCAGATGGAGAATACACTTTGCTGGCACCTGTGAATAATGCATTTTCTGATGATACTCTCAGCATGGATCAGCGCCTCCTTAAATTAATTCTGCAGAATCACATATTGAAAGTAAAAGTTGGCCTTAATGAGCTTTACAACGGGCAAATACTGGAAACCATCGGAGGCAAACAGCTCAGAGTCTTCGTATATCGTACAGCTGTCTGCATTGAAAATTCATGCATGGAGAAAGGGAGTAAGCAAGGGAGAAACGGTGCGATTCACATATTCCGCGAGATCATCAAGCCAGCAGAGAAATCCCTCCATGAAAAGTTAAAACAAGATAAGCGCTTTAGCACCTTCCTCAGCCTACTTGAAGCTGCAGACTTGAAAGAGCTCCTGACACAACCTGGAGACTGGACATTATTTGTGCCAACCAATGATGCTTTTAAGGGAATGACTAGTGAAGAAAAAGAAATTCTGATACGGGACAAAAATGCTCTTCAAAACATCATTCTTTATCACCTGACACCAGGAGTTTTCATTGGAAAAGGATTTGAACCTGGTGTTACTAACATTTTAAAGACCACACAAGGAAGCAAAATCTTTCTGAAAGAAGTAAATGATACACTTCTGGTGAATGAATTGAAATCAAAAGAATCTGACATCATGACAACAAATGGTGTAATTCATGTTGTAGATAAACTCCTCTATCCAGCAGACACACCTGTTGGAAATGATCAACTGCTGGAAATACTTAATAAATTAATCAAATACATCCAAATTAAGTTTGTTCGTGGTAGCACCTTCAAAGAAATCCCCGTGACTGTCTATAAGCCAATTATTAAAAAATACACCAAAATCATTGATGGAGTGCCTGTGGAAATAACTGAAAAAGAGACACGAGAAGAACGAATCATTACAGGTCCTGAAATAAAATACACTAGGATTTCTACTGGAGGTGGAGAAACAGAAGAAACTCTGAAGAAATTGTTACAAGAAGACACACCCGTGAGGAAGTTGCAAGCCAACAAAAAAGTTCAAGGATCTAGAAGACGATTAAGGGAAGGTCGTTCTCAGTGA-3' Human periostin gene mRNA variant 4 CDS (SEQ ID NO: 133)
5 '-3'
ヒトペリオスチン遺伝子ｍＲＮＡ　ｖａｒｉａｎｔ　５　ＣＤＳ（配列番号１３４）
5'-ATGATTCCCTTTTTACCCATGTTTTCTCTACTATTGCTGCTTATTGTTAACCCTATAAACGCCAACAATCATTATGACAAGATCTTGGCTCATAGTCGTATCAGGGGTCGGGACCAAGGCCCAAATGTCTGTGCCCTTCAACAGATTTTGGGCACCAAAAAGAAATACTTCAGCACTTGTAAGAACTGGTATAAAAAGTCCATCTGTGGACAGAAAACGACTGTGTTATATGAATGTTGCCCTGGTTATATGAGAATGGAAGGAATGAAAGGCTGCCCAGCAGTTTTGCCCATTGACCATGTTTATGGCACTCTGGGCATCGTGGGAGCCACCACAACGCAGCGCTATTCTGACGCCTCAAAACTGAGGGAGGAGATCGAGGGAAAGGGATCCTTCACTTACTTTGCACCGAGTAATGAGGCTTGGGACAACTTGGATTCTGATATCCGTAGAGGTTTGGAGAGCAACGTGAATGTTGAATTACTGAATGCTTTACATAGTCACATGATTAATAAGAGAATGTTGACCAAGGACTTAAAAAATGGCATGATTATTCCTTCAATGTATAACAATTTGGGGCTTTTCATTAACCATTATCCTAATGGGGTTGTCACTGTTAATTGTGCTCGAATCATCCATGGGAACCAGATTGCAACAAATGGTGTTGTCCATGTCATTGACCGTGTGCTTACACAAATTGGTACCTCAATTCAAGACTTCATTGAAGCAGAAGATGACCTTTCATCTTTTAGAGCAGCTGCCATCACATCGGACATATTGGAGGCCCTTGGAAGAGACGGTCACTTCACACTCTTTGCTCCCACCAATGAGGCTTTTGAGAAACTTCCACGAGGTGTCCTAGAAAGGATCATGGGAGACAAAGTGGCTTCCGAAGCTCTTATGAAGTACCACATCTTAAATACTCTCCAGTGTTCTGAGTCTATTATGGGAGGAGCAGTCTTTGAGACGCTGGAAGGAAATACAATTGAGATAGGATGTGACGGTGACAGTATAACAGTAAATGGAATCAAAATGGTGAACAAAAAGGATATTGTGACAAATAATGGTGTGATCCATTTGATTGATCAGGTCCTAATTCCTGATTCTGCCAAACAAGTTATTGAGCTGGCTGGAAAACAGCAAACCACCTTCACGGATCTTGTGGCCCAATTAGGCTTGGCATCTGCTCTGAGGCCAGATGGAGAATACACTTTGCTGGCACCTGTGAATAATGCATTTTCTGATGATACTCTCAGCATGGATCAGCGCCTCCTTAAATTAATTCTGCAGAATCACATATTGAAAGTAAAAGTTGGCCTTAATGAGCTTTACAACGGGCAAATACTGGAAACCATCGGAGGCAAACAGCTCAGAGTCTTCGTATATCGTACAGCTGTCTGCATTGAAAATTCATGCATGGAGAAAGGGAGTAAGCAAGGGAGAAACGGTGCGATTCACATATTCCGCGAGATCATCAAGCCAGCAGAGAAATCCCTCCATGAAAAGTTAAAACAAGATAAGCGCTTTAGCACCTTCCTCAGCCTACTTGAAGCTGCAGACTTGAAAGAGCTCCTGACACAACCTGGAGACTGGACATTATTTGTGCCAACCAATGATGCTTTTAAGGGAATGACTAGTGAAGAAAAAGAAATTCTGATACGGGACAAAAATGCTCTTCAAAACATCATTCTTTATCACCTGACACCAGGAGTTTTCATTGGAAAAGGATTTGAACCTGGTGTTACTAACATTTTAAAGACCACACAAGGAAGCAAAATCTTTCTGAAAGAAGTAAATGATACACTTCTGGTGAATGAATTGAAATCAAAAGAATCTGACATCATGACAACAAATGGTGTAATTCATGTTGTAGATAAACTCCTCTATCCAGCAGACACACCTGTTGGAAATGATCAACTGCTGGAAATACTTAATAAATTAATCAAATACATCCAAATTAAGTTTGTTCGTGGTAGCACCTTCAAAGAAATCCCCGTGACTGTCTATAGACCCACACTAACAAAAGTCAAAATTGAAGGTGAACCTGAATTCAGACTGATTAAAGAAGGTGAAACAATAACTGAAGTGATCCATGGAGAGCCAATTATTAAAAAATACACCAAAATCATTGATGGAGTGCCTGTGGAAATAACTGAAAAAGAGACACGAGAAGAACGAATCATTACAGGTCCTGAAATAAAATACACTAGGATTTCTACTGGAGGTGGAGAAACAGAAGAAACTCTGAAGAAATTGTTACAAGAAGAGGTCACCAAGGTCACCAAATTCATTGAAGGTGGTGATGGTCATTTATTTGAAGATGAAGAAATTAAAAGACTGCTTCAGGGAGACACACCCGTGAGGAAGTTGCAAGCCAACAAAAAAGTTCAAGGATCTAGAAGACGATTAAGGGAAGGTCGTTCTCAGTGA-3' Human periostin gene mRNA variant 5 CDS (SEQ ID NO: 134)
5 '-3'
ヒトペリオスチン遺伝子ｍＲＮＡ　ｖａｒｉａｎｔ　６　ＣＤＳ（配列番号１３５）
5'-ATGATTCCCTTTTTACCCATGTTTTCTCTACTATTGCTGCTTATTGTTAACCCTATAAACGCCAACAATCATTATGACAAGATCTTGGCTCATAGTCGTATCAGGGGTCGGGACCAAGGCCCAAATGTCTGTGCCCTTCAACAGATTTTGGGCACCAAAAAGAAATACTTCAGCACTTGTAAGAACTGGTATAAAAAGTCCATCTGTGGACAGAAAACGACTGTGTTATATGAATGTTGCCCTGGTTATATGAGAATGGAAGGAATGAAAGGCTGCCCAGCAGTTTTGCCCATTGACCATGTTTATGGCACTCTGGGCATCGTGGGAGCCACCACAACGCAGCGCTATTCTGACGCCTCAAAACTGAGGGAGGAGATCGAGGGAAAGGGATCCTTCACTTACTTTGCACCGAGTAATGAGGCTTGGGACAACTTGGATTCTGATATCCGTAGAGGTTTGGAGAGCAACGTGAATGTTGAATTACTGAATGCTTTACATAGTCACATGATTAATAAGAGAATGTTGACCAAGGACTTAAAAAATGGCATGATTATTCCTTCAATGTATAACAATTTGGGGCTTTTCATTAACCATTATCCTAATGGGGTTGTCACTGTTAATTGTGCTCGAATCATCCATGGGAACCAGATTGCAACAAATGGTGTTGTCCATGTCATTGACCGTGTGCTTACACAAATTGGTACCTCAATTCAAGACTTCATTGAAGCAGAAGATGACCTTTCATCTTTTAGAGCAGCTGCCATCACATCGGACATATTGGAGGCCCTTGGAAGAGACGGTCACTTCACACTCTTTGCTCCCACCAATGAGGCTTTTGAGAAACTTCCACGAGGTGTCCTAGAAAGGATCATGGGAGACAAAGTGGCTTCCGAAGCTCTTATGAAGTACCACATCTTAAATACTCTCCAGTGTTCTGAGTCTATTATGGGAGGAGCAGTCTTTGAGACGCTGGAAGGAAATACAATTGAGATAGGATGTGACGGTGACAGTATAACAGTAAATGGAATCAAAATGGTGAACAAAAAGGATATTGTGACAAATAATGGTGTGATCCATTTGATTGATCAGGTCCTAATTCCTGATTCTGCCAAACAAGTTATTGAGCTGGCTGGAAAACAGCAAACCACCTTCACGGATCTTGTGGCCCAATTAGGCTTGGCATCTGCTCTGAGGCCAGATGGAGAATACACTTTGCTGGCACCTGTGAATAATGCATTTTCTGATGATACTCTCAGCATGGATCAGCGCCTCCTTAAATTAATTCTGCAGAATCACATATTGAAAGTAAAAGTTGGCCTTAATGAGCTTTACAACGGGCAAATACTGGAAACCATCGGAGGCAAACAGCTCAGAGTCTTCGTATATCGTACAGCTGTCTGCATTGAAAATTCATGCATGGAGAAAGGGAGTAAGCAAGGGAGAAACGGTGCGATTCACATATTCCGCGAGATCATCAAGCCAGCAGAGAAATCCCTCCATGAAAAGTTAAAACAAGATAAGCGCTTTAGCACCTTCCTCAGCCTACTTGAAGCTGCAGACTTGAAAGAGCTCCTGACACAACCTGGAGACTGGACATTATTTGTGCCAACCAATGATGCTTTTAAGGGAATGACTAGTGAAGAAAAAGAAATTCTGATACGGGACAAAAATGCTCTTCAAAACATCATTCTTTATCACCTGACACCAGGAGTTTTCATTGGAAAAGGATTTGAACCTGGTGTTACTAACATTTTAAAGACCACACAAGGAAGCAAAATCTTTCTGAAAGAAGTAAATGATACACTTCTGGTGAATGAATTGAAATCAAAAGAATCTGACATCATGACAACAAATGGTGTAATTCATGTTGTAGATAAACTCCTCTATCCAGCAGACACACCTGTTGGAAATGATCAACTGCTGGAAATACTTAATAAATTAATCAAATACATCCAAATTAAGTTTGTTCGTGGTAGCACCTTCAAAGAAATCCCCGTGACTGTCTATAGTCCTGAAATAAAATACACTAGGATTTCTACTGGAGGTGGAGAAACAGAAGAAACTCTGAAGAAATTGTTACAAGAAGAGGTCACCAAGGTCACCAAATTCATTGAAGGTGGTGATGGTCATTTATTTGAAGATGAAGAAATTAAAAGACTGCTTCAGGGAGACACACCCGTGAGGAAGTTGCAAGCCAACAAAAAAGTTCAAGGATCTAGAAGACGATTAAGGGAAGGTCGTTCTCAGTGA-3' Human periostin gene mRNA variant 6 CDS (SEQ ID NO: 135)
5 '-3'
ヒトペリオスチン遺伝子ｍＲＮＡ　ｖａｒｉａｎｔ　７　ＣＤＳ（配列番号１３６）
5'-ATGATTCCCTTTTTACCCATGTTTTCTCTACTATTGCTGCTTATTGTTAACCCTATAAACGCCAACAATCATTATGACAAGATCTTGGCTCATAGTCGTATCAGGGGTCGGGACCAAGGCCCAAATGTCTGTGCCCTTCAACAGATTTTGGGCACCAAAAAGAAATACTTCAGCACTTGTAAGAACTGGTATAAAAAGTCCATCTGTGGACAGAAAACGACTGTGTTATATGAATGTTGCCCTGGTTATATGAGAATGGAAGGAATGAAAGGCTGCCCAGCAGTTTTGCCCATTGACCATGTTTATGGCACTCTGGGCATCGTGGGAGCCACCACAACGCAGCGCTATTCTGACGCCTCAAAACTGAGGGAGGAGATCGAGGGAAAGGGATCCTTCACTTACTTTGCACCGAGTAATGAGGCTTGGGACAACTTGGATTCTGATATCCGTAGAGGTTTGGAGAGCAACGTGAATGTTGAATTACTGAATGCTTTACATAGTCACATGATTAATAAGAGAATGTTGACCAAGGACTTAAAAAATGGCATGATTATTCCTTCAATGTATAACAATTTGGGGCTTTTCATTAACCATTATCCTAATGGGGTTGTCACTGTTAATTGTGCTCGAATCATCCATGGGAACCAGATTGCAACAAATGGTGTTGTCCATGTCATTGACCGTGTGCTTACACAAATTGGTACCTCAATTCAAGACTTCATTGAAGCAGAAGATGACCTTTCATCTTTTAGAGCAGCTGCCATCACATCGGACATATTGGAGGCCCTTGGAAGAGACGGTCACTTCACACTCTTTGCTCCCACCAATGAGGCTTTTGAGAAACTTCCACGAGGTGTCCTAGAAAGGATCATGGGAGACAAAGTGGCTTCCGAAGCTCTTATGAAGTACCACATCTTAAATACTCTCCAGTGTTCTGAGTCTATTATGGGAGGAGCAGTCTTTGAGACGCTGGAAGGAAATACAATTGAGATAGGATGTGACGGTGACAGTATAACAGTAAATGGAATCAAAATGGTGAACAAAAAGGATATTGTGACAAATAATGGTGTGATCCATTTGATTGATCAGGTCCTAATTCCTGATTCTGCCAAACAAGTTATTGAGCTGGCTGGAAAACAGCAAACCACCTTCACGGATCTTGTGGCCCAATTAGGCTTGGCATCTGCTCTGAGGCCAGATGGAGAATACACTTTGCTGGCACCTGTGAATAATGCATTTTCTGATGATACTCTCAGCATGGATCAGCGCCTCCTTAAATTAATTCTGCAGAATCACATATTGAAAGTAAAAGTTGGCCTTAATGAGCTTTACAACGGGCAAATACTGGAAACCATCGGAGGCAAACAGCTCAGAGTCTTCGTATATCGTACAGCTGTCTGCATTGAAAATTCATGCATGGAGAAAGGGAGTAAGCAAGGGAGAAACGGTGCGATTCACATATTCCGCGAGATCATCAAGCCAGCAGAGAAATCCCTCCATGAAAAGTTAAAACAAGATAAGCGCTTTAGCACCTTCCTCAGCCTACTTGAAGCTGCAGACTTGAAAGAGCTCCTGACACAACCTGGAGACTGGACATTATTTGTGCCAACCAATGATGCTTTTAAGGGAATGACTAGTGAAGAAAAAGAAATTCTGATACGGGACAAAAATGCTCTTCAAAACATCATTCTTTATCACCTGACACCAGGAGTTTTCATTGGAAAAGGATTTGAACCTGGTGTTACTAACATTTTAAAGACCACACAAGGAAGCAAAATCTTTCTGAAAGAAGTAAATGATACACTTCTGGTGAATGAATTGAAATCAAAAGAATCTGACATCATGACAACAAATGGTGTAATTCATGTTGTAGATAAACTCCTCTATCCAGCAGACACACCTGTTGGAAATGATCAACTGCTGGAAATACTTAATAAATTAATCAAATACATCCAAATTAAGTTTGTTCGTGGTAGCACCTTCAAAGAAATCCCCGTGACTGTCTATAGTCCTGAAATAAAATACACTAGGATTTCTACTGGAGGTGGAGAAACAGAAGAAACTCTGAAGAAATTGTTACAAGAAGACACACCCGTGAGGAAGTTGCAAGCCAACAAAAAAGTTCAAGGATCTAGAAGACGATTAAGGGAAGGTCGTTCTCAGTGA-3' Human periostin gene mRNA variant 7 CDS (SEQ ID NO: 136)
5 '-3'
　前記配列番号１２１の塩基配列において、前記ハイブリダイズする位置は、ペリオスチン遺伝子の発現をより抑制できることから、好ましくは、前記ＣＤＳおよび前記３’ＵＴＲの少なくとも一方であり、より好ましくは、下記位置があげられる。
（ＣＤＳ－３’ＵＴＲ）
前記配列番号１２１の塩基配列における１２２９～３１３２番目の塩基配列（配列番号１２９の塩基配列における１１１１～２５１１番目の塩基配列および配列番号１３０の塩基配列における１～５０３番目の塩基配列）
（ＣＤＳ）
前記配列番号１２１の塩基配列における８１５～１１２７番目の塩基配列（配列番号１２９の塩基配列における６９７～１００９番目の塩基配列）
前記配列番号１２１の塩基配列における９６５～９８８番目の塩基配列（配列番号１２９の塩基配列における８４７～８７０番目の塩基配列）
前記配列番号１２１の塩基配列における１１０５～１１２７番目の塩基配列（配列番号１２９の塩基配列における９８７～１００９番目の塩基配列）
前記配列番号１２１の塩基配列における１２２９～１２５７番目の塩基配列（配列番号１２９の塩基配列における１１１１～１１３９番目の塩基配列）
前記配列番号１２１の塩基配列における１２７０～１３０１番目の塩基配列（配列番号１２９の塩基配列における１１５２～１１８３番目の塩基配列）
前記配列番号１２１の塩基配列における１４２６～１４４５番目の塩基配列（配列番号１２９の塩基配列における１３０８～１３２７番目の塩基配列）
前記配列番号１２１の塩基配列における１４８８～１５０９番目の塩基配列（配列番号１２９の塩基配列における１３７０～１３９１番目の塩基配列）
前記配列番号１２１の塩基配列における１７０６～１９６５番目の塩基配列（配列番号１２９の塩基配列における１５８８～１８４７番目の塩基配列）
前記配列番号１２１の塩基配列における２０７８～２１０３番目の塩基配列（配列番号１２９の塩基配列における１９６０～１９８５番目の塩基配列）
（３’ＵＴＲ）
前記配列番号１２１の塩基配列における２６３０～３３９０番目の塩基配列（配列番号１３０の塩基配列における１～７６１番目の塩基配列）
前記配列番号１２１の塩基配列における２９２１～３１３２番目の塩基配列（配列番号１３０の塩基配列における２９２～５０３番目の塩基配列）
前記配列番号１２１の塩基配列における２９２２～３０７８番目の塩基配列（配列番号１３０の塩基配列における２９３～４４９番目の塩基配列） In the base sequence of SEQ ID NO: 121, the hybridizing position is more preferably at least one of the CDS and the 3′UTR, and more preferably the following position because the expression of the periostin gene can be further suppressed. It is done.
(CDS-3'UTR)
The 1229th to 3132rd base sequence in the base sequence of SEQ ID NO: 121 (the 1111st to 2511th base sequence in the base sequence of SEQ ID NO: 129 and the 1st to 503rd base sequence in the base sequence of SEQ ID NO: 130)
(CDS)
815 to 1127th nucleotide sequence in the nucleotide sequence of SEQ ID NO: 121 (697 to 1009th nucleotide sequence in the nucleotide sequence of SEQ ID NO: 129)
The 965th to 988th base sequences in the base sequence of SEQ ID NO: 121 (the 847th to 870th base sequences in the base sequence of SEQ ID NO: 129)
1105th to 1127th base sequences in the base sequence of SEQ ID NO: 121 (the 987th to 1009th base sequences in the base sequence of SEQ ID NO: 129)
The 1229th to 1257th base sequence in the base sequence of SEQ ID NO: 121 (the 1111st to 1139th base sequence in the base sequence of SEQ ID NO: 129)
1270 to 1301 base sequence in the base sequence of SEQ ID NO: 121 (1152 to 1183 base sequences in the base sequence of SEQ ID NO: 129)
The 1426th to 1445th base sequences in the base sequence of SEQ ID NO: 121 (the 1308th to 1327th base sequences in the base sequence of SEQ ID NO: 129)
1488th to 1509th base sequence in the base sequence of SEQ ID NO: 121 (1370th to 1391st base sequence in the base sequence of SEQ ID NO: 129)
The 1706th to 1965th base sequences in the base sequence of SEQ ID NO: 121 (the 1588th to 1847th base sequences in the base sequence of SEQ ID NO: 129)
The 2078th to 2103rd base sequence in the base sequence of SEQ ID NO: 121 (the 1960th to 1985th base sequence in the base sequence of SEQ ID NO: 129)
(3'UTR)
2630th to 3390th base sequence in the base sequence of SEQ ID NO: 121 (1st to 761st base sequence in the base sequence of SEQ ID NO: 130)
The 2921st to 3132rd base sequence in the base sequence of SEQ ID NO: 121 (the 292th to 503rd base sequence in the base sequence of SEQ ID NO: 130)
The 2922th to 3078th base sequences in the base sequence of SEQ ID NO: 121 (the 293th to 449th base sequences in the base sequence of SEQ ID NO: 130)
　前記配列番号１２２の塩基配列において、前記ハイブリダイズする位置は、ペリオスチン遺伝子の発現をより抑制できることから、好ましくは、前記ＣＤＳおよび前記３’ＵＴＲの少なくとも一方であり、より好ましくは、下記位置、ならびに前記配列番号１２１におけるハイブリダイズする位置の前記ＣＤＳおよび前記３’ＵＴＲの例示と対応する位置である。前記対応する位置は、例えば、前記配列番号１２１の塩基配列および前記配列番号１２２の塩基配列を比較することにより特定できる。
（ＣＤＳ－３’ＵＴＲ）
前記配列番号１２２の塩基配列における１２２９～２９６１番目の塩基配列（配列番号１３１の塩基配列における１１１１～２３４０番目の塩基配列および配列番号１３０の塩基配列における１～５０３番目の塩基配列） In the base sequence of SEQ ID NO: 122, the position to hybridize is more preferably at least one of the CDS and the 3′UTR, more preferably the following position, since the expression of the periostin gene can be further suppressed. This is a position corresponding to the illustration of the CDS and the 3′UTR at the position to be hybridized in SEQ ID NO: 121. The corresponding position can be identified, for example, by comparing the base sequence of SEQ ID NO: 121 and the base sequence of SEQ ID NO: 122.
(CDS-3'UTR)
The 1229 to 2961st base sequence in the base sequence of SEQ ID NO: 122 (the 1111 to 2340th base sequence in the base sequence of SEQ ID NO: 131 and the 1st to 503rd base sequence in the base sequence of SEQ ID NO: 130)
　前記配列番号１２３の塩基配列において、前記ハイブリダイズする位置は、ペリオスチン遺伝子の発現をより抑制できることから、好ましくは、前記ＣＤＳおよび前記３’ＵＴＲの少なくとも一方であり、より好ましくは、下記位置、ならびに前記配列番号１２１におけるハイブリダイズする位置の前記ＣＤＳおよび前記３’ＵＴＲの例示と対応する位置である。前記対応する位置は、例えば、前記配列番号１２１の塩基配列および前記配列番号１２３の塩基配列を比較することにより特定できる。
（ＣＤＳ－３’ＵＴＲ）
前記配列番号１２３の塩基配列における１２２９～２９６７番目の塩基配列（配列番号１３２の塩基配列における１１１１～２３４６番目の塩基配列および配列番号１３０の塩基配列における１～５０３番目の塩基配列） In the base sequence of SEQ ID NO: 123, the hybridizing position is preferably at least one of the CDS and the 3′UTR, more preferably the following position, and more preferably, since the expression of the periostin gene can be further suppressed. This is a position corresponding to the illustration of the CDS and the 3′UTR at the position to be hybridized in SEQ ID NO: 121. The corresponding position can be identified by comparing the base sequence of SEQ ID NO: 121 and the base sequence of SEQ ID NO: 123, for example.
(CDS-3'UTR)
The 1229 to 2967th base sequence in the base sequence of SEQ ID NO: 123 (the 1111 to 2346th base sequence in the base sequence of SEQ ID NO: 132 and the 1st to 503rd base sequence in the base sequence of SEQ ID NO: 130)
　前記配列番号１２４の塩基配列において、前記ハイブリダイズする位置は、ペリオスチン遺伝子の発現をより抑制できることから、好ましくは、前記ＣＤＳおよび前記３’ＵＴＲの少なくとも一方であり、より好ましくは、下記位置、ならびに前記配列番号１２１におけるハイブリダイズする位置の前記ＣＤＳおよび前記３’ＵＴＲの例示と対応する位置である。前記対応する位置は、例えば、前記配列番号１２１の塩基配列および前記配列番号１２４の塩基配列を比較することにより特定できる。
（ＣＤＳ－３’ＵＴＲ）
前記配列番号１２４の塩基配列における１２２９～２８７７番目の塩基配列（配列番号１３３の塩基配列における１１１１～２２５６番目の塩基配列および配列番号１３０の塩基配列における１～５０３番目の塩基配列） In the base sequence of SEQ ID NO: 124, the hybridizing position is preferably at least one of the CDS and the 3′UTR, more preferably the following position, since the expression of the periostin gene can be further suppressed. This is a position corresponding to the illustration of the CDS and the 3′UTR at the position to be hybridized in SEQ ID NO: 121. The corresponding position can be specified, for example, by comparing the base sequence of SEQ ID NO: 121 and the base sequence of SEQ ID NO: 124.
(CDS-3'UTR)
The 1229 to 2877th base sequence in the base sequence of SEQ ID NO: 124 (the 1111 to 2256th base sequence in the base sequence of SEQ ID NO: 133 and the 1st to 503rd base sequence in the base sequence of SEQ ID NO: 130)
　前記配列番号１２５の塩基配列において、前記ハイブリダイズする位置は、ペリオスチン遺伝子の発現をより抑制できることから、好ましくは、前記ＣＤＳおよび前記３’ＵＴＲの少なくとも一方であり、より好ましくは、下記位置、ならびに前記配列番号１２１におけるハイブリダイズする位置の前記ＣＤＳおよび前記３’ＵＴＲの例示と対応する位置である。前記対応する位置は、例えば、前記配列番号１２１の塩基配列および前記配列番号１２５の塩基配列を比較することにより特定できる。
（ＣＤＳ－３’ＵＴＲ）
前記配列番号１２５の塩基配列における１２２９～３０５１番目の塩基配列（配列番号１３４の塩基配列における１１１１～２４３０番目の塩基配列および配列番号１３０の塩基配列における１～５０３番目の塩基配列） In the base sequence of SEQ ID NO: 125, the hybridizing position is preferably at least one of the CDS and the 3′UTR, more preferably the following position, and more preferably, since the expression of the periostin gene can be further suppressed. This is a position corresponding to the illustration of the CDS and the 3′UTR at the position to be hybridized in SEQ ID NO: 121. The corresponding position can be identified by comparing the base sequence of SEQ ID NO: 121 and the base sequence of SEQ ID NO: 125, for example.
(CDS-3'UTR)
The 1229-3051st base sequence in the base sequence of SEQ ID NO: 125 (the 1111-2430th base sequence in the base sequence of SEQ ID NO: 134 and the 1st to 503rd base sequence in the base sequence of SEQ ID NO: 130)
　前記配列番号１２６の塩基配列において、前記ハイブリダイズする位置は、ペリオスチン遺伝子の発現をより抑制できることから、好ましくは、前記ＣＤＳおよび前記３’ＵＴＲの少なくとも一方であり、より好ましくは、下記位置、ならびに前記配列番号１２１におけるハイブリダイズする位置の前記ＣＤＳおよび前記３’ＵＴＲの例示と対応する位置である。前記対応する位置は、例えば、前記配列番号１２１の塩基配列および前記配列番号１２６の塩基配列を比較することにより特定できる。
（ＣＤＳ－３’ＵＴＲ）
前記配列番号１２６の塩基配列における１２２９～２８７１番目の塩基配列（配列番号１３５の塩基配列における１１１１～２２５０番目の塩基配列および配列番号１３０の塩基配列における１～５０３番目の塩基配列） In the base sequence of SEQ ID NO: 126, the hybridizing position is preferably at least one of the CDS and the 3′UTR, more preferably the following position, since the expression of the periostin gene can be further suppressed. This is a position corresponding to the illustration of the CDS and the 3′UTR at the position to be hybridized in SEQ ID NO: 121. The corresponding position can be specified by comparing the base sequence of SEQ ID NO: 121 and the base sequence of SEQ ID NO: 126, for example.
(CDS-3'UTR)
The 1229 to 2871st base sequence in the base sequence of SEQ ID NO: 126 (the 1111 to 2250th base sequence in the base sequence of SEQ ID NO: 135 and the 1st to 503rd base sequence in the base sequence of SEQ ID NO: 130)
　前記配列番号１２７の塩基配列において、前記ハイブリダイズする位置は、ペリオスチン遺伝子の発現をより抑制できることから、好ましくは、前記ＣＤＳおよび前記３’ＵＴＲの少なくとも一方であり、より好ましくは、下記位置、ならびに前記配列番号１２１におけるハイブリダイズする位置の前記ＣＤＳおよび前記３’ＵＴＲの例示と対応する位置である。前記対応する位置は、例えば、前記配列番号１２１の塩基配列および前記配列番号１２７の塩基配列を比較することにより特定できる。
（ＣＤＳ－３’ＵＴＲ）
前記配列番号１２７の塩基配列における１２２９～２７８７番目の塩基配列（配列番号１３６の塩基配列における１１１１～２１６６番目の塩基配列および配列番号１３０の塩基配列における１～５０３番目の塩基配列） In the base sequence of SEQ ID NO: 127, the hybridizing position is preferably at least one of the CDS and the 3′UTR, more preferably the following position, since the expression of the periostin gene can be further suppressed. This is a position corresponding to the illustration of the CDS and the 3′UTR at the position to be hybridized in SEQ ID NO: 121. The corresponding position can be identified, for example, by comparing the base sequence of SEQ ID NO: 121 and the base sequence of SEQ ID NO: 127.
(CDS-3'UTR)
The 1229 to 2787th base sequence in the base sequence of SEQ ID NO: 127 (the 1111 to 2166th base sequence in the base sequence of SEQ ID NO: 136 and the 1st to 503rd base sequence in the base sequence of SEQ ID NO: 130)
　前記ａｓ４ヌクレオチドの長さは、特に制限されず、例えば、前記発現抑制配列の長さの説明を援用できる。前記ａｓ４ヌクレオチドの長さは、例えば、１９塩基長を除く。 The length of the as4 nucleotide is not particularly limited, and for example, the description of the length of the expression suppressing sequence can be used. The length of the as4 nucleotide excludes, for example, 19 bases.
　前記ａｓ４ヌクレオチドの具体例としては、例えば、前記ａｓ１ヌクレオチド、前記ａｓ２ヌクレオチド、および前記ａｓ３ヌクレオチドがあげられる。 Specific examples of the as4 nucleotide include the as1 nucleotide, the as2 nucleotide, and the as3 nucleotide.
　本発明の核酸分子は、前記核酸分子が３０ｎｍｏｌ／Ｌで存在する場合において、ヒトグリア芽腫細胞おけるペリオスチン遺伝子の発現を、例えば、４５％以上、５０％以上、５５％以上、６０％以上、６５％以上、７０％以上、７５％以上、８０％以上、８５％以上、９０％以上、９５％以上抑制してもよい。前記発現の抑制は、例えば、前記核酸分子が０ｎｍｏｌ／Ｌの場合における前記ヒトグリア芽腫細胞のペリオスチン遺伝子の発現量を基準とし、前記基準と前記核酸分子が３０ｎｍｏｌ／Ｌで存在する場合の前記ヒトグリア芽腫細胞のペリオスチン遺伝子の発現量とを比較することにより算出できる。前記ヒトグリア芽腫細胞は、例えば、Ａ１７２細胞（ATCC（登録商標） CRL-1620（商標））等があげられる。 When the nucleic acid molecule of the present invention is present at 30 nmol / L, the expression of the periostin gene in human glioblastoma cells is, for example, 45% or more, 50% or more, 55% or more, 60% or more, 65 % Or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. The suppression of the expression is based on, for example, the expression level of the periostin gene of the human glioblastoma cell when the nucleic acid molecule is 0 nmol / L, and the human glia when the reference and the nucleic acid molecule are present at 30 nmol / L. It can be calculated by comparing the expression level of periostin gene in blastoma cells. Examples of the human glioblastoma cells include A172 cells (ATCC (registered trademark) CRL-1620 (trademark)) and the like.
　本発明の核酸分子は、前記核酸分子が２０ｎｍｏｌ／Ｌで存在する場合において、マウス線維芽細胞おけるペリオスチン遺伝子の発現を、例えば、４５％以上、５０％以上、５５％以上、６０％以上、６５％以上、７０％以上、７５％以上、８０％以上、８５％以上、９０％以上、９５％以上抑制してもよい。前記発現の抑制は、例えば、前記核酸分子が０ｎｍｏｌ／Ｌの場合における前記マウス線維芽細胞のペリオスチン遺伝子の発現量を基準とし、前記基準と前記核酸分子が２０ｎｍｏｌ／Ｌで存在する場合の前記マウス線維芽細胞のペリオスチン遺伝子の発現量とを比較することにより算出できる。前記マウス線維芽細胞は、例えば、ＮＩＨ－３Ｔ３細胞（ATCC（登録商標） CRL-1658（商標））等があげられる。 When the nucleic acid molecule of the present invention is present at 20 nmol / L, the expression of the periostin gene in mouse fibroblasts is, for example, 45% or more, 50% or more, 55% or more, 60% or more, 65 % Or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. The suppression of the expression is based on, for example, the expression level of the periostin gene of the mouse fibroblast when the nucleic acid molecule is 0 nmol / L, and the mouse when the reference and the nucleic acid molecule are present at 20 nmol / L. It can be calculated by comparing the expression level of the periostin gene in fibroblasts. Examples of the mouse fibroblasts include NIH-3T3 cells (ATCC (registered trademark) CRL-1658 (trademark)) and the like.
　本発明の核酸分子は、さらに、付加配列を有してもよい。本発明の核酸分子が前記付加配列を有する場合、前記付加配列は、例えば、前記発現抑制配列において、前記ａｓヌクレオチドの３’末端および５’末端の少なくとも一方に付加されてもよく、前記核酸分子の製造が容易であることから、好ましくは前記５’末端に付加されている。 The nucleic acid molecule of the present invention may further have an additional sequence. When the nucleic acid molecule of the present invention has the additional sequence, the additional sequence may be added to at least one of the 3 ′ end and the 5 ′ end of the as nucleotide in the expression suppression sequence, for example. Is preferably added to the 5 ′ end.
　前記付加配列は、特に制限されず、例えば、長さおよび配列も、特に制限されない。前記付加配列は、例えば、（Ｎ）ｎで表わすことができる。Ｎは、塩基であり、例えば、天然の塩基でもよいし、人工塩基でもよい。前記天然塩基は、例えば、Ａ、Ｃ、Ｇ、ＵおよびＴがあげられる。前記ｎは、正の整数であり、付加配列の塩基長を示す。前記付加配列（Ｎ）ｎの長さ（ｎ）は、例えば、１、２、３塩基長であり、好ましくは１または２塩基長であり、より好ましくは２塩基長である。前記付加配列（Ｎ）ｎの長さが２塩基長以上の場合（ｎ≧２）、連続する塩基（Ｎ）は、例えば、同じ塩基でもよいし、異なる塩基でもよい。前記（Ｎ）ｎは、例えば、３’側または５’側から、ＵＵ、ＣＵ、ＵＣ、ＧＡ、ＡＧ、ＧＣ、ＵＡ、ＡＡ、ＣＣ、ＧＵ、ＵＧ、ＣＧ、ＡＵ、ＴＴ等が例示できる。 The additional sequence is not particularly limited, and for example, the length and sequence are not particularly limited. The additional sequence can be represented by (N) n , for example. N is a base, and may be a natural base or an artificial base, for example. Examples of the natural base include A, C, G, U, and T. The n is a positive integer and indicates the base length of the additional sequence. The additional sequence (N) n has a length (n) of, for example, 1, 2, 3 bases, preferably 1 or 2 bases, and more preferably 2 bases. When the additional sequence (N) n has a length of 2 bases or more (n ≧ 2), the consecutive bases (N) may be, for example, the same base or different bases. Examples of (N) n include UU, CU, UC, GA, AG, GC, UA, AA, CC, GU, UG, CG, AU, and TT from the 3 ′ side or the 5 ′ side.
　前記発現抑制配列が前記付加配列を有する場合、前記発現抑制配列は、例えば、前記ａｓ１ヌクレオチドと前記付加配列とが連結された配列があげられる。 When the expression suppression sequence has the additional sequence, examples of the expression suppression sequence include a sequence in which the as1 nucleotide and the additional sequence are linked.
　前記核酸分子は、例えば、保護基を有する核酸分子であることが好ましい。前記保護基は、例えば、前記核酸分子に対して、エキソヌクレアーゼ等のヌクレアーゼに対し耐性を付与する修飾基である。前記保護基を有する核酸分子は、例えば、後述する修飾ヌクレオチド残基を含む核酸分子があげられる。 The nucleic acid molecule is preferably a nucleic acid molecule having a protecting group, for example. The protecting group is, for example, a modifying group that imparts resistance to a nuclease such as exonuclease with respect to the nucleic acid molecule. Examples of the nucleic acid molecule having the protecting group include a nucleic acid molecule containing a modified nucleotide residue described later.
　本発明の核酸分子の構成単位は、特に制限されず、例えば、ヌクレオチド残基があげられる。前記ヌクレオチド残基は、例えば、リボヌクレオチド残基およびデオキシリボヌクレオチド残基があげられる。前記ヌクレオチド残基は、例えば、修飾されていない非修飾ヌクレオチド残基および修飾された修飾ヌクレオチド残基があげられる。 The structural unit of the nucleic acid molecule of the present invention is not particularly limited, and examples thereof include nucleotide residues. Examples of the nucleotide residue include a ribonucleotide residue and a deoxyribonucleotide residue. Examples of the nucleotide residue include an unmodified unmodified nucleotide residue and a modified modified nucleotide residue.
　本発明の核酸分子は、例えば、ＤＮＡ分子でもよいし、ＲＮＡ分子でもよいし、ＤＮＡ分子とＲＮＡ分子とを含む分子でもよい。本発明の核酸分子がＤＮＡ分子の場合、前記核酸分子は、例えば、デオキシリボヌクレオチド残基のみからなるＤＮＡ分子でもよいし、デオキシリボヌクレオチド残基の他に、リボヌクレオチド残基および／または非ヌクレオチド残基を含むＤＮＡ分子でもよい。本発明の核酸分子がＲＮＡ分子の場合、前記核酸分子は、例えば、リボヌクレオチド残基のみからなるＲＮＡ分子でもよいし、リボヌクレオチド残基の他に、デオキシリボヌクレオチド残基および／または非ヌクレオチド残基を含むＲＮＡ分子でもよい。 The nucleic acid molecule of the present invention may be, for example, a DNA molecule, an RNA molecule, or a molecule containing a DNA molecule and an RNA molecule. When the nucleic acid molecule of the present invention is a DNA molecule, the nucleic acid molecule may be, for example, a DNA molecule consisting of only deoxyribonucleotide residues, and in addition to deoxyribonucleotide residues, ribonucleotide residues and / or non-nucleotide residues It may be a DNA molecule containing When the nucleic acid molecule of the present invention is an RNA molecule, the nucleic acid molecule may be, for example, an RNA molecule consisting of only ribonucleotide residues, and in addition to ribonucleotide residues, deoxyribonucleotide residues and / or non-nucleotide residues RNA molecules containing
　前記ヌクレオチド残基は、例えば、構成要素として、糖、塩基およびリン酸を含む。前記ヌクレオチド残基は、前述のように、例えば、リボヌクレオチド残基およびデオキシリボヌクレオチド残基があげられる。前記リボヌクレオチド残基は、例えば、糖としてリボース残基を有し、塩基として、アデニン（Ａ）、グアニン（Ｇ）、シトシン（Ｃ）およびウラシル（Ｕ）を有し、前記デオキシリボヌクレオチド残基は、例えば、糖としてデオキシリボース残基を有し、塩基として、アデニン（Ａ）、グアニン（Ｇ）、シトシン（Ｃ）およびチミン（Ｔ）を有する。 The nucleotide residue includes, for example, a sugar, a base and a phosphate as constituent elements. Examples of the nucleotide residue include a ribonucleotide residue and a deoxyribonucleotide residue as described above. The ribonucleotide residue has, for example, a ribose residue as a sugar, and has adenine (A), guanine (G), cytosine (C) and uracil (U) as bases, and the deoxyribonucleotide residue is For example, it has a deoxyribose residue as a sugar and has adenine (A), guanine (G), cytosine (C) and thymine (T) as bases.
　前記ヌクレオチド残基は、非修飾ヌクレオチド残基および修飾ヌクレオチド残基があげられる。前記非修飾ヌクレオチド残基は、前記各構成要素が、例えば、天然に存在するものと同一または実質的に同一であり、好ましくは、人体において天然に存在するものと同一または実質的に同一である。 The nucleotide residue includes an unmodified nucleotide residue and a modified nucleotide residue. In the unmodified nucleotide residue, each of the constituent elements is, for example, the same or substantially the same as that existing in nature, preferably the same or substantially the same as that naturally occurring in the human body. .
　前記修飾ヌクレオチド残基は、例えば、前記非修飾ヌクレオチド残基を修飾したヌクレオチド残基である。前記修飾ヌクレオチド残基は、例えば、前記非修飾ヌクレオチド残基の構成要素のいずれが修飾されてもよい。本発明において、「修飾」は、例えば、前記構成要素の置換、付加および／または欠失、前記構成要素における原子および／または官能基の置換、付加および／または欠失であり、「改変」ということができる。前記修飾ヌクレオチド残基は、例えば、天然に存在するヌクレオチド残基、人工的に修飾したヌクレオチド残基等があげられる。前記天然由来の修飾ヌクレオチド残基は、例えば、リンバックら（Limbach et al．、1994、Summary：the modified nucleosides of RNA、Nucleic Acids Res．22：2183～2196）を参照できる。また、前記修飾ヌクレオチド残基は、例えば、前記ヌクレオチドの代替物の残基でもよい。 The modified nucleotide residue is, for example, a nucleotide residue obtained by modifying the unmodified nucleotide residue. In the modified nucleotide residue, for example, any of the constituent elements of the unmodified nucleotide residue may be modified. In the present invention, “modification” refers to, for example, substitution, addition and / or deletion of the component, substitution, addition and / or deletion of atoms and / or functional groups in the component, and is referred to as “modification”. be able to. Examples of the modified nucleotide residue include naturally occurring nucleotide residues, artificially modified nucleotide residues, and the like. See, for example, Limbach et al. (Limbach et al., 1994, Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183-2196) for the naturally occurring modified nucleotide residues. The modified nucleotide residue may be, for example, a residue of the nucleotide substitute.
　前記ヌクレオチド残基の修飾は、例えば、糖－リン酸骨格（以下、「糖リン酸骨格」ともいう。）の修飾があげられ、具体的には、糖残基の修飾およびリン酸の修飾があげられる。 Examples of the modification of the nucleotide residue include modification of a sugar-phosphate skeleton (hereinafter also referred to as “sugar phosphate skeleton”). Specifically, the modification of a sugar residue and the modification of a phosphate are possible. can give.
　前記糖リン酸骨格において、例えば、前記核酸分子の安定性を向上できることから、前記デオキシリボース残基および／またはリボース残基を修飾してもよい。前記デオキシリボース残基は、２’位炭素を修飾でき、具体的には、例えば、２’位炭素に結合する水素を、フルオロ等のハロゲンに置換できる。また、前記デオキシリボース残基は、例えば、前記２’位炭素の水素を水酸基に置換することで、デオキシリボース残基をリボース残基に置換できる。前記リボース残基は、例えば、２’位炭素を修飾でき、具体的には、例えば、２’位炭素に結合する水酸基を、水素またはフルオロ等のハロゲンに置換できる。また、前記リボース残基は、例えば、２’位炭素に結合する水酸基の水素を置換してもよい。具体例として、例えば、２’－Ｏ－メチルリボース残基、２’－Ｏ－メトキシエチルリボース残基、２’－Ｏ－ジメチルアミノエチルリボース残基、２’－Ｏ－ジメチルアミノエトキシエチルリボース残基等があげられる。前記リボース残基は、例えば、前記２’位炭素の水酸基を水素に置換することで、リボース残基をデオキシリボース残基に置換できる。前記リボース残基は、例えば、立体異性体に置換でき、例えば、アラビノース残基に置換してもよい。前記修飾ヌクレオチド残基は、この他に、例えば、前記ヌクレオチド残基を構成する糖残基において、前記糖残基の環状構造を架橋した二環式の糖残基としてもよい。前記二環式の糖残基を含む修飾ヌクレオチド残基の具体例は、特に制限されず、公知の二環式人工核酸モノマー残基があげられる。前記二環式人工核酸モノマー残基は、例えば、ｃＥｔ（ｃｏｎｓｔｒａｉｎｅｄ　ｅｔｈｙｌ　ｂｉｃｙｃｌｉｃ　ｎｕｃｌｅｉｃ　ａｃｉｄ、Ionis Pharmaceuticals社製）、ＬＮＡ（（商標）、Ｌｏｃｋｅｄ　Ｎｕｃｌｅｉｃ　Ａｃｉｄ）、ＥＮＡ（登録商標、２’－Ｏ，４’－Ｃ－Ｅｔｈｙｌｅｎｅｂｒｉｄｇｅｄ　Ｎｕｃｌｅｉｃ　Ａｃｉｄ）等があげられ、好ましくは、ＬＮＡである。 In the sugar phosphate skeleton, for example, since the stability of the nucleic acid molecule can be improved, the deoxyribose residue and / or ribose residue may be modified. The deoxyribose residue can modify the 2'-position carbon. Specifically, for example, hydrogen bonded to the 2'-position carbon can be substituted with a halogen such as fluoro. The deoxyribose residue can be substituted with a ribose residue by, for example, substituting the hydrogen at the 2'-position with a hydroxyl group. The ribose residue can be modified, for example, at the 2′-position carbon. Specifically, for example, a hydroxyl group bonded to the 2′-position carbon can be replaced with hydrogen or a halogen such as fluoro. In addition, the ribose residue may replace, for example, a hydroxyl group hydrogen bonded to the 2'-position carbon. As a specific example, for example, 2′-O-methylribose residue, 2′-O-methoxyethylribose residue, 2′-O-dimethylaminoethylribose residue, 2′-O-dimethylaminoethoxyethylribose residue Group and the like. The ribose residue can be substituted with a deoxyribose residue by, for example, substituting the hydroxyl group at the 2'-position with hydrogen. The ribose residue can be substituted with, for example, a stereoisomer, and can be substituted with, for example, an arabinose residue. In addition to this, the modified nucleotide residue may be, for example, a bicyclic sugar residue obtained by crosslinking a cyclic structure of the sugar residue in the sugar residue constituting the nucleotide residue. Specific examples of the modified nucleotide residue containing the bicyclic sugar residue are not particularly limited, and examples thereof include known bicyclic artificial nucleic acid monomer residues. The bicyclic artificial nucleic acid monomer residues include, for example, cEt (constrained ethyl bicyclic acid, manufactured by Ionis Pharmaceuticals), LNA (TM), Locked Nucleic Acid, ENA (registered trademark, 2'-O, 4 '). -C-Ethylenebridged Nucleic Acid) and the like, preferably LNA.
　前記糖リン酸骨格は、例えば、非デオキシリボース残基、非リボース残基および／または非リン酸を有する非糖リン酸骨格に置換してもよい。前記非糖リン酸骨格は、例えば、前記糖リン酸骨格の非荷電体があげられる。前記非糖リン酸骨格に置換された、前記ヌクレオチドの代替物は、例えば、モルホリノ、シクロブチル、ピロリジン、ＰＮＡ（ペプチド核酸）等があげられる。 The sugar phosphate skeleton may be substituted with a non-sugar phosphate skeleton having a non-deoxyribose residue, a non-ribose residue and / or a non-phosphate, for example. Examples of the non-sugar phosphate skeleton include uncharged bodies of the sugar phosphate skeleton. Examples of the substitute for the nucleotide substituted with the non-sugar phosphate skeleton include morpholino, cyclobutyl, pyrrolidine, PNA (peptide nucleic acid) and the like.
　前記糖リン酸骨格において、例えば、リン酸基を修飾できる。前記糖リン酸骨格において、糖残基に最も隣接するリン酸基は、αリン酸基と呼ばれる。前記αリン酸基は、負に荷電し、その電荷は、糖残基に非結合の２つの酸素原子にわたって、均一に分布している。前記αリン酸基における４つの酸素原子のうち、ヌクレオチド残基間のホスホジエステル結合において、糖残基と非結合である２つの酸素原子は、以下、「非結合（ｎｏｎ－ｌｉｎｋｉｎｇ）酸素」ともいう。他方、前記ヌクレオチド残基間のホスホジエステル結合において、糖残基と結合している２つの酸素原子は、以下、「結合（ｌｉｎｋｉｎｇ）酸素」という。前記αリン酸基は、例えば、非荷電となる修飾、または、前記非結合酸素における電荷分布が非対称型となる修飾を行うことが好ましい。 In the sugar phosphate skeleton, for example, a phosphate group can be modified. In the sugar phosphate skeleton, the phosphate group closest to the sugar residue is called an α-phosphate group. The α-phosphate group is negatively charged, and the charge is evenly distributed over two oxygen atoms that are not bound to a sugar residue. Of the four oxygen atoms in the α-phosphate group, in the phosphodiester bond between nucleotide residues, the two oxygen atoms that are non-bonded to the sugar residue are hereinafter referred to as “non-linking oxygen”. Say. On the other hand, in the phosphodiester bond between the nucleotide residues, the two oxygen atoms bonded to the sugar residue are hereinafter referred to as “linking oxygen”. The α-phosphate group is preferably subjected to, for example, a modification that makes it uncharged or a modification that makes the charge distribution in the unbound oxygen asymmetric.
　前記リン酸基は、例えば、前記非結合酸素を置換してもよい。前記酸素は、例えば、Ｓ（硫黄）、Ｓｅ（セレン）、Ｂ（ホウ素）、Ｃ（炭素）、Ｈ（水素）、Ｎ（窒素）およびＯＲ（Ｒは、アルキル基またはアリール基）のいずれかの原子で置換でき、好ましくは、Ｓで置換される。前記非結合酸素は、例えば、両方が置換されていることが好ましく、より好ましくは、両方がＳで置換される。前記修飾リン酸基は、例えば、ホスホロチオエート、ホスホロジチオエート、ホスホロセレネート、ボラノホスフェート、ボラノホスフェートエステル、ホスホネート水素、ホスホロアミデート、アルキルまたはアリールホスホネート、およびホスホトリエステル等があげられ、中でも、ホスホロチオエート、前記２つの非結合酸素が両方ともＳで置換されているホスホロジチオエートが好ましい。 The phosphate group may replace the non-bonded oxygen, for example. The oxygen is, for example, one of S (sulfur), Se (selenium), B (boron), C (carbon), H (hydrogen), N (nitrogen), and OR (R is an alkyl group or an aryl group). And is preferably substituted with S. In the non-bonded oxygen, for example, both are preferably substituted, and more preferably, both are substituted with S. Examples of the modified phosphate group include phosphorothioate, phosphorodithioate, phosphoroselenate, boranophosphate, boranophosphate ester, phosphonate hydrogen, phosphoramidate, alkyl or arylphosphonate, and phosphotriester. Among them, phosphorothioate and phosphorodithioate in which the two non-bonded oxygens are both substituted with S are preferable.
　前記リン酸基は、例えば、前記結合酸素を置換してもよい。前記酸素は、例えば、Ｓ（硫黄）、Ｃ（炭素）およびＮ（窒素）のいずれかの原子で置換でき、前記修飾リン酸基は、例えば、Ｎで置換した架橋ホスホロアミデート、Ｓで置換した架橋ホスホロチオエート、およびＣで置換した架橋メチレンホスホネート等があげられる。前記結合酸素の置換は、例えば、本発明の核酸分子の５’末端ヌクレオチド残基および３’末端ヌクレオチド残基の少なくとも一方において行うことが好ましく、５’側の場合、Ｃによる置換が好ましく、３’側の場合、Ｎによる置換が好ましい。 The phosphate group may substitute, for example, the bonded oxygen. The oxygen can be substituted, for example, with any atom of S (sulfur), C (carbon) and N (nitrogen), and the modified phosphate group is, for example, a bridged phosphoramidate, S substituted with N Substituted bridged phosphorothioates, bridged methylene phosphonates substituted with C, and the like. The binding oxygen substitution is preferably performed, for example, on at least one of the 5 ′ terminal nucleotide residue and the 3 ′ terminal nucleotide residue of the nucleic acid molecule of the present invention. For the 'side, substitution with N is preferred.
　前記リン酸基は、例えば、前記リン非含有のリンカーに置換してもよい。前記リンカーは、例えば、シロキサン、カーボネート、カルボキシメチル、カルバメート、アミド、チオエーテル、エチレンオキサイドリンカー、スルホネート、スルホンアミド、チオホルムアセタール、ホルムアセタール、オキシム、メチレンイミノ、メチレンメチルイミノ、メチレンヒドラゾ、メチレンジメチルヒドラゾ、およびメチレンオキシメチルイミノ等を含み、好ましくは、メチレンカルボニルアミノ基およびメチレンメチルイミノ基を含む。 The phosphate group may be substituted with, for example, the phosphorus-free linker. Examples of the linker include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioform acetal, form acetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethyl. Hydrazo, methyleneoxymethylimino and the like, preferably methylenecarbonylamino group and methylenemethylimino group.
　本発明の核酸分子は、例えば、３’末端および５’末端の少なくとも一方のヌクレオチド残基が修飾されてもよい。前記修飾は、例えば、３’末端および５’末端のいずれか一方でもよいし、両方でもよい。前記修飾は、例えば、前述の通りであり、好ましくは、末端のリン酸基に行うことが好ましい。前記リン酸基は、例えば、全体を修飾してもよいし、前記リン酸基における１つ以上の原子を修飾してもよい。前者の場合、例えば、リン酸基全体の置換でもよいし、欠失でもよい。 In the nucleic acid molecule of the present invention, for example, at least one nucleotide residue at the 3 'end and the 5' end may be modified. The modification may be, for example, either the 3 'end or the 5' end, or both. The modification is, for example, as described above, and is preferably performed on the terminal phosphate group. For example, the phosphate group may be modified entirely, or one or more atoms in the phosphate group may be modified. In the former case, for example, the entire phosphate group may be substituted or deleted.
　前記末端のヌクレオチド残基の修飾は、例えば、他の分子の付加があげられる。前記他の分子は、例えば、後述するような標識物質、保護基等の機能性分子があげられる。前記保護基は、例えば、Ｓ（硫黄）、Ｓｉ（ケイ素）、Ｂ（ホウ素）、エステル含有基等があげられる。前記標識物質等の機能性分子は、例えば、本発明の核酸分子の検出等に利用できる。 Examples of the modification of the terminal nucleotide residue include addition of other molecules. Examples of the other molecule include functional molecules such as a labeling substance and a protecting group as described later. Examples of the protecting group include S (sulfur), Si (silicon), B (boron), ester-containing groups, and the like. The functional molecule such as the labeling substance can be used for detecting the nucleic acid molecule of the present invention, for example.
　前記他の分子は、例えば、前記ヌクレオチド残基のリン酸基に付加してもよいし、スペーサーを介して、前記リン酸基または前記糖残基に付加してもよい。前記スペーサーの末端原子は、例えば、前記リン酸基の前記結合酸素、または、糖残基のＯ、Ｎ、ＳもしくはＣに、付加または置換できる。前記糖残基の結合部位は、例えば、３’位のＣもしくは５’位のＣ、またはこれらに結合する原子が好ましい。前記スペーサーは、例えば、前記ＰＮＡ等のヌクレオチド代替物の末端原子に、付加または置換することもできる。 The other molecule may be added to the phosphate group of the nucleotide residue, for example, or may be added to the phosphate group or the sugar residue via a spacer. The terminal atom of the spacer can be added or substituted, for example, to the binding oxygen of the phosphate group or O, N, S or C of the sugar residue. The binding site of the sugar residue is preferably, for example, C at the 3 'position or C at the 5' position, or an atom bonded thereto. The spacer can be added or substituted at a terminal atom of a nucleotide substitute such as PNA.
　前記スペーサーは、特に制限されず、例えば、－（ＣＨ２）ｎ－、－（ＣＨ２）ｎＮ－、－（ＣＨ２）ｎＯ－、－（ＣＨ２）ｎＳ－、Ｏ（ＣＨ２ＣＨ２Ｏ）ｎＣＨ２ＣＨ２ＯＨ、無塩基糖、アミド、カルボキシ、アミン、オキシアミン、オキシイミン、チオエーテル、ジスルフィド、チオ尿素、スルホンアミド、およびモルホリノ等、ならびに、ビオチン試薬およびフルオレセイン試薬等を含んでもよい。前記式において、ｎは、正の整数であり、ｎ＝３または６が好ましい。 The spacer is not particularly limited. For example, — (CH 2 ) n —, — (CH 2 ) n N—, — (CH 2 ) n O—, — (CH 2 ) n S—, O (CH 2 CH 2 O) n CH 2 CH 2 OH, abasic sugar, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, morpholino and the like, and biotin reagent and fluorescein reagent Good. In the above formula, n is a positive integer, and n = 3 or 6 is preferable.
　前記末端に付加する分子は、これらの他に、例えば、色素、インターカレート剤（例えば、アクリジン）、架橋剤（例えば、ソラレン、マイトマイシンＣ）、抗がん剤、ポルフィリン（ＴＰＰＣ４、テキサフィリン、サッフィリン）、多環式芳香族炭化水素（例えば、フェナジン、ジヒドロフェナジン）、人工エンドヌクレアーゼ（例えば、ＥＤＴＡ）、親油性担体（例えば、コレステロール、コール酸、アダマンタン酢酸、１－ピレン酪酸、ジヒドロテストステロン、１，３－ビス－Ｏ（ヘキサデシル）グリセロール、ゲラニルオキシヘキシル基、ヘキサデシルグリセロール、ボルネオール、メントール、１，３－プロパンジオール、ヘプタデシル基、パルミチン酸、ミリスチン酸、Ｏ３－（オレオイル）リトコール酸、Ｏ３－（オレオイル）コール酸、ジメトキシトリチル、またはフェノキサジン）およびペプチド複合体（例えば、アンテナペディアペプチド、Ｔａｔペプチド、ＲＧＤペプチド）、アルキル化剤、リン酸、アミノ、メルカプト、ＰＥＧ（例えば、ＰＥＧ－４０Ｋ）、ＭＰＥＧ、［ＭＰＥＧ］２、ポリアミノ、アルキル、置換アルキル、放射線標識マーカー、酵素、ハプテン（例えば、ビオチン）、抗体、輸送／吸収促進剤（例えば、アスピリン、ビタミンＡ、ビタミンＥ、葉酸）、合成リボヌクレアーゼ（例えば、イミダゾール、ビスイミダゾール、ヒスタミン、イミダゾールクラスター、アクリジン－イミダゾール複合体、テトラアザマクロ環のＥｕ３＋複合体）等、糖（例えば、Ｎ－アセチルガラクトサミン、ガラクトース、マンノース）があげられる。 In addition to these, the molecule added to the terminal includes, for example, a dye, an intercalating agent (for example, acridine), a crosslinking agent (for example, psoralen, mitomycin C), an anticancer agent, a porphyrin (TPPC4, texaphyrin, suffirin). ), Polycyclic aromatic hydrocarbons (eg phenazine, dihydrophenazine), artificial endonucleases (eg EDTA), lipophilic carriers (eg cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1 , 3-bis-O (hexadecyl) glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3 -(Oleoi ) Cholic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (eg, antennapedia peptide, Tat peptide, RGD peptide), alkylating agents, phosphate, amino, mercapto, PEG (eg, PEG-40K), MPEG , [MPEG] 2 , polyamino, alkyl, substituted alkyl, radiolabeled marker, enzyme, hapten (eg, biotin), antibody, transport / absorption enhancer (eg, aspirin, vitamin A, vitamin E, folic acid), synthetic ribonuclease ( for example, imidazole, bisimidazole, histamine, imidazole clusters, acridine - imidazole complexes, Eu 3+ complex of tetra-aza macrocycles), etc., sugars (e.g., N- acetylgalactosamine, galactose, mannose) and the like
　本発明の核酸分子は、前記５’末端が、例えば、リン酸基またはリン酸基アナログで修飾されてもよい。前記リン酸基は、例えば、５’一リン酸（(HO)2(O)P-O-5’）、５’二リン酸（(HO)2(O)P-O-P(HO)(O)-O-5’）、５’三リン酸（(HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5’）、５’－グアノシンキャップ（７－メチル化または非メチル化、7m-G-O-5’-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5’）、５’－アデノシンキャップ（Ａｐｐｐ）、任意の修飾または非修飾ヌクレオチドキャップ構造（N-O-5’-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5’）、５’一チオリン酸（ホスホロチオエート：(HO)2(S)P-O-5’）、５’一ジチオリン酸（ホスホロジチオエート：(HO)(HS)(S)P-O-5’）、５’－ホスホロチオール酸（(HO)2(O)P-S-5’）、硫黄置換の一リン酸、二リン酸および三リン酸（例えば、５’－α－チオ三リン酸、５’－γ－チオ三リン酸等）、５’－ホスホルアミデート（(HO)2(O)P-NH-5’、(HO)(NH2)(O)P-O-5’）、５’－アルキルホスホン酸（例えば、RP(OH)(O)-O-5’、(OH)2(O)P-5’-CH2、Ｒはアルキル（例えば、メチル、エチル、イソプロピル、プロピル等））、５’－アルキルエーテルホスホン酸（例えば、RP(OH)(O)-O-5’、Ｒはアルキルエーテル（例えば、メトキシメチル、エトキシメチル等））等があげられる。 In the nucleic acid molecule of the present invention, the 5 ′ end may be modified with, for example, a phosphate group or a phosphate group analog. The phosphate group is, for example, 5 ′ monophosphate ((HO) 2 (O) PO-5 ′), 5 ′ diphosphate ((HO) 2 (O) POP (HO) (O) —O— 5 '), 5' triphosphate ((HO) 2 (O) PO- (HO) (O) POP (HO) (O) -O-5 '), 5'-guanosine cap (7-methylated or Unmethylated, 7m-GO-5 '-(HO) (O) PO- (HO) (O) POP (HO) (O) -O-5'), 5'-adenosine cap (Appp), optional Modified or unmodified nucleotide cap structure (NO-5 '-(HO) (O) PO- (HO) (O) POP (HO) (O) -O-5'), 5 'monothiophosphate (phosphorothioate: ( HO) 2 (S) PO-5 ′), 5 ′ monodithiophosphoric acid (phosphorodithioate: (HO) (HS) (S) PO-5 ′), 5′-phosphorothiolic acid ((HO) 2 (O) PS-5 ′), sulfur-substituted monophosphate, diphosphate and triphosphate (eg, 5′-α-thiotriphosphate, 5′-γ-thiotriphosphate, etc.), 5 ′ Phosphoramidates ((HO) 2 (O) P—NH-5 ′, (HO) (NH 2 ) (O) PO-5 ′), 5′-alkylphosphonic acids (eg RP (OH) ( O) -O-5 ' (OH) 2 (O) P -5'-CH 2, R is alkyl (e.g., methyl, ethyl, isopropyl, propyl, etc.)), 5'-alkyl ether phosphonic acid (e.g., RP (OH) (O) - O-5 ′ and R include alkyl ethers (for example, methoxymethyl, ethoxymethyl, etc.).
　前記ヌクレオチド残基において、前記塩基は、特に制限されない。前記塩基は、例えば、天然の塩基でもよいし、非天然の塩基でもよい。前記塩基は、例えば、天然由来でもよいし、合成品でもよい。前記塩基は、例えば、一般的な塩基、その修飾アナログ等が使用できる。 In the nucleotide residue, the base is not particularly limited. The base may be, for example, a natural base or a non-natural base. The base may be, for example, naturally derived or a synthetic product. As the base, for example, a general base or a modified analog thereof can be used.
　前記塩基は、例えば、アデニンおよびグアニン等のプリン塩基、シトシン、ウラシルおよびチミン等のピリミジン塩基があげられる。前記塩基は、この他に、イノシン、キサンチン、ヒポキサンチン、プリン、イソグアニン、イソシトシン、７‐デアザアデニン等があげられる。前記塩基は、例えば、２－アミノアデニン、６－メチル化プリン等のアルキル誘導体；２－プロピル化プリン等のアルキル誘導体；５－ハロウラシルおよび５－ハロシトシン；５－プロピニルウラシルおよび５－プロピニルシトシン；６－アゾウラシル、６－アゾシトシンおよび６－アゾチミン；５－ウラシル（プソイドウラシル）、４－チオウラシル、５－ハロウラシル、５－（２－アミノプロピル）ウラシル、５－アミノアリルウラシル；８－ハロ化、アミノ化、チオール化、チオアルキル化、ヒドロキシル化および他の８－置換プリン；５－トリフルオロメチル化および他の５－置換ピリミジン；７－メチルグアニン；５－置換ピリミジン；６－アザピリミジン；Ｎ－２、Ｎ－６、およびＯ－６置換プリン（２－アミノプロピルアデニンを含む）；５－プロピニルウラシルおよび５－プロピニルシトシン；ジヒドロウラシル；３－デアザ－５－アザシトシン；２－アミノプリン；５－アルキルウラシル；７－アルキルグアニン；５－アルキルシトシン；７－デアザアデニン；Ｎ６，Ｎ６－ジメチルアデニン；２，６－ジアミノプリン；５－アミノ－アリル－ウラシル；Ｎ３－メチルウラシル；置換１，２，４－トリアゾール；２－ピリジノン；５－ニトロインドール；３－ニトロピロール；５－メトキシウラシル；ウラシル－５－オキシ酢酸；５－メトキシカルボニルメチルウラシル；５－メチル－２－チオウラシル；５－メトキシカルボニルメチル－２－チオウラシル；５－メチルアミノメチル－２－チオウラシル；３－（３－アミノ－３－カルボキシプロピル）ウラシル；３－メチルシトシン；５－メチルシトシン；Ｎ４－アセチルシトシン；２－チオシトシン；Ｎ６－メチルアデニン；Ｎ６－イソペンチルアデニン；２－メチルチオ－Ｎ６－イソペンテニルアデニン；Ｎ－メチルグアニン；Ｏ－アルキル化塩基等があげられる。また、プリン塩基およびピリミジン塩基は、例えば、米国特許第３，６８７，８０８号、「Concise　Encyclopedia　Of　Polymer　Science　And　Engineering」、８５８～８５９頁、クロシュビッツ　ジェー　アイ（Kroschwitz　J．I．）編、John　Wiley　＆　Sons、１９９０、およびイングリッシュら（Englischら）、Angewandte　Chemie、International　Edition、１９９１、３０巻、ｐ．６１３に開示されるものが含まれる。 Examples of the base include purine bases such as adenine and guanine, and pyrimidine bases such as cytosine, uracil and thymine. Other examples of the base include inosine, xanthine, hypoxanthine, purine, isoguanine, isocytosine, and 7-deazaadenine. Examples of the base include alkyl derivatives such as 2-aminoadenine and 6-methylated purine; alkyl derivatives such as 2-propylated purine; 5-halouracil and 5-halocytosine; 5-propynyluracil and 5-propynylcytosine; -Azouracil, 6-azocytosine and 6-azothymine; 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5- (2-aminopropyl) uracil, 5-aminoallyluracil; 8-halogenated, aminated, Thiolated, thioalkylated, hydroxylated and other 8-substituted purines; 5-trifluoromethylated and other 5-substituted pyrimidines; 7-methylguanine; 5-substituted pyrimidines; 6-azapyrimidines; N-2, N -6 and O-6 substituted purines (2-aminopropyladenyl 5-propynyluracil and 5-propynylcytosine; dihydrouracil; 3-deaza-5-azacytosine; 2-aminopurine; 5-alkyluracil; 7-alkylguanine; 5-alkylcytosine; 7-deazaadenine; N6 2,6-diaminopurine; 5-amino-allyl-uracil; N3-methyluracil; substituted 1,2,4-triazole; 2-pyridinone; 5-nitroindole; 3-nitropyrrole; -Methoxyuracil; uracil-5-oxyacetic acid; 5-methoxycarbonylmethyluracil; 5-methyl-2-thiouracil; 5-methoxycarbonylmethyl-2-thiouracil; 5-methylaminomethyl-2-thiouracil; 3- (3 -Amino-3-carboxypropyl) uracil 3-methylcytosine; 5-methylcytosine; N4-acetylcytosine; 2-thiocytosine; N6-methyladenine; N6-isopentyladenine; 2-methylthio-N6-isopentenyladenine; N-methylguanine; O-alkylated base Etc. Purine bases and pyrimidine bases are described in, for example, US Pat. No. 3,687,808, “Concise Encyclopedia Of Polymer Science And Engineering”, pages 858-859, edited by Kroschwitz J.I., John. Wiley & Sons, 1990, and Englisch et al., Angewandte Chemie, International Edition, 1991, 30, p. 613 is included.
　前記修飾ヌクレオチド残基は、これらの他に、例えば、塩基を欠失する残基、すなわち、無塩基の糖リン酸骨格を含んでもよい。また、前記修飾ヌクレオチド残基は、例えば、米国仮出願第６０／４６５，６６５号（出願日：２００３年４月２５日）、および国際出願第ＰＣＴ／ＵＳ０４／０７０７０号（出願日：２００４年３月８日）に記載される残基が使用でき、本発明は、これらの文献を援用できる。 In addition to these, the modified nucleotide residue may include, for example, a residue lacking a base, that is, an abasic sugar phosphate skeleton. The modified nucleotide residues are, for example, US Provisional Application No. 60 / 465,665 (filing date: April 25, 2003) and International Application No. PCT / US04 / 07070 (filing date: 2004/3). The residues described on the 8th of May) can be used, and the present invention can incorporate these documents.
　本発明の核酸分子は、例えば、標識物質を含み、前記標識物質で標識化されてもよい。前記標識物質は、特に制限されず、例えば、蛍光物質、色素、同位体等があげられる。前記標識物質は、例えば、ピレン、ＴＡＭＲＡ、フルオレセイン、Ｃｙ（登録商標）３色素、Ｃｙ（登録商標）５色素等の蛍光団があげられ、前記色素は、例えば、Ａｌｅｘａ４８８等のＡｌｅｘａ色素等があげられる。前記同位体は、例えば、安定同位体および放射性同位体があげられ、好ましくは安定同位体である。前記安定同位体は、例えば、被ばくの危険性が少なく、専用の施設も不要であることから取り扱い性に優れ、また、コストも低減できる。また、前記安定同位体は、例えば、標識した化合物の物性変化がなく、トレーサーとしての性質にも優れる。前記安定同位体は、特に制限されず、例えば、２Ｈ、１３Ｃ、１５Ｎ、１７Ｏ、１８Ｏ、３３Ｓ、３４Ｓおよび３６Ｓがあげられる。 The nucleic acid molecule of the present invention may contain, for example, a labeling substance and be labeled with the labeling substance. The labeling substance is not particularly limited, and examples thereof include fluorescent substances, dyes, isotopes and the like. Examples of the labeling substance include fluorophores such as pyrene, TAMRA, fluorescein, Cy (registered trademark) 3 dye, and Cy (registered trademark) 5 dye, and examples of the dye include Alexa dyes such as Alexa488. It is done. Examples of the isotope include a stable isotope and a radioactive isotope, and preferably a stable isotope. For example, the stable isotope has a low risk of exposure and does not require a dedicated facility, so that it is easy to handle and the cost can be reduced. In addition, the stable isotope does not change the physical properties of the labeled compound, for example, and is excellent in properties as a tracer. The stable isotope is not particularly limited, and examples thereof include 2 H, 13 C, 15 N, 17 O, 18 O, 33 S, 34 S, and 36 S.
　本発明の核酸分子は、例えば、一本鎖核酸分子であり、５’側から３’側にかけて、５’側領域、内部領域、および３’側領域を前記順序で有していてもよい。前記発現抑制配列は、例えば、５’側領域、内部領域、および３’側領域のいずれか１つの領域にに配置されてもよいし、２つ以上の領域にわたり配置されてもよいし、全ての領域にわたり配置されてもよい。具体的に、本発明の核酸分子がａｓ１ヌクレオチドを含む場合、ａｓ１ヌクレオチドにおいて、例えば、５’側の小文字の塩基（ａ、ｕ、ｇ、ｃ、ｔ）を前記５’側領域とし、３’側の小文字の塩基（ａ、ｕ、ｇ、ｃ、ｔ）を前記３’側領域とし、前記５’側の小文字の塩基および前記３’側の小文字の塩基に挟まれた大文字の塩基（Ａ、Ｔ、Ｇ、Ｃ）を前記内部領域としてもよい。また、本発明の核酸分子がａｓ２ヌクレオチドまたはａｓ３ヌクレオチドを含む場合、例えば、ａｓ１ヌクレオチドにおける前記５’側の小文字の塩基、前記５’側の小文字の塩基および前記３’側の小文字の塩基に挟まれた大文字の塩基、ならびに前記３’側の小文字の塩基と対応する塩基を、それぞれ、前記５’側領域、前記内部領域、および前記３’側領域としてもよい。 The nucleic acid molecule of the present invention is, for example, a single-stranded nucleic acid molecule, and may have a 5 'side region, an internal region, and a 3' side region in the above order from the 5 'side to the 3' side. For example, the expression suppression sequence may be arranged in any one of the 5 ′ region, the internal region, and the 3 ′ region, or may be arranged over two or more regions, or all It may be arranged over the area. Specifically, when the nucleic acid molecule of the present invention contains an as1 nucleotide, in the as1 nucleotide, for example, the 5 ′ lower-case base (a, u, g, c, t) is the 5 ′ region, and 3 ′ Lower-case bases (a, u, g, c, t) on the side as the 3′-side region, and upper-case bases (A) sandwiched between the lower-case base on the 5′-side and the lower-case base on the 3′-side , T, G, C) may be the internal region. When the nucleic acid molecule of the present invention contains as2 nucleotides or as3 nucleotides, for example, it is sandwiched between the 5 ′ lower case base, the 5 ′ lower case base and the 3 ′ lower case base in the as1 nucleotide. The upper case base and the base corresponding to the 3 'side lower case base may be the 5' side region, the internal region, and the 3 'side region, respectively.
　前記５’側領域、前記内部領域、および前記３’側領域を構成するヌクレオチド残基は、特に制限されず、例えば、リボヌクレオチド残基でもよいし、デオキシリボヌクレオチド残基でもよい。前記内部領域は、例えば、ＲＮａｓｅＨを介したペリオスチン遺伝子のｍＲＮＡの分解により発現抑制を誘導できることから、デオキシリボヌクレオチド残基であることが好ましく、前記修飾デオキシリボヌクレオチド残基がより好ましく、前記リン酸基が修飾された修飾デオキシリボヌクレオチド残基がさらに好ましい。また、前記５’側領域および前記３’側領域を構成するヌクレオチド残基は、リボヌクレオチド残基であることが好ましく、前記糖残基が修飾された修飾リボヌクレオチド残基、または前記リン酸基が修飾された修飾リボヌクレオチド残基であることがより好ましく、前記糖残基および前記リン酸基が修飾されたリボヌクレオチド残基であることがさらに好ましい。前記核酸分子は、具体的には、前記５’側領域および前記３’側領域を構成するヌクレオチド残基が、リボヌクレオチド残基であり、前記内部領域を構成するヌクレオチド残基が、デオキシリボヌクレオチド残基であることが好ましい。 The nucleotide residues constituting the 5 'region, the internal region, and the 3' region are not particularly limited, and may be, for example, ribonucleotide residues or deoxyribonucleotide residues. The internal region is preferably a deoxyribonucleotide residue, more preferably the modified deoxyribonucleotide residue, and more preferably the phosphate group because expression suppression can be induced by degradation of mRNA of the periostin gene via RNaseH. More preferred are modified deoxyribonucleotide residues that have been modified. The nucleotide residues constituting the 5′-side region and the 3′-side region are preferably ribonucleotide residues, the modified ribonucleotide residue in which the sugar residue is modified, or the phosphate group Is more preferably a modified ribonucleotide residue, more preferably a ribonucleotide residue in which the sugar residue and the phosphate group are modified. Specifically, in the nucleic acid molecule, nucleotide residues constituting the 5 ′ region and the 3 ′ region are ribonucleotide residues, and nucleotide residues constituting the internal region are deoxyribonucleotide residues. It is preferably a group.
　前記５’側領域および前記３’側領域を構成するヌクレオチド残基は、例えば、前記非修飾ヌクレオチド残基でもよいし、前記修飾ヌクレオチド残基でもよい。前記５’側領域および前記３’側領域は、例えば、前記核酸分子のヌクレアーゼ耐性を向上できることから、前記５’側領域および前記３’側領域の少なくとも一方を構成するヌクレオチド残基が、前記修飾ヌクレオチド残基であることが好ましく、前記核酸分子のヌクレアーゼ耐性をより向上できることから、前記５’側領域および前記３’側領域を構成するヌクレオチド残基が、前記修飾ヌクレオチド残基であることがより好ましい。 The nucleotide residues constituting the 5'-side region and the 3'-side region may be, for example, the unmodified nucleotide residue or the modified nucleotide residue. Since the 5′-side region and the 3′-side region can improve, for example, the nuclease resistance of the nucleic acid molecule, the nucleotide residues constituting at least one of the 5′-side region and the 3′-side region are modified as described above. Since it is preferably a nucleotide residue, and the nuclease resistance of the nucleic acid molecule can be further improved, the nucleotide residues constituting the 5 ′ side region and the 3 ′ side region are more preferably the modified nucleotide residues. preferable.
　前記５’側領域が修飾ヌクレオチド残基を含む場合、前記５’側領域は、例えば、前記５’側領域を構成するヌクレオチド残基の全部が、前記修飾ヌクレオチド残基であってもよいし、その一部が、前記修飾ヌクレオチド残基であってもよい。後者の場合、前記５’側領域において、連続的なヌクレオチド残基が、前記修飾ヌクレオチド残基であってもよいし、非連続的なヌクレオチド残基が、前記修飾ヌクレオチド残基であってもよい。前記連続的なヌクレオチド残基が前記修飾ヌクレオチド残基である場合、前記５’側領域における５’末端のヌクレオチド残基から連続的なヌクレオチド残基が、前記修飾ヌクレオチド残基であることが好ましい。前記修飾ヌクレオチド残基は、例えば、前述の通りであり、好ましくは、前記糖残基が修飾された修飾リボヌクレオチド残基、または前記リン酸基が修飾された修飾リボヌクレオチド残基であり、より好ましくは、前記糖残基および前記リン酸基が修飾されたリボヌクレオチド残基である。 When the 5 ′ region includes a modified nucleotide residue, the 5 ′ region may be, for example, all of the nucleotide residues constituting the 5 ′ region may be the modified nucleotide residue, A part thereof may be the modified nucleotide residue. In the latter case, in the 5 ′ region, a continuous nucleotide residue may be the modified nucleotide residue, or a non-continuous nucleotide residue may be the modified nucleotide residue. . When the continuous nucleotide residue is the modified nucleotide residue, it is preferable that the nucleotide residue continuous from the 5 'terminal nucleotide residue in the 5' side region is the modified nucleotide residue. The modified nucleotide residue is, for example, as described above, preferably a modified ribonucleotide residue in which the sugar residue is modified, or a modified ribonucleotide residue in which the phosphate group is modified, and more Preferably, the sugar residue and the phosphate group are modified ribonucleotide residues.
　前記３’側領域が修飾ヌクレオチド残基を含む場合、前記３’側領域は、例えば、前記３’側領域を構成するヌクレオチド残基の全部が、前記修飾ヌクレオチド残基であってもよいし、その一部が、前記修飾ヌクレオチド残基であってもよい。後者の場合、前記３’側領域において、連続的なヌクレオチド残基が、前記修飾ヌクレオチド残基であってもよいし、非連続的なヌクレオチド残基が、前記修飾ヌクレオチド残基であってもよい。前記連続的なヌクレオチド残基が前記修飾ヌクレオチド残基である場合、前記３’側領域における３’末端のヌクレオチド残基から連続的なヌクレオチド残基が、前記修飾ヌクレオチド残基であることが好ましい。前記修飾ヌクレオチド残基は、例えば、前述の通りであり、好ましくは、前記糖残基が修飾された修飾リボヌクレオチド残基、または前記リン酸基が修飾された修飾リボヌクレオチド残基であり、より好ましくは、前記糖残基および前記リン酸基が修飾されたリボヌクレオチド残基である。 When the 3 ′ side region includes a modified nucleotide residue, the 3 ′ side region, for example, all of the nucleotide residues constituting the 3 ′ side region may be the modified nucleotide residue, A part thereof may be the modified nucleotide residue. In the latter case, in the 3 ′ region, a continuous nucleotide residue may be the modified nucleotide residue, or a non-continuous nucleotide residue may be the modified nucleotide residue. . When the continuous nucleotide residue is the modified nucleotide residue, it is preferable that the nucleotide residue continuous from the 3 'terminal nucleotide residue in the 3' side region is the modified nucleotide residue. The modified nucleotide residue is, for example, as described above, preferably a modified ribonucleotide residue in which the sugar residue is modified, or a modified ribonucleotide residue in which the phosphate group is modified, and more Preferably, the sugar residue and the phosphate group are modified ribonucleotide residues.
　前記内部領域は、例えば、非修飾ヌクレオチド残基でもよいし、修飾ヌクレオチド残基でもよい。前記内部領域が修飾ヌクレオチド残基を含む場合、前記内部領域は、例えば、前記内部領域を構成するヌクレオチド残基の全部が、前記修飾ヌクレオチド残基であってもよいし、一部が、前記修飾ヌクレオチド残基であってもよい。前記内部領域が修飾ヌクレオチド残基を含む場合、前記内部領域は、前記リン酸基が修飾された修飾デオキシリボヌクレオチド残基が好ましい。 The internal region may be, for example, an unmodified nucleotide residue or a modified nucleotide residue. When the internal region includes a modified nucleotide residue, for example, all of the nucleotide residues constituting the internal region may be the modified nucleotide residue, or part of the internal region may be the modified nucleotide residue. It may be a nucleotide residue. When the internal region includes a modified nucleotide residue, the internal region is preferably a modified deoxyribonucleotide residue in which the phosphate group is modified.
　本発明の核酸分子は、具体的には、前記５’側領域および前記３’側領域を構成するヌクレオチド残基が、修飾リボヌクレオチド残基であり、前記内部領域を構成するヌクレオチド残基が、デオキシリボヌクレオチド残基であることが好ましく、前記５’側領域および前記３’側領域を構成するヌクレオチド残基が、前記修飾リボヌクレオチド残基であり、前記内部領域を構成するヌクレオチド残基が、修飾デオキシリボヌクレオチド残基であることがより好ましく、前記５’側領域および前記３’側領域を構成するヌクレオチド残基が、２’－Ｏ－メチルリボースを含み、且つリン酸基が修飾された修飾リボヌクレオチド残基およびＬＮＡの少なくとも一方であり、前記内部領域を構成するヌクレオチド残基が、リン酸基が修飾された修飾デオキシリボヌクレオチド残基であることがさらに好ましい。前記５’側領域および前記３’側領域を構成するヌクレオチド残基がＬＮＡの場合、前記修飾ヌクレオチド残基は、リン酸基が修飾された修飾リボヌクレオチド残基であることが好ましい。 Specifically, in the nucleic acid molecule of the present invention, the nucleotide residues constituting the 5 ′ region and the 3 ′ region are modified ribonucleotide residues, and the nucleotide residues constituting the internal region are: It is preferably a deoxyribonucleotide residue, the nucleotide residues constituting the 5 ′ region and the 3 ′ region are the modified ribonucleotide residues, and the nucleotide residues constituting the internal region are modified More preferably, it is a deoxyribonucleotide residue, and the nucleotide residues constituting the 5′-side region and the 3′-side region contain 2′-O-methylribose and have a phosphate group modified. At least one of a nucleotide residue and LNA, and the nucleotide residue constituting the internal region is modified with a phosphate group modified More preferably a deoxyribonucleotide residues. When the nucleotide residues constituting the 5'-side region and the 3'-side region are LNA, the modified nucleotide residue is preferably a modified ribonucleotide residue having a phosphate group modified.
　各領域の塩基数は、特に制限されない。前記５’側領域の塩基数は、例えば、１～１４個、１～１２個、２～１１個、３～８個、３～６個、３～５個、２～５個、２～４個である。前記内部領域の塩基数は、例えば、５～１６個、６～１４個、８～１４個、６～１２個、７～１２個である。前記３’側領域の塩基数は、例えば、１～１４個、１～１２個、２～１１個、３～８個、３～６個、３～５個、２～５個、２～４個である。前記５’側領域の塩基数と前記３’側領域の塩基数とは、例えば、同じでもよいし、異なってもよいが、前者が好ましい。 The number of bases in each region is not particularly limited. The number of bases in the 5′-side region is, for example, 1 to 14, 1 to 12, 2 to 11, 3 to 8, 3 to 6, 3 to 5, 2 to 5, 2 to 4 It is a piece. The number of bases in the internal region is, for example, 5 to 16, 6 to 14, 8 to 14, 6 to 12, or 7 to 12. The number of bases in the 3 ′ side region is, for example, 1 to 14, 1 to 12, 2 to 11, 3 to 8, 3 to 6, 3 to 5, 2 to 5, 2 to 4 It is a piece. The number of bases in the 5 'side region and the number of bases in the 3' side region may be the same or different, for example, but the former is preferred.
　前記核酸分子の全長、前記５’側領域の塩基数、前記内部領域の塩基数、および前記３’側領域の塩基数の組合せは、特に制限されない。具体例として、前記核酸分子において、前記５’側領域および前記３’側領域を構成するヌクレオチド残基が、修飾リボヌクレオチド残基であり、前記内部領域を構成するヌクレオチド残基が、デオキシリボヌクレオチド残基および修飾デオキシリボヌクレオチド残基の少なくとも一方である場合、例えば、下記の組合せがあげられる。前記修飾リボヌクレオチド残基は、２’－Ｏ－メチルリボースを含み、且つリン酸基が修飾された修飾リボヌクレオチド残基が好ましい。
核酸分子の全長：１２～４０塩基長、１５～３０塩基長、１８～２０塩基長
５’側領域の塩基数：１～１４個、２～８個、３～６個
内部領域の塩基数：５～１６個、７～１５個、８～１４個
３’側領域の塩基数：１～１４個、２～８個、３～６個 The combination of the total length of the nucleic acid molecule, the number of bases in the 5 ′ side region, the number of bases in the internal region, and the number of bases in the 3 ′ side region is not particularly limited. As a specific example, in the nucleic acid molecule, the nucleotide residues constituting the 5 ′ side region and the 3 ′ side region are modified ribonucleotide residues, and the nucleotide residues constituting the internal region are deoxyribonucleotide residues. When it is at least one of a group and a modified deoxyribonucleotide residue, for example, the following combinations can be mentioned. The modified ribonucleotide residue is preferably a modified ribonucleotide residue containing 2′-O-methyl ribose and having a phosphate group modified.
Total length of nucleic acid molecule: 12 to 40 base length, 15 to 30 base length, 18 to 20 base length 5 ′ base number: 1 to 14, 2 to 8, 3 to 6 internal region base number: 5-16, 7-15, 8-14, 3 ′ base number: 1-14, 2-8, 3-6
　また、別の具体例として、前記核酸分子において、前記５’側領域および前記３’側領域を構成するヌクレオチド残基が、二環式人工核酸モノマー残基であり、前記内部領域を構成するヌクレオチド残基が、デオキシリボヌクレオチド残基および修飾デオキシリボヌクレオチド残基の少なくとも一方である場合、例えば、下記の組合せがあげられる。
核酸分子の全長：１２～４０塩基長、１３～４０塩基長、１４～３０塩基長、１６～２０塩基長
５’側領域の塩基数：１～１４個、２～８個、２～５個
内部領域の塩基数：５～１６個、７～１５個、８～１４個
３’側領域の塩基数：１～１４個、２～８個、２～５個 As another specific example, in the nucleic acid molecule, the nucleotide residues constituting the 5 ′ side region and the 3 ′ side region are bicyclic artificial nucleic acid monomer residues, and the nucleotides constituting the internal region When the residue is at least one of a deoxyribonucleotide residue and a modified deoxyribonucleotide residue, examples thereof include the following combinations.
Total length of nucleic acid molecule: 12 to 40 base length, 13 to 40 base length, 14 to 30 base length, 16 to 20 base length 5 ′ base number of bases: 1 to 14, 2 to 8, 2 to 5 Number of bases in internal region: 5 to 16, 7 to 15, 8 to 14 Number of bases in 3 'side region: 1 to 14, 2 to 8, 2 to 5
　本発明の核酸分子は、前述のように、ペリオスチン遺伝子の発現抑制ができる。このため、本発明の核酸分子は、例えば、ペリオスチン遺伝子の発現が原因となる疾患（以下、「ペリオスチン遺伝子関連疾患」ともいう。）の治療剤として使用できる。本発明において、「治療」は、例えば、前記疾患の予防、前記疾患の改善、前記疾患の予後の改善の意味を含み、いずれでもよい。 As described above, the nucleic acid molecule of the present invention can suppress the expression of the periostin gene. For this reason, the nucleic acid molecule of the present invention can be used, for example, as a therapeutic agent for a disease caused by expression of the periostin gene (hereinafter also referred to as “periostin gene-related disease”). In the present invention, “treatment” includes, for example, the meaning of prevention of the disease, improvement of the disease, and improvement of the prognosis of the disease.
　前記ペリオスチン遺伝子関連疾患は、特に制限されず、例えば、眼疾患、皮膚疾患、呼吸器疾患、腎疾患、肝臓疾患、消化器疾患、耳鼻咽喉科疾患、循環器疾患、血液疾患、骨関節疾患、がん、炎症性疾患、および線維化疾患等があげられる。前記眼疾患は、特に制限されず、網膜症、黄斑変性症、翼状片、結膜炎、眼内血管新生、眼手術後の線維瘢痕等があげられ、前記網膜症は、例えば、増殖性糖尿病網膜症、増殖硝子体網膜症等の増殖性網膜症等があげられる。前記皮膚疾患は、特に制限されず、例えば、アトピー性皮膚炎、創傷治癒、肥厚性瘢痕、ケロイド、全身性強皮症等があげられる。前記呼吸器疾患は、特に制限されず、例えば、気管支喘息、気道炎症、肺線維症等があげられる。前記腎疾患は、特に制限されず、例えば、慢性腎臓病、多発性嚢胞腎等があげられる。前記肝臓疾患は、特に制限されず、例えば、非アルコール性脂肪性肝炎、非アルコール性脂肪性肝疾患等があげられる。前記消化器疾患は、特に制限されず、例えば、被嚢性腹膜硬化症等があげられる。前記耳鼻咽喉科疾患は、特に制限されず、例えば、好酸球中耳炎、アレルギー性鼻炎、慢性副鼻腔炎、ＩｇＧ４関連硬化性唾液腺炎、鼻ポリープ等があげられる。前記循環器疾患は、特に制限されず、例えば、急性心筋梗塞、アテローム性動脈硬化、腹部大動脈瘤、リウマチ性弁膜疾患等があげられる。前記血液疾患は、特に制限されず、例えば、骨髄線維症等があげられる。前記骨関節疾患は、特に制限されず、例えば、膝関節症等があげられる。前記がんは、特に制限されず、例えば、肝がん、乳がん、胃がん、食道がん、頭頸部がん、膵臓がん、肺がん、骨肉腫、悪性黒色腫、神経膠細胞腫、髄膜腫、大腸がん、前立腺がん、卵巣がん、腎臓がん等があげられる。前記炎症性疾患は、特に制限されず、例えば、アトピー性皮膚炎、気管支喘息、アレルギー性鼻炎、慢性副鼻腔炎、好酸球性中耳炎、間質性肺炎、全身性エリテマトーデス等があげられる。前記線維化疾患は、特に制限されず、例えば、特発性肺線維症、慢性腎臓病、肝硬変、被嚢性腹膜硬化症、骨髄線維症、強皮症、デュピュイトラン拘縮等があげられる。 The periostin gene-related disease is not particularly limited, for example, eye disease, skin disease, respiratory disease, kidney disease, liver disease, gastrointestinal disease, otolaryngology disease, cardiovascular disease, blood disease, bone joint disease, Examples include cancer, inflammatory diseases, and fibrotic diseases. The eye disease is not particularly limited, and includes retinopathy, macular degeneration, pterygium, conjunctivitis, intraocular neovascularization, fiber scar after eye surgery, etc. The retinopathy is, for example, proliferative diabetic retinopathy And proliferative retinopathy such as proliferative vitreoretinopathy. The skin disease is not particularly limited, and examples thereof include atopic dermatitis, wound healing, hypertrophic scar, keloid, systemic scleroderma and the like. The respiratory disease is not particularly limited, and examples thereof include bronchial asthma, airway inflammation, and pulmonary fibrosis. The renal disease is not particularly limited, and examples thereof include chronic kidney disease and polycystic kidney disease. The liver disease is not particularly limited, and examples thereof include nonalcoholic steatohepatitis and nonalcoholic fatty liver disease. The digestive system disease is not particularly limited, and examples thereof include capsular peritoneal sclerosis. The otolaryngology disease is not particularly limited, and examples thereof include eosinophil otitis media, allergic rhinitis, chronic sinusitis, IgG4-related sclerosing salivary glanditis, and nasal polyp. The cardiovascular disease is not particularly limited, and examples thereof include acute myocardial infarction, atherosclerosis, abdominal aortic aneurysm, rheumatic valvular disease and the like. The blood disease is not particularly limited, and examples thereof include myelofibrosis. The bone joint disease is not particularly limited, and examples thereof include knee arthropathy. The cancer is not particularly limited. For example, liver cancer, breast cancer, stomach cancer, esophageal cancer, head and neck cancer, pancreatic cancer, lung cancer, osteosarcoma, malignant melanoma, glioma, meningioma Colorectal cancer, prostate cancer, ovarian cancer, kidney cancer and the like. The inflammatory disease is not particularly limited, and examples thereof include atopic dermatitis, bronchial asthma, allergic rhinitis, chronic sinusitis, eosinophilic otitis media, interstitial pneumonia, and systemic lupus erythematosus. The fibrotic disease is not particularly limited, and examples thereof include idiopathic pulmonary fibrosis, chronic kidney disease, cirrhosis, encapsulating peritoneal sclerosis, myelofibrosis, scleroderma, and Dupuytren's contracture.
　本発明の核酸分子の使用方法は、特に制限されず、例えば、前記投与対象に、前記核酸分子を投与すればよい。 The method for using the nucleic acid molecule of the present invention is not particularly limited, and for example, the nucleic acid molecule may be administered to the administration subject.
　前記投与対象は、例えば、細胞、組織または器官があげられる。前記投与対象は、例えば、ヒト、ヒトを除く非ヒト動物があげられる。前記非ヒト動物は、例えば、マウス、ラット、ウサギ、ヒツジ、ウシ、ウマ、イヌ、ブタ、サル等の非ヒト哺乳類動物等があげられる。前記投与は、例えば、ｉｎ　ｖｉｖｏでもｉｎ　ｖｉｔｒｏでもよい。 Examples of the administration subject include cells, tissues, and organs. Examples of the administration subject include humans and non-human animals other than humans. Examples of the non-human animals include non-human mammals such as mice, rats, rabbits, sheep, cows, horses, dogs, pigs, monkeys, and the like. The administration may be, for example, in vivo or in vitro .
　前記細胞は、特に制限されず、例えば、ヒトおよびマウス等の、ＡＲＰＥ－１９等の網膜色素上皮細胞、ＮＩＨ３Ｔ３等の線維芽細胞、Ａ１７２細胞等のグリア芽腫細胞等の各種培養細胞、ＥＳ細胞、造血幹細胞等の幹細胞、初代培養細胞等の生体から単離した細胞等があげられる。前記細胞は、例えば、ヒト受精卵、ならびに、ヒト胚およびヒト個体内の細胞を除く。 The cells are not particularly limited, for example, various cultured cells such as human and mouse retinal pigment epithelial cells such as ARPE-19, fibroblasts such as NIH3T3, glioblastoma cells such as A172 cells, ES cells And stem cells such as hematopoietic stem cells, cells isolated from living organisms such as primary cultured cells, and the like. The cells exclude, for example, human fertilized eggs and cells in human embryos and human individuals.
　本発明の核酸分子に関しては、後述する本発明の組成物、医薬品、ペリオスチン遺伝子の発現抑制方法およびペリオスチン遺伝子関連疾患の治療方法等の記載を参照できる。 Referring to the nucleic acid molecule of the present invention, the description of the composition of the present invention, pharmaceuticals, periostin gene expression suppression method, periostin gene-related disease treatment method and the like described later can be referred to.
　本発明の核酸分子は、前述のように、ペリオスチン遺伝子の発現を抑制できることから、例えば、医薬品として有用である。 The nucleic acid molecule of the present invention is useful as, for example, a pharmaceutical because it can suppress the expression of the periostin gene as described above.
　本発明の核酸分子は、例えば、遺伝子工学的手法または有機合成的手法によって合成でき、合成ＤＮＡ分子、合成ＲＮＡ分子、合成ＤＮＡ／ＲＮＡ分子ということもできる。 The nucleic acid molecule of the present invention can be synthesized by, for example, a genetic engineering technique or an organic synthetic technique, and can also be referred to as a synthetic DNA molecule, a synthetic RNA molecule, or a synthetic DNA / RNA molecule.
＜組成物＞
　本発明の組成物は、本発明の発現抑制核酸分子を含むことを特徴とする。本発明の組成物は、前記本発明の発現抑制核酸分子を含むことが特徴であり、その他の構成は、何ら制限されない。 <Composition>
The composition of the present invention comprises the expression-inhibiting nucleic acid molecule of the present invention. The composition of the present invention is characterized by including the expression-suppressing nucleic acid molecule of the present invention, and other configurations are not limited at all.
　本発明の組成物によれば、ペリオスチン遺伝子の発現を抑制できるため、本発明の組成物は、例えば、発現抑制用試薬ということもできる。本発明によれば、例えば、ペリオスチン遺伝子が存在する対象、特に、ペリオスチン遺伝子の発現が相対的に高い対象、相対的に高くなると予測される対象に投与することで、ペリオスチン遺伝子の発現を抑制できる。投与対象は、例えば、前述の通りである。 Since the expression of the periostin gene can be suppressed according to the composition of the present invention, the composition of the present invention can also be referred to as, for example, an expression suppression reagent. According to the present invention, for example, administration of a periostin gene can be suppressed by administering to a subject in which the periostin gene is present, particularly a subject having a relatively high periostin gene expression, or a subject that is predicted to be relatively high. . The administration target is, for example, as described above.
　また、本発明の発現抑制核酸分子は、前述のように、眼疾患の治療に使用できることから、本発明の組成物は、ペリオスチン遺伝子関連疾患用の薬学的組成物、ペリオスチン遺伝子関連疾患の治療薬、ペリオスチン遺伝子関連疾患用医薬ともいえる。 In addition, since the expression-suppressing nucleic acid molecule of the present invention can be used for the treatment of eye diseases as described above, the composition of the present invention is a pharmaceutical composition for a periostin gene-related disease, a therapeutic agent for a periostin gene-related disease. It can also be said to be a drug for periostin gene-related diseases.
　本発明によれば、例えば、ペリオスチン遺伝子関連疾患の患者に投与することで、ペリオスチン遺伝子の発現を抑制し、前記疾患を治療できる。前記ペリオスチン遺伝子関連疾患は、例えば、前述の通りである。本発明において、「治療」は、前述のように、例えば、前記疾患の予防、前記疾患の改善、前記疾患の予後の改善の意味を含み、いずれでもよい。 According to the present invention, for example, by administering to a patient with a periostin gene-related disease, expression of the periostin gene can be suppressed and the disease can be treated. The periostin gene-related disease is, for example, as described above. In the present invention, as described above, “treatment” includes, for example, the meaning of prevention of the disease, improvement of the disease, and improvement of the prognosis of the disease.
　前記投与方法は、特に制限されず、例えば、投与対象に応じて適宜決定できる。前記投与対象が、生体から分離された細胞等の場合、例えば、トランスフェクション試薬を使用する方法、エレクトロポレーション法、ナノバブル法等があげられる。前記投与対象が生体の場合、例えば、非経口投与、経口投与等があげられる。非経口投与は、例えば、局所投与、皮下投与、静脈内投与等があげられる。前記眼疾患に対する投与部位は、例えば、眼、血管等があげられる。眼に直接投与する場合、その投与方法は、特に制限されず、例えば、点眼、点入、硝子体内注射、結膜下注射、テノン嚢下注射、前房内投与等があげられる。本発明の組成物の投与条件、例えば、投与回数、投与量等は、特に制限されない。 The administration method is not particularly limited, and can be appropriately determined according to the administration subject, for example. When the administration target is a cell or the like separated from a living body, examples thereof include a method using a transfection reagent, an electroporation method, and a nanobubble method. When the administration subject is a living body, examples thereof include parenteral administration and oral administration. Examples of parenteral administration include local administration, subcutaneous administration, and intravenous administration. Examples of the administration site for the eye disease include eyes and blood vessels. In the case of direct administration to the eye, the administration method is not particularly limited, and examples thereof include instillation, instillation, intravitreal injection, subconjunctival injection, subtenon injection, and intraanterior administration. There are no particular restrictions on the administration conditions of the composition of the present invention, for example, the number of administrations, the dosage and the like.
　本発明の組成物の形態は、特に制限されず、例えば、注射液、点滴静注液、点眼液、眼軟膏、皮膚用軟膏、貼付剤、吸入剤、液剤、エアゾール剤、ポンプスプレー剤、経口剤等である。 The form of the composition of the present invention is not particularly limited, and examples thereof include injections, intravenous infusions, eye drops, eye ointments, skin ointments, patches, inhalants, liquids, aerosols, pump sprays, oral Agents.
　本発明の組成物において、前記発現抑制核酸分子の配合量は、特に制限されない。前記発現抑制核酸分子の投与条件は、特に制限されない。具体例として、本発明の組成物を皮下注射または静脈注射等によりヒトに全身投与する場合、１回あたりの投与量（合計）は、例えば、５～５０００ｍｇ、５０～５００ｍｇであり、投与回数は、例えば、２週間～８週間に１回である。また、硝子体注射の場合、例えば、ヒト成人男子の眼球１個に対する１回あたりの投与量（合計）は、例えば、０．０１～１０ｍｇであり、好ましくは０．１～１ｍｇであり、投与回数は、例えば、２週間～８週間に１回である。本発明の組成物において、前記核酸分子の配合量は、例示した投与条件を実現できるような濃度で含まれていることが好ましい。 In the composition of the present invention, the amount of the expression-suppressing nucleic acid molecule is not particularly limited. The administration conditions for the expression-suppressing nucleic acid molecule are not particularly limited. As a specific example, when the composition of the present invention is systemically administered to a human by subcutaneous injection or intravenous injection, the dose (total) per administration is, for example, 5 to 5000 mg, 50 to 500 mg, and the number of administrations is For example, once every 2 to 8 weeks. In the case of vitreous injection, for example, the dose (total) per dose for one eyeball of a human adult male is, for example, 0.01 to 10 mg, preferably 0.1 to 1 mg. The number of times is, for example, once every 2 to 8 weeks. In the composition of the present invention, the compounding amount of the nucleic acid molecule is preferably contained at a concentration that can realize the exemplified administration conditions.
　本発明の組成物は、例えば、本発明の発現抑制核酸分子のみを含んでもよいし、さらにその他の添加物を含んでもよい。前記添加物の配合量は、前記発現抑制核酸分子の機能を妨げるものでなければ、特に制限されない。前記添加物は、特に制限されず、例えば、薬学的に許容された添加物が好ましい。前記添加物の種類は、特に制限されず、例えば、投与対象の種類に応じて適宜選択できる。 The composition of the present invention may contain, for example, only the expression-suppressing nucleic acid molecule of the present invention, or may further contain other additives. The amount of the additive is not particularly limited as long as it does not interfere with the function of the expression suppressing nucleic acid molecule. The additive is not particularly limited, and for example, a pharmaceutically acceptable additive is preferable. The type of the additive is not particularly limited, and can be appropriately selected depending on, for example, the type of administration target.
　本発明の組成物において、前記添加物は、例えば、前記発現抑制核酸分子と複合体を形成するものでもよい。この場合、前記添加物は、例えば、複合化剤ともいえる。本発明の組成物において、前記発現抑制核酸分子を複合体とすることによって、例えば、前記発現抑制核酸分子を効率よくデリバリーできる。前記発現抑制核酸分子と前記複合化剤との結合は、特に制限されず、例えば、非共有結合があげられる。前記複合体は、例えば、包接複合体があげられる。 In the composition of the present invention, the additive may form, for example, a complex with the expression-suppressing nucleic acid molecule. In this case, the additive can be said to be a complexing agent, for example. In the composition of the present invention, the expression-suppressing nucleic acid molecule can be efficiently delivered, for example, by making the expression-suppressing nucleic acid molecule a complex. The binding between the expression-suppressing nucleic acid molecule and the complexing agent is not particularly limited, and examples thereof include non-covalent binding. Examples of the complex include an inclusion complex.
　前記複合化剤は、特に制限されず、ポリマー、シクロデキストリン、アダマンチン等があげられる。前記シクロデキストリンは、例えば、線状シクロデキストリンコポリマー、線状酸化シクロデキストリンコポリマー等があげられる。 The complexing agent is not particularly limited, and examples thereof include a polymer, cyclodextrin, adamantine and the like. Examples of the cyclodextrin include a linear cyclodextrin copolymer and a linear oxidized cyclodextrin copolymer.
　前記添加剤は、この他に、例えば、担体、標的細胞への結合物質、縮合剤、融合剤、賦形剤、基剤、安定化剤、保存剤等があげられる。 Examples of the additive include a carrier, a binding substance to a target cell, a condensing agent, a fusing agent, an excipient, a base, a stabilizer, a preservative, and the like.
＜ペリオスチン遺伝子の発現抑制試薬＞
　本発明のペリオスチン遺伝子の発現抑制試薬は、前記本発明の発現抑制核酸分子を含むことを特徴とする。本発明の発現抑制試薬は、前記本発明の発現抑制核酸分子を含むことが特徴であり、その他の構成および条件は、何ら制限されない。本発明の発現抑制試薬は、例えば、前記本発明の発現抑制核酸分子等を援用できる。 <Periostin gene expression suppression reagent>
The periostin gene expression-suppressing reagent of the present invention comprises the expression-suppressing nucleic acid molecule of the present invention. The expression-suppressing reagent of the present invention is characterized by including the expression-suppressing nucleic acid molecule of the present invention, and other configurations and conditions are not limited at all. For example, the expression-suppressing nucleic acid molecule of the present invention can be used as the expression-suppressing reagent of the present invention.
　本発明の発現抑制試薬は、例えば、さらに、後述する本発明の発現抑制方法において使用できる成分を備えてもよい。前記成分は、例えば、緩衝液等があげられる The expression suppression reagent of the present invention may further include, for example, components that can be used in the expression suppression method of the present invention described later. Examples of the component include a buffer solution.
　本発明の発現抑制試薬は、例えば、一つの容器に各成分が収容されてもよいし、複数の容器に各成分が別個に収容されてもよい。後者の場合、本発明の発現抑制試薬は、例えば、発現抑制用キットともいう。本発明の発現抑制用キットは、例えば、さらに、使用説明書を備えてもよい。 In the expression suppression reagent of the present invention, for example, each component may be stored in one container, or each component may be stored separately in a plurality of containers. In the latter case, the expression suppression reagent of the present invention is also referred to as an expression suppression kit, for example. The expression suppression kit of the present invention may further include instructions for use, for example.
＜ペリオスチン遺伝子関連疾患用医薬品＞
　本発明のペリオスチン遺伝子関連疾患用医薬品は、前記本発明の発現抑制核酸分子を含むことを特徴とする。本発明のペリオスチン遺伝子関連疾患用医薬品は、前記本発明の発現抑制核酸分子を含むことが特徴であり、その他の構成および条件は、何ら制限されない。 <Pharmaceuticals for periostin gene-related diseases>
The medicinal product for periostin gene-related disease of the present invention comprises the nucleic acid molecule for suppressing expression of the present invention. The medicinal product for periostin gene-related disease of the present invention is characterized by including the expression-suppressing nucleic acid molecule of the present invention, and other configurations and conditions are not limited at all.
　本発明のペリオスチン遺伝子関連疾患用医薬品は、例えば、前記本発明の発現抑制核酸分子等を援用できる。本発明が対象とする疾患は、例えば、前述の通りである。 For the periostin gene-related disease drug of the present invention, for example, the expression-suppressing nucleic acid molecule of the present invention can be used. The diseases targeted by the present invention are, for example, as described above.
＜ペリオスチン遺伝子の発現抑制方法＞
　本発明の抑制方法は、前述のように、ペリオスチン遺伝子の発現を抑制する方法であって、前記本発明の発現抑制核酸分子、前記本発明の組成物または前記ペリオスチン遺伝子関連疾患用医薬を使用することを特徴とする。本発明の抑制方法は、本発明の発現抑制核酸分子を使用することが特徴であって、その他の工程および条件は、何ら制限されない。 <Periostin gene expression suppression method>
As described above, the suppression method of the present invention is a method of suppressing expression of a periostin gene, and uses the expression-suppressing nucleic acid molecule of the present invention, the composition of the present invention, or the drug for a periostin gene-related disease. It is characterized by that. The suppression method of the present invention is characterized by using the expression-suppressing nucleic acid molecule of the present invention, and other steps and conditions are not limited at all.
　本発明の抑制方法は、例えば、ペリオスチン遺伝子が存在する対象、特に、ペリオスチン遺伝子の発現が相対的に高い対象または相対的に高くなると予測される対象に、前記発現抑制核酸分子を投与する工程を含む。前記投与工程により、例えば、前記投与対象に前記発現抑制核酸分子を接触させる。前記投与対象は、例えば、前述のような、細胞、組織または器官があげられる。前記投与対象は、例えば、前述と同様に、ヒト、前記非ヒト動物があげられる。前記投与は、例えば、ｉｎ　ｖｉｖｏでもｉｎ　ｖｉｔｒｏでもよい。 The suppression method of the present invention includes, for example, a step of administering the expression-suppressing nucleic acid molecule to a subject in which a periostin gene is present, particularly to a subject in which periostin gene expression is relatively high or predicted to be relatively high. Including. By the administration step, for example, the expression-suppressing nucleic acid molecule is brought into contact with the administration subject. Examples of the administration target include cells, tissues or organs as described above. Examples of the administration subject include humans and non-human animals as described above. The administration may be, for example, in vivo or in vitro .
　本発明の抑制方法は、例えば、前記発現抑制核酸分子を単独で投与してもよいし、前記発現抑制核酸分子を含む前記本発明の組成物を投与してもよい。前記投与方法は、特に制限されず、例えば、投与対象の種類に応じて適宜選択でき、前述の記載が援用できる。 In the suppression method of the present invention, for example, the expression-suppressing nucleic acid molecule may be administered alone, or the composition of the present invention containing the expression-suppressing nucleic acid molecule may be administered. The administration method is not particularly limited, and can be appropriately selected depending on, for example, the type of administration target, and the above description can be used.
＜治療方法＞
　本発明のペリオスチン遺伝子関連疾患の治療方法は、前述のように、本発明の発現抑制核酸分子を、患者に投与する工程を含むことを特徴とする。本発明の治療方法は、ペリオスチン遺伝子関連疾患の治療に、本発明の発現抑制核酸分子を使用することが特徴であって、その他の工程および条件は、何ら制限されない。本発明が対象とする疾患は、例えば、前述の通りである。 <Treatment method>
As described above, the method for treating a periostin gene-related disease of the present invention includes a step of administering the expression-suppressing nucleic acid molecule of the present invention to a patient. The treatment method of the present invention is characterized by using the expression-suppressing nucleic acid molecule of the present invention for the treatment of a periostin gene-related disease, and other steps and conditions are not limited at all. The diseases targeted by the present invention are, for example, as described above.
　本発明の治療方法は、例えば、前記本発明の抑制方法等を援用できる。前記投与方法は、特に制限されず、例えば、前述のように、非経口投与および経口投与のいずれでもよい。 For the treatment method of the present invention, for example, the suppression method of the present invention can be used. The administration method is not particularly limited, and for example, as described above, either parenteral administration or oral administration may be used.
＜発現抑制核酸分子の使用＞
　本発明の発現抑制核酸分子は、ペリオスチン遺伝子の発現またはペリオスチンタンパク質の機能の抑制のための核酸分子、または、ペリオスチン遺伝子関連疾患の治療のための核酸分子である。また、本発明の発現抑制核酸分子は、ペリオスチン遺伝子の発現抑制剤またはペリオスチン遺伝子関連疾患用医薬の製造のための核酸分子である。 <Use of expression-suppressing nucleic acid molecules>
The expression suppressing nucleic acid molecule of the present invention is a nucleic acid molecule for suppressing periostin gene expression or periostin protein function, or a nucleic acid molecule for treating periostin gene-related diseases. The expression-suppressing nucleic acid molecule of the present invention is a nucleic acid molecule for producing a periostin gene expression inhibitor or a periostin gene-related disease drug.
　以下、実施例等により、本発明を詳しく説明するが、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.
（実施例１）
　本発明の核酸分子を合成し、ｉｎ　ｖｉｔｒｏにおけるヒトペリオスチン遺伝子の発現抑制を確認した。 Example 1
The nucleic acid molecule of the present invention was synthesized, and the suppression of human periostin gene expression in vitro was confirmed.
（１）核酸分子
　実施例の核酸分子として、下記表２ＡおよびＢの核酸分子Ｘ１３７０７、Ｘ１３７０８、Ｘ１３７１０～Ｘ１３７１２、Ｘ１３７１４、Ｘ１３７１５、Ｘ１３７１７～Ｘ１３７２７、Ｘ１３７３０、Ｘ１３７３４～Ｘ１３７４３、Ｘ１３７４５～Ｘ１３７５７、Ｘ１３７５９～Ｘ１３７６３、Ｘ１３７６５～Ｘ１３７７６、Ｘ１３７７８、Ｘ１３７８１、Ｘ１３７８３～Ｘ１３７８８、Ｘ１３７９１、Ｘ１３７９２、Ｘ１３７９５～Ｘ１３８００、およびＸ１３８０２～Ｘ１３８２７を取得した。具体的には、核酸合成装置（ABI3900、Applied Biosystems社製）を用いて、前記核酸分子を含む合成物を合成後、得られた合成物をＨＰＬＣにより精製することで、前記核酸分子を取得した。前記核酸分子の精製は、２６０ｎｍにおける吸光度と、エレクトロスプレーイオン化質量分析とにより確認した。なお、実施例の核酸分子は、下記表２ＡおよびＢの各核酸分子の塩基配列において、「a、u、ｇ、ｃ」を、２’－Ｏ－メチルリボースおよびホスホロチオエートを含む修飾リボヌクレオチド残基とし、「Ａ、Ｔ、Ｇ、Ｃ」を、ホスホロチオエートを含む修飾デオキシリボヌクレオチド残基とした。また、下記表２ＡおよびＢにおいて、「5’end position」は、前記配列番号１２１の塩基配列において、各核酸分子がハイブリダイズする５’端側の塩基の位置を示し、「3’end position」は、前記配列番号１２１の塩基配列において、各核酸分子がハイブリダイズする３’端側の塩基の位置を示す。 (1) Nucleic acid molecules As the nucleic acid molecules of Examples, the nucleic acid molecules X13707, X13708, X13710 to X13712, X13714, X13715, X13717 to X13727, X13730, X13734 to X13743, X13745 to X13757, X13759 to X13763, Table 2A and B below X13765 to X13776, X13778, X13781, X13783 to X13788, X13791, X13792, X13795 to X13800, and X13802 to X13827 were obtained. Specifically, using a nucleic acid synthesizer (ABI3900, manufactured by Applied Biosystems), the nucleic acid molecule was obtained by synthesizing a synthetic product containing the nucleic acid molecule and then purifying the resulting synthetic product by HPLC. . Purification of the nucleic acid molecule was confirmed by absorbance at 260 nm and electrospray ionization mass spectrometry. In the nucleic acid molecules of the examples, “a, u, g, c” in the nucleotide sequences of the nucleic acid molecules in the following Tables 2A and B are modified ribonucleotide residues containing 2′-O-methylribose and phosphorothioate. And “A, T, G, C” were modified deoxyribonucleotide residues containing phosphorothioate. In Tables 2A and B below, “5 ′ end position” indicates the position of the base on the 5 ′ end side where each nucleic acid molecule hybridizes in the base sequence of SEQ ID NO: 121, and “3 ′ end position” Indicates the position of the base on the 3 ′ end side where each nucleic acid molecule hybridizes in the base sequence of SEQ ID NO: 121.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
（２）ペリオスチン遺伝子の発現量の測定
　細胞は、ヒトグリア芽腫細胞Ａ１７２細胞（American　Type　Culture　Collection：ＡＴＣＣ）を使用した。培養条件は、３７℃、５％ＣＯ２とした。培地は、１０％ＦＢＳ（Biochrom AG社製）、２ｍｍｏｌ／Ｌ　Ｌ－グルタミン（Biochrom AG社製）、１ｍｍｏｌ／Ｌ　ピルビン酸ナトリウム（Biochrom AG社製）、１００Ｕ／ｍＬ　ペニシリン（Biochrom AG社製）、および１００ｍｇ／ｍＬ　ストレプトマイシン（Biochrom AG社製）を含むＤＭＥＭ（Biochrom AG社製）を使用した。 (2) Measurement of expression level of periostin gene Human glioblastoma cells A172 cells (American Type Culture Collection: ATCC) were used as cells. The culture conditions were 37 ° C. and 5% CO 2 . The medium is 10% FBS (manufactured by Biochrom AG), 2 mmol / L L-glutamine (manufactured by Biochrom AG), 1 mmol / L sodium pyruvate (manufactured by Biochrom AG), 100 U / mL penicillin (manufactured by Biochrom AG), And DMEM (Biochrom AG) containing 100 mg / mL streptomycin (Biochrom AG) was used.
　まず、細胞を、前記培地中で培養し、その培養液を、９６穴プレートに、１．５×１０4細胞／ウェルとなるように播種した。さらに、トランスフェクション試薬Lipofectamine（登録商標）2000（Invitrogen社製）を用いて、添付プロトコールに従って、前記核酸分子をそれぞれトランスフェクションした。具体的には、前記ウェルあたり、前記核酸分子の最終濃度が、３または３０ｎｍｏｌ／Ｌとなるようにトランスフェクションした。 First, the cells were cultured in the medium, and the culture was seeded in a 96-well plate at 1.5 × 10 4 cells / well. Further, each of the nucleic acid molecules was transfected using a transfection reagent Lipofectamine (registered trademark) 2000 (Invitrogen) according to the attached protocol. Specifically, transfection was performed so that the final concentration of the nucleic acid molecule was 3 or 30 nmol / L per well.
　トランスフェクション後、前記ウェル中の細胞を２４時間培養した。前記培養後、各ウェルの細胞を回収し、ｍＲＮＡ定量試薬（QuantiGene 2.0、Affymetrix/Panomics社製（Axolabs社カスタマイズ製品））を用い、細胞の溶解を５３℃の条件下で行なった以外は、添付のプロトコールにしたがって、ペリオスチン遺伝子の発現量を測定した。具体的には、前記ｍＲＮＡ定量試薬を用い、細胞溶解液を調製した。そして、５０μＬの前記細胞溶解液とヒトペリオスチン遺伝子用プローブセットとを反応させ、光学測定機器（Victor2-Light, Perkin Elmer社製）を用い、ＲＬＵ（relative light unit）を算出することにより、ヒトペリオスチン遺伝子の発現量を測定した。また、ヒトＧＡＰＤＨ遺伝子の発現量は、前記ｍＲＮＡ定量試薬として、Quantigene Explore Kit（Panomics社製）を用い、１０μＬの前記細胞溶解液とヒトＧＡＰＤＨ遺伝子用プローブセットとを反応させた以外は同様にして、測定した。そして、前記ヒトペリオスチン遺伝子の発現量は、前記ヒトＧＡＰＤＨ遺伝子の発現量により補正した。さらに、発現量は、前記トランスフェクション試薬を添加し、前記核酸分子を添加していない非添加細胞群を１として、発現量の相対値を算出した。また、コントロールは、前記核酸分子および前記トランスフェクション試薬を添加しない以外は、同様にして測定した。なお、前記ヒトペリオスチン遺伝子用プローブセットにおいて、各プローブは、ヒトペリオスチン遺伝子のｍＲＮＡにハイブリダイズするポリヌクレオチドとして、下記表３Ａの塩基配列からなるポリヌクレオチドを含むプローブとした。また、前記ヒトＧＡＰＤＨ遺伝子用プローブセットにおいて、各プローブは、ヒトＧＡＰＤＨ遺伝子のｍＲＮＡにハイブリダイズするポリヌクレオチドとして、下記表３Ｂの塩基配列からなるポリヌクレオチドを含むプローブとした。 After transfection, the cells in the wells were cultured for 24 hours. After culturing, the cells in each well were collected and attached except that the cells were lysed at 53 ° C. using an mRNA quantification reagent (QuantiGene 2.0, Affymetrix / Panomics (Axolabs customized product)). The expression level of periostin gene was measured according to the protocol. Specifically, a cell lysate was prepared using the mRNA quantification reagent. Then, 50 μL of the cell lysate is reacted with the probe set for human periostin gene, and human periostin is calculated by calculating RLU (relative light unit) using an optical measuring instrument (Victor2-Light, manufactured by Perkin® Elmer). The expression level of the gene was measured. The expression level of the human GAPDH gene is the same as that described above except that 10 μL of the cell lysate and the probe set for human GAPDH gene were reacted using Quantigene Explore Kit (Panomics) as the mRNA quantification reagent. ,It was measured. The expression level of the human periostin gene was corrected by the expression level of the human GAPDH gene. Furthermore, the expression level was calculated as the relative value of the expression level, assuming that the transfection reagent was added and the non-added cell group to which the nucleic acid molecule was not added was 1. The control was measured in the same manner except that the nucleic acid molecule and the transfection reagent were not added. In the human periostin gene probe set, each probe was a probe containing a polynucleotide having the base sequence shown in Table 3A below as a polynucleotide hybridizing to the mRNA of the human periostin gene. In the probe set for human GAPDH gene, each probe was a probe containing a polynucleotide having the nucleotide sequence shown in Table 3B below as a polynucleotide hybridizing to mRNA of the human GAPDH gene.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
（３）結果
　これらの結果を、図１Ａ～Ｄに示す。図１Ａ～Ｄは、発現量の相対値を示すグラフであり、縦軸は、発現量の相対値であり、図中の白色のバーが３ｎｍｏｌ／Ｌの結果を示し、黒色のバーが３０ｎｍｏｌ／Ｌの結果を示す。図１Ａ～Ｄに示すように、前記実施例の核酸分子は、コントロールよりも低い値を示したことから、いずれも発現抑制活性を有することが確認できた。中でも、Ｘ１３７１８、Ｘ１３７１９、Ｘ１３７３７、Ｘ１３７５２、Ｘ１３７５６、Ｘ１３７６６、Ｘ１３７８６、Ｘ１３７９２、Ｘ１３７９７、Ｘ１３７９８、Ｘ１３８０２、Ｘ１３８０３、Ｘ１３８０６、Ｘ１３８０８、Ｘ１３８１０、Ｘ１３８１６、Ｘ１３８１７、Ｘ１３８１８、Ｘ１３８２１、およびＸ１３８２７は、極めて強い発現抑制活性を示した。これらの結果から、これらのペリオスチンの発現抑制核酸分子が、優れた発現抑制活性を有することがわかった。 (3) Results These results are shown in FIGS. 1A to 1D. 1A to 1D are graphs showing the relative value of the expression level, the vertical axis is the relative value of the expression level, the white bar in the figure indicates the result of 3 nmol / L, and the black bar is 30 nmol / L The result of L is shown. As shown in FIGS. 1A to 1D, the nucleic acid molecules of the above examples showed lower values than the control, so that it was confirmed that all of them had expression suppressing activity. Among them, X13718, X13719, X13737, X13752, X13756, X13766, X13786, X13792, X13797, X13798, X13802, X13803, X13806, X13808, X13810, X13816, X13817, X13818, X13818, X13818, X13818, X13818, X13818, X13818, Indicated. From these results, it was found that these periostin expression-suppressing nucleic acid molecules have excellent expression-suppressing activity.
（実施例２）
　本発明の核酸分子を合成し、ｉｎ　ｖｉｔｒｏにおけるペリオスチン遺伝子の発現抑制を確認した。 (Example 2)
The nucleic acid molecule of the present invention was synthesized, and the suppression of periostin gene expression in vitro was confirmed.
（１）核酸分子
　実施例の核酸分子として、前記実施例１（１）と同様にして、下記表４の核酸分子Ｘ１３４６４～Ｘ１３４６７、Ｘ１３４６９～Ｘ１３４７１、Ｘ１３４７３、Ｘ１３４７４、およびＸ１３４７６～Ｘ１３４８０を取得した。なお、実施例の核酸分子は、下記表４の各核酸分子の塩基配列において、「a、u、ｇ、ｃ」を、２’－Ｏ－メチルリボースおよびホスホロチオエートを含む修飾リボヌクレオチド残基とし、「Ａ、Ｔ、Ｇ、Ｃ」を、ホスホロチオエートを含む修飾デオキシリボヌクレオチド残基とした。また、下記表４において、「5’end position」は、前記配列番号１２１の塩基配列において、各核酸分子がハイブリダイズする５’端側の塩基の位置を示し、「3’end position」は、前記配列番号１２１の塩基配列において、各核酸分子がハイブリダイズする３’端側の塩基の位置を示す。 (1) Nucleic acid molecules The nucleic acid molecules X13464 to X13467, X13469 to X13471, X13473, X13474, and X13476 to X13480 shown in Table 4 below were obtained as the nucleic acid molecules of Examples in the same manner as in Example 1 (1). In the nucleic acid molecules of the examples, “a, u, g, c” in the base sequence of each nucleic acid molecule shown in Table 4 below is a modified ribonucleotide residue containing 2′-O-methylribose and phosphorothioate, “A, T, G, C” were modified deoxyribonucleotide residues including phosphorothioate. In Table 4 below, “5 ′ end position” indicates the position of the base on the 5 ′ end side where each nucleic acid molecule hybridizes in the base sequence of SEQ ID NO: 121, and “3 ′ end position” In the base sequence of SEQ ID NO: 121, the position of the 3 ′ end base to which each nucleic acid molecule hybridizes is shown.
Figure JPOXMLDOC01-appb-T000006
Figure JPOXMLDOC01-appb-T000006
（２）ペリオスチン遺伝子の発現量の測定
　Ａ１７２細胞に代えて、マウス線維芽細胞ＮＩＨ－３Ｔ３細胞（ＡＴＣＣ）を用い、培地として、１０％ＦＢＳ、２ｍｍｏｌ／Ｌ　Ｌ－グルタミン、１００Ｕ／ｍＬ　ペニシリン、および１００ｍｇ／ｍＬ　ストレプトマイシンを含むＤＭＥＭを用い、前記核酸分子の最終濃度を２０ｎｍｏｌ／Ｌとし、前記トランスフェクション後の培養時間を４８時間とし、前記ヒトペリオスチン遺伝子用プローブセットおよび前記ヒトＧＡＰＤＨ遺伝子用プローブセットに代えて、マウスペリオスチン遺伝子用プローブセットおよびマウスペリオスチン遺伝子用プローブセットを用いた以外は、前記実施例１（２）と同様にして、発現量の相対値を測定した。なお、前記マウスペリオスチン遺伝子用プローブセットにおいて、各プローブは、マウスペリオスチン遺伝子のｍＲＮＡにハイブリダイズするポリヌクレオチドとして、下記表５Ａの塩基配列からなるポリヌクレオチドを含むプローブとした。また、前記マウスＧＡＰＤＨ遺伝子用プローブセットにおいて、各プローブは、マウスＧＡＰＤＨ遺伝子のｍＲＮＡにハイブリダイズするポリヌクレオチドとして、下記表５Ｂの塩基配列からなるポリヌクレオチドを含むプローブとした。 (2) Measurement of expression level of periostin gene Instead of A172 cells, mouse fibroblast NIH-3T3 cells (ATCC) were used as a medium, 10% FBS, 2 mmol / L L-glutamine, 100 U / mL penicillin, and Using DMEM containing 100 mg / mL streptomycin, the final concentration of the nucleic acid molecule is 20 nmol / L, the culture time after the transfection is 48 hours, and the probe set for the human periostin gene and the probe set for the human GAPDH gene are used. Instead, the relative value of the expression level was measured in the same manner as in Example 1 (2) except that the mouse periostin gene probe set and the mouse periostin gene probe set were used. In the mouse periostin gene probe set, each probe was a probe containing a polynucleotide consisting of the nucleotide sequence shown in Table 5A below as a polynucleotide hybridizing to the mRNA of the mouse periostin gene. In the mouse GAPDH gene probe set, each probe was a probe containing a polynucleotide having the nucleotide sequence shown in Table 5B below as a polynucleotide hybridizing to the mRNA of the mouse GAPDH gene.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
（３）結果
　これらの結果を、図２に示す。図２は、発現量の相対値を示すグラフであり、縦軸は、発現量の相対値である。図２に示すように、前記実施例の核酸分子は、非添加細胞群よりも低い値を示したことから、いずれも発現抑制活性を有することが確認できた。中でも、Ｘ１３４７０、Ｘ１３４７８、Ｘ１３４６６、およびＸ１３４７９は、極めて強い発現抑制活性を示した。これらの結果から、本発明の核酸分子が優れた発現抑制活性を有することがわかった。 (3) Results These results are shown in FIG. FIG. 2 is a graph showing the relative value of the expression level, and the vertical axis represents the relative value of the expression level. As shown in FIG. 2, since the nucleic acid molecule of the said Example showed a value lower than a non-added cell group, it has confirmed that all have expression suppression activity. Among them, X13470, X13478, X13466, and X13479 showed extremely strong expression suppressing activity. From these results, it was found that the nucleic acid molecule of the present invention has an excellent expression suppressing activity.
（実施例３）
　本発明の核酸分子の半数阻害濃度を確認した。 (Example 3)
The half-inhibitory concentration of the nucleic acid molecule of the present invention was confirmed.
　前記核酸分子に代えて、Ｘ１３７１８、Ｘ１３７１９、Ｘ１３７３７、Ｘ１３７５２、Ｘ１３７５６、Ｘ１３７６６、Ｘ１３７８６、Ｘ１３７９２、Ｘ１３７９７、Ｘ１３７９８、Ｘ１３８０２、Ｘ１３８０３、Ｘ１３８０６、Ｘ１３８０８、Ｘ１３８１０、Ｘ１３８１６、Ｘ１３８１７、Ｘ１３８１８、Ｘ１３８２１、およびＸ１３８２７を使用し、前記核酸分子の最終濃度を０．１５６、０．６２５、２．５、１０または４０ｎｍｏｌ／Ｌとした以外は、前記実施例１（２）と同様にして、発現量の相対値を測定した。また、前記核酸分子に代えて、Ｘ１３４７０、Ｘ１３４７８、Ｘ１３４６６、およびＸ１３４７９を使用し、前記核酸分子の最終濃度を０．０１０２、０．０３０５、０．０９１４、０．２７４、０．８２３、２．４７、７．４１、２２．２、６６．７、または２００ｎｍｏｌ／Ｌとした以外は、前記実施例２（２）と同様にして、発現量の相対値を測定した。 In place of the nucleic acid molecule, X13718, X13719, X13737, X13752, X13756, X13766, X13786, X13792, X13797, X13798, X13802, X13803, X13806, X13808, X13810, X13817, X13818, X13818, X13818, X13818 The relative value of the expression level was measured in the same manner as in Example 1 (2) except that the final concentration of the nucleic acid molecule was 0.156, 0.625, 2.5, 10 or 40 nmol / L. . Further, instead of the nucleic acid molecule, X13470, X13478, X13466, and X13479 are used, and the final concentration of the nucleic acid molecule is 0.0102, 0.0305, 0.0914, 0.274, 0.823, 2.. Except for 47, 7.41, 22.2, 66.7, or 200 nmol / L, the relative value of the expression level was measured in the same manner as in Example 2 (2).
　そして、得られた発現量の相対値に基づき、各核酸分子の半数阻害濃度を算出した。この結果を表６に示す。表６に示すように、いずれの核酸分子も十分に低い半数阻害濃度であり、優れた発現抑制活性を有することが確認できた。中でも、Ｘ１３７９８、Ｘ１３８０２、Ｘ１３８０３、およびＸ１３８１０の半数阻害濃度は、１ｎｍｏｌ／Ｌ未満であり、極めて優れた発現抑制活性を有することが確認できた。これらの結果から、本発明の核酸分子が優れた発現抑制活性を有することがわかった。 And based on the relative value of the obtained expression level, the half-inhibitory concentration of each nucleic acid molecule was calculated. The results are shown in Table 6. As shown in Table 6, it was confirmed that all the nucleic acid molecules had sufficiently low half-inhibitory concentrations and had excellent expression suppression activity. Among them, the half-inhibitory concentrations of X13798, X13802, X13803, and X13810 were less than 1 nmol / L, and it was confirmed that they had extremely excellent expression suppressing activity. From these results, it was found that the nucleic acid molecule of the present invention has an excellent expression suppressing activity.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
（実施例４）
　ブレオマイシン誘発肺線維症モデルマウスを用いて、ペリオスチンの発現抑制核酸分子の投与により、線維化が抑制されることを確認した。 Example 4
Using a bleomycin-induced pulmonary fibrosis model mouse, it was confirmed that fibrosis was suppressed by administration of periostin expression-suppressing nucleic acid molecule.
（１）核酸分子
　ペリオスチンの発現抑制核酸分子として、配列番号２１７の核酸分子（Ｘ１７７５２）を使用し、核酸溶液を作製した。前記核酸分子は、前記実施例１の方法により作製した。なお、Ｘ１７７５２において、「a、u、ｇ、ｃ」を、２’－Ｏ－メチルリボースおよびホスホロチオエートを含む修飾リボヌクレオチド残基とし、「Ａ、Ｔ、Ｇ、Ｃ」を、ホスホロチオエートを含む修飾デオキシリボヌクレオチド残基とした。また、「ｃ」および「Ｃ」は、５－メチルシトシンとした。
Ｘ１７７５２（配列番号２１７）
5'-caccaCTGTTCGTAAuuugg-3’ (1) Nucleic acid molecule The nucleic acid molecule of SEQ ID NO: 217 (X17752) was used as a periostin expression-suppressing nucleic acid molecule to prepare a nucleic acid solution. The nucleic acid molecule was prepared by the method of Example 1. In X17752, “a, u, g, c” is a modified ribonucleotide residue containing 2′-O-methylribose and phosphorothioate, and “A, T, G, C” is a modified deoxyribonucleotide containing phosphorothioate. Nucleotide residues. “C” and “C” were 5-methylcytosine.
X17752 (SEQ ID NO: 217)
5'-caccaCTGTTCGTAAuuugg-3 '
（２）ブレオマイシン誘発肺線維症モデルマウスの作製
　試験開始日（０日）に、マウス（C57BL/6、雌、７～９週齢、ｎ＝４）にペントバルビタールを腹腔内投与し、麻酔した。つぎに、２００μＬのブレオマイシン塩酸塩水溶液（１０ｍｇ／ｍＬ）をあらかじめ注入したＡＬＺＥＴ浸透圧ミニポンプ（model 2001、DURECT Corporation社製）を、前記マウスの背部皮下に埋め込んだ。そして、前記試験開始後２日目、７日目および１２日目に、イソフルラン麻酔下で、所定濃度（０．４ｍｇ／ｍＬまたは２ｍｇ／ｍＬ（それぞれ、実施例４（１）および（２）））の前記核酸溶液５０μＬを、前記マウスに経鼻投与した。また、ネガティブコントロールは、ブレオマイシン塩酸塩水溶液および前記核酸溶液に代えて、生理食塩水を用いた以外は、同様にして、コントロール１は、前記核酸溶液に代えて、生理食塩水を用いた以外は、同様にして、コントロール２は、前記核酸溶液に代えて、下記コントロールの核酸分子を２ｍｇ／ｍＬで含むコントロールの核酸溶液を用いた以外は、同様に処置した。なお、コントロールの核酸分子において、「a、u、ｇ、ｃ」を、２’－Ｏ－メチルリボースおよびホスホロチオエートを含む修飾リボヌクレオチド残基とし、「Ａ、Ｔ、Ｇ、Ｃ」を、ホスホロチオエートを含む修飾デオキシリボヌクレオチド残基とした。また、「ｃ」および「Ｃ」は、５－メチルシトシンとした。 (2) Preparation of bleomycin-induced pulmonary fibrosis model mice On the day of the study (day 0), mice (C57BL / 6, female, 7-9 weeks old, n = 4) were intraperitoneally administered with pentobarbital and anesthetized. . Next, an ALZET osmotic minipump (model 2001, manufactured by DURECT Corporation) into which 200 μL of bleomycin hydrochloride aqueous solution (10 mg / mL) had been previously injected was implanted subcutaneously in the back of the mouse. Then, on the 2nd, 7th and 12th days after the start of the test, under isoflurane anesthesia, a predetermined concentration (0.4 mg / mL or 2 mg / mL (Examples 4 (1) and (2), respectively)). 50 μL of the nucleic acid solution was administered nasally to the mice. The negative control was similarly used except that physiological saline was used in place of the bleomycin hydrochloride aqueous solution and the nucleic acid solution. Similarly, the control 1 was except that physiological saline was used in place of the nucleic acid solution. Similarly, Control 2 was treated in the same manner except that a control nucleic acid solution containing 2 mg / mL of the following control nucleic acid molecule was used instead of the nucleic acid solution. In the control nucleic acid molecule, “a, u, g, c” is a modified ribonucleotide residue containing 2′-O-methylribose and phosphorothioate, and “A, T, G, C” is phosphorothioate. The modified deoxyribonucleotide residues were included. “C” and “C” were 5-methylcytosine.
コントロールの核酸分子（配列番号２３３）
5'-cgacaTCGTGCGTCGuauau-3’ Control nucleic acid molecule (SEQ ID NO: 233)
5'-cgacaTCGTGCGTCGuauau-3 '
（３）試料調製
　前記試験開始２１日目に、全マウスにペントバルビタールを腹腔内投与し、麻酔した。つぎに、前記麻酔下、各マウスの頸部皮膚および筋肉を剥離し、気管を露出させた。つぎに、頸動脈から全血採取して安楽死させた。さらに、留置針を用いて３回に分けて合計２ｍＬの生理食塩水を気管内に挿入し、気管支肺胞洗浄液（bronchoalveolar lavage fluid：BALF）を採取した。そして、開胸し、肺を摘出した。摘出した肺のうち、右肺（下葉の一部）を、後述する肺組織中のマウスペリオスチン遺伝子の発現量の測定に用いた。また、摘出した肺のうち、右肺（上葉、中葉、および副葉）をホルマリン固定し、後述する組織病理解析に用いた。さらに、摘出した肺のうち、左肺は、後述するヒドロキシプロリン量の測定に用いた。 (3) Sample preparation On the 21st day from the start of the test, all mice were intraperitoneally administered with pentobarbital and anesthetized. Next, under the anesthesia, the neck skin and muscles of each mouse were peeled off to expose the trachea. Next, whole blood was collected from the carotid artery and euthanized. Furthermore, a total of 2 mL of physiological saline was inserted into the trachea in 3 times using an indwelling needle, and bronchoalveolar lavage fluid (BALF) was collected. The chest was opened and the lungs were removed. Among the extracted lungs, the right lung (a part of the lower lobe) was used for measurement of the expression level of the mouse periostin gene in the lung tissue described later. Further, among the extracted lungs, the right lung (upper lobe, middle lobe, and accessory lobe) was fixed in formalin and used for histopathological analysis described later. Furthermore, among the extracted lungs, the left lung was used for measurement of the amount of hydroxyproline described later.
（４）ペリオスチン遺伝子の発現量の測定
　前記右肺（下葉の一部）組織から、TRIzol（登録商標、Invitrogen社製）を用い、添付のプロトコールに従って、Ｔｏｔａｌ　ＲＮＡを回収した。つぎに、逆転写酵素（ReverTra Ace（登録商標）、東洋紡社製、カタログ番号TRT-101）と、ｏｌｉｇｏ（ｄＴ）とを用い、回収したＲＮＡからｃＤＮＡを合成した。マウスペリオスチンｍＲＮＡまたはマウスＧＡＰＤＨ遺伝子に特異的な下記プライマーセットと、AmpliTaq（登録商標）Gol（Applied Biosystems社製）とを用い、Applied Biosystems（登録商標） StepOne（商標）リアルタイムPCRシステムにより、それぞれのｃＤＮＡを増幅することにより、各遺伝子の発現量を測定した。ペリオスチン遺伝子の発現量は、マウスＧＡＰＤＨ遺伝子で補正した補正値とした。 (4) Measurement of expression level of periostin gene Total RNA was collected from the right lung (part of lower lobe) tissue using TRIzol (registered trademark, manufactured by Invitrogen) according to the attached protocol. Next, cDNA was synthesized from the recovered RNA using reverse transcriptase (ReverTra Ace (registered trademark), manufactured by Toyobo Co., Ltd., catalog number TRT-101) and oligo (dT). Using the following primer set specific to mouse periostin mRNA or mouse GAPDH gene and AmpliTaq (registered trademark) Gol (Applied Biosystems), each cDNA was analyzed by Applied Biosystems (registered trademark) StepOne (registered trademark) real-time PCR system. Was used to measure the expression level of each gene. The expression level of the periostin gene was a correction value corrected with the mouse GAPDH gene.
ペリオスチン遺伝子増幅用プライマーセット
　　　（配列番号２３４）5’-CACGGCATGGTTATTCCTTCA-3’
　　　（配列番号２３５）5’-TCAGGACACGGTCAATGACAT-3’
ＧＡＰＤＨ遺伝子増幅用プライマーセット
　　　（配列番号２３６）5’-TGGCCTTCCGTGTTCCTAC-3’
　　　（配列番号２３７）5’-GAGTTGCTGTTGAAGTCGCA-3’ Primer set for periostin gene amplification (SEQ ID NO: 234) 5'-CACGGCATGGTTATTCCTTCA-3 '
(SEQ ID NO: 235) 5'-TCAGGACACGGTCAATGACAT-3 '
GAPDH gene amplification primer set (SEQ ID NO: 236) 5'-TGGCCTTCCGTGTTCCTAC-3 '
(SEQ ID NO: 237) 5'-GAGTTGCTGTTGAAGTCGCA-3 '
　ペリオスチン遺伝子の発現量の測定結果を図３に示す。図３において、横軸は、サンプルの種類を示し、縦軸は、ペリオスチン遺伝子の発現量を示す。図３に示すように、ネガティブコントロールと比較して、ブレオマイシンにより肺線維症を誘発し、且つ本発明の発現抑制核酸分子を投与していないコントロール１および２では、ペリオスチン遺伝子の発現量が増加した。また、図３に示すように、本発明の発現抑制核酸分子を投与した場合（実施例４（１）および（２））、コントロールの核酸分子（コントロール２）と比較して、ブレオマイシンによる肺線維症誘発時のペリオスチン遺伝子の発現上昇が有意に抑制され、それぞれ、コントロール２と比較して、６９．５％および６５．１％の発現量であった。 The measurement result of the expression level of periostin gene is shown in FIG. In FIG. 3, the horizontal axis indicates the type of sample, and the vertical axis indicates the expression level of the periostin gene. As shown in FIG. 3, the expression level of periostin gene was increased in controls 1 and 2 in which pulmonary fibrosis was induced by bleomycin and the expression-suppressing nucleic acid molecule of the present invention was not administered as compared with the negative control. . Also, as shown in FIG. 3, when the expression-suppressing nucleic acid molecule of the present invention was administered (Examples 4 (1) and (2)), lung fibers caused by bleomycin were compared with the control nucleic acid molecule (Control 2). The expression increase of the periostin gene at the time of induction of the disease was significantly suppressed, and the expression levels were 69.5% and 65.1%, respectively, compared with the control 2.
（５）ＢＡＬＦ中のタンパク質の測定
　前記（３）で採取したBALF中のペリオスチンタンパク質量を、捕捉用抗体として、ラット抗マウスペリオスチンモノクローナル抗体（R&D Systems社製、カタログ番号：MAB2955）、検出用抗体として、ビオチン標識ヤギ抗マウスペリオスチンポリクローナル抗体（R&D Systems社製、カタログ番号：BAF2955）を用い、サンドイッチＥＬＩＳＡ法により測定した。また、コラーゲンタンパク質量を、捕捉用抗体として、ウサギポリクローナル抗Ｉ型コラーゲン抗体（Rockland Immunochemicals社製、カタログ番号：600-401-103）、検出用抗体として、ビオチン標識ウサギポリクローナル抗Ｉ型コラーゲン抗体（Rockland Immunochemicals社製、カタログ番号600-406-103）を用い、サンドイッチＥＬＩＳＡ法により測定した。 (5) Measurement of protein in BALF Rat anti-mouse periostin monoclonal antibody (manufactured by R & D Systems, catalog number: MAB2955), detection antibody, using the amount of periostin protein in BALF collected in (3) as a capture antibody As a measurement, a biotin-labeled goat anti-mouse periostin polyclonal antibody (manufactured by R & D Systems, catalog number: BAF2955) was used and measured by sandwich ELISA. In addition, the amount of collagen protein was determined as a rabbit polyclonal anti-type I collagen antibody (manufactured by Rockland Immunochemicals, catalog number: 600-401-103) as a capture antibody, and as a detection antibody, a biotin-labeled rabbit polyclonal anti-type I collagen antibody ( Using Rockland Immunochemicals, catalog number 600-406-103), the measurement was performed by sandwich ELISA.
　これらの結果を図４に示す。図４は、ＢＡＬＦ中のタンパク質量を示すグラフであり、（Ａ）は、ペリオスチンタンパク質量を示し、（Ｂ）は、Ｉ型コラーゲンタンパク質量を示す。図４（Ａ）および（Ｂ）において、横軸は、サンプルの種類を示し、縦軸は、各タンパク質量を示す。図４（Ａ）に示すように、ネガティブコントロールと比較して、ブレオマイシンにより肺線維症を誘発し、且つ本発明の発現抑制核酸分子を投与していないコントロール１および２では、ペリオスチンタンパク質量が増加した。また、図４（Ａ）に示すように、本発明の発現抑制核酸分子を投与した場合（実施例４（１）および（２））、コントロールの核酸分子（コントロール２）と比較して、ブレオマイシンによる肺線維症誘発時のペリオスチンタンパク質量の増加が有意に抑制され、それぞれ、コントロール２と比較して、４４．０％および５０．０％のタンパク質量であった。さらに、図４（Ｂ）に示すように、ネガティブコントロールと比較して、ブレオマイシンにより肺線維症を誘発し、且つ本発明の発現抑制核酸分子を投与していないコントロール１および２では、Ｉ型コラーゲンタンパク質量が増加した。また、図４（Ａ）に示すように、本発明の発現抑制核酸分子を投与した場合、コントロールの核酸分子（コントロール２）と比較して、ブレオマイシンによる肺線維症誘発時のＩ型コラーゲンタンパク質量の増加が有意に抑制され、それぞれ、コントロール２と比較して、８２．０％および５２．９％のタンパク質量であった。これらの結果から、ブレオマイシン誘発肺線維症モデルマウスにおいて、本発明の発現抑制核酸分子により、ペリオスチン遺伝子の発現量およびペリオスチンタンパク質量を抑制でき、これにより、Ｉ型コラーゲンの産生も抑制できることがわかった。 These results are shown in FIG. FIG. 4 is a graph showing the amount of protein in BALF, where (A) shows the amount of periostin protein and (B) shows the amount of type I collagen protein. 4 (A) and 4 (B), the horizontal axis indicates the type of sample, and the vertical axis indicates the amount of each protein. As shown in FIG. 4 (A), the amount of periostin protein is increased in controls 1 and 2 in which pulmonary fibrosis is induced by bleomycin and the expression-suppressing nucleic acid molecule of the present invention is not administered as compared with the negative control. did. In addition, as shown in FIG. 4A, when the expression-suppressing nucleic acid molecule of the present invention was administered (Examples 4 (1) and (2)), bleomycin was compared with the control nucleic acid molecule (Control 2). The increase in the amount of periostin protein at the time of inducing pulmonary fibrosis was significantly suppressed, and the amounts of protein were 44.0% and 50.0%, respectively, compared with control 2. Furthermore, as shown in FIG. 4 (B), compared with the negative control, in control 1 and 2 in which pulmonary fibrosis is induced by bleomycin and the expression-suppressing nucleic acid molecule of the present invention is not administered, type I collagen The amount of protein increased. In addition, as shown in FIG. 4 (A), when the expression-suppressing nucleic acid molecule of the present invention is administered, the amount of type I collagen protein when pulmonary fibrosis is induced by bleomycin, compared with the control nucleic acid molecule (control 2) Increase was significantly suppressed, with 82.0% and 52.9% protein content compared to Control 2, respectively. From these results, it was found that in the bleomycin-induced pulmonary fibrosis model mouse, the expression-inhibited nucleic acid molecule of the present invention can suppress the expression level of periostin gene and the amount of periostin protein, thereby suppressing the production of type I collagen. .
（６）ヒドロキシプロリンの測定
　前記（３）で採取した左肺（左葉）中のヒドロキシプロリン量を、市販のヒドロキシプロリン測定キット（Hydroxyproline colorimetric assay kit、BIOVISION社製、カタログ番号：K555-100）を用い、比色分析法により測定した。 (6) Measurement of hydroxyproline The amount of hydroxyproline in the left lung (left lobe) collected in (3) above was measured using a commercially available hydroxyproline measurement kit (Hydroxyproline colorimetric assay kit, manufactured by BIOVISION, catalog number: K555-100). Was measured by a colorimetric analysis method.
　これらの結果を図５に示す。図５は、ヒドロキシプロリン量を示すグラフである。図５において、横軸は、サンプルの種類を示し、縦軸は、ヒドロキシプロリン量を示す。図５に示すように、ネガティブコントロールと比較して、ブレオマイシンにより肺線維症を誘発し、且つ本発明の発現抑制核酸分子を投与していないコントロール１および２では、ヒドロキシプロリン量が増加した。また、図５に示すように、本発明の発現抑制核酸分子を投与した場合（実施例４（１）および（２））、コントロールの核酸分子（コントロール２）と比較して、ブレオマイシンによる肺線維症誘発時のヒドロキシプロリン量の増加が有意に抑制され、それぞれ、コントロール２と比較して、５７．０％および５２．７％のヒドロキシプロリン量であった。これらの結果から、ブレオマイシン誘発肺線維症モデルマウスにおいて、本発明の発現抑制核酸分子により、コラーゲンの産生を抑制できることがわかった。 These results are shown in FIG. FIG. 5 is a graph showing the amount of hydroxyproline. In FIG. 5, the horizontal axis indicates the type of sample, and the vertical axis indicates the amount of hydroxyproline. As shown in FIG. 5, in comparison with the negative control, pulmonary fibrosis was induced by bleomycin and the amount of hydroxyproline was increased in Controls 1 and 2 to which the expression suppressing nucleic acid molecule of the present invention was not administered. In addition, as shown in FIG. 5, when the expression-suppressing nucleic acid molecule of the present invention was administered (Examples (1) and (2)), lung fibers caused by bleomycin were compared with the control nucleic acid molecule (Control 2). The increase in the amount of hydroxyproline at the time of induction of the disease was significantly suppressed, and the amounts of hydroxyproline were 57.0% and 52.7%, respectively, as compared with Control 2. From these results, it was found that the production of collagen can be suppressed in the bleomycin-induced pulmonary fibrosis model mouse by the expression-suppressing nucleic acid molecule of the present invention.
（７）病理組織解析
　前記（３）で採取した右肺（上葉、中葉、および副葉）に、ホルマリンを気管から注入することにより、前記右肺を固定した。前記固定後、前記右肺からパラフィン切片を作製し、マッソントリクローム染色を行った。得られた染色後の組織切片のマッソントリクローム染色像について、下記参考文献のアシュクロフトスコアに基づき、線維化の程度を評価した。
参考文献：Ashcroft T. et al., “Simple method of estimating severity of pulmonary fibrosis on a numerical scale.”, J Clin Pathol, 1988, vol.41, pp.467-470 (7) Histopathological analysis The right lung was fixed by injecting formalin from the trachea into the right lung (upper lobe, middle lobe, and accessory lobe) collected in (3). After the fixation, a paraffin section was prepared from the right lung and stained with Masson trichrome. The degree of fibrosis was evaluated on the Masson trichrome stained image of the obtained tissue section after staining based on the Ashcroft score of the following reference.
Reference: Ashcroft T. et al., “Simple method of controlling severity of pulmonary fibrosis on a numerical scale.”, J Clin Pathol, 1988, vol.41, pp.467-470
　マッソントリクローム染色の結果を図６に示す。図６は、マッソントリクローム染色後の組織切片の写真である。図６において、（Ａ）が、ネガティブコントロールの結果、（Ｂ）が、コントロール１の結果、（Ｃ）が、コントロール２の結果、（Ｄ）が、実施例４（１）の結果、（Ｅ）が、実施例４（２）の結果を示す。図６に示すように、ブレオマイシンにより肺線維症を誘発し、且つ本発明の発現抑制核酸分子を投与していないコントロール１および２では、肺胞の欠落が顕著に認められた。これに対し、本発明の発現抑制核酸分子を投与した場合（実施例４（１）および（２））、コントロールの核酸分子（コントロール２）と比較して、肺胞の欠落が抑制された。 Fig. 6 shows the results of Masson trichrome staining. FIG. 6 is a photograph of a tissue section after Masson trichrome staining. In FIG. 6, (A) is the result of negative control, (B) is the result of control 1, (C) is the result of control 2, and (D) is the result of Example 4 (1), (E ) Shows the result of Example 4 (2). As shown in FIG. 6, in the control 1 and 2 in which pulmonary fibrosis was induced by bleomycin and the expression-suppressing nucleic acid molecule of the present invention was not administered, the absence of alveoli was remarkably observed. In contrast, when the expression-suppressing nucleic acid molecule of the present invention was administered (Examples 4 (1) and (2)), alveolar omission was suppressed as compared to the control nucleic acid molecule (Control 2).
　つぎに、アシュクロフトスコアの結果を図７に示す。図７は、アシュクロフトスコアを示すグラフである。図７において、横軸は、サンプルの種類を示し、縦軸は、アシュクロフトスコアを示す。図７に示すように、ブレオマイシンにより肺線維症を誘発し、且つ本発明の発現抑制核酸分子を投与していないコントロール１および２では、アシュクロフトスコアが上昇した。これに対し、本発明の発現抑制核酸分子を投与した場合（実施例４（１）および（２））、コントロールの核酸分子（コントロール２）と比較して、アシュクロフトスコアが有意に低減した。また、本発明の核酸分子を投与した場合、アシュクロフトスコアが４以下となり、肺の線維化が大きく抑制されていることがわかった。 Next, the results of the Ashcroft score are shown in FIG. FIG. 7 is a graph showing the Ashcroft score. In FIG. 7, the horizontal axis indicates the sample type, and the vertical axis indicates the Ashcroft score. As shown in FIG. 7, the ashcroft score increased in controls 1 and 2 in which pulmonary fibrosis was induced by bleomycin and the expression-suppressing nucleic acid molecule of the present invention was not administered. On the other hand, when the expression-suppressing nucleic acid molecule of the present invention was administered (Examples 4 (1) and (2)), the Ashcroft score was significantly reduced as compared with the control nucleic acid molecule (Control 2). Moreover, when the nucleic acid molecule of this invention was administered, the Ashcroft score became 4 or less, and it turned out that the fibrosis of the lung is suppressed greatly.
　以上のことから、本発明の発現抑制核酸分子により、肺線維症における線維化を抑制できることがわかった。 From the above, it was found that the expression-suppressing nucleic acid molecule of the present invention can suppress fibrosis in pulmonary fibrosis.
（実施例５）
　術後癒着モデルマウスを作製し、癒着の形成とペリオスチンの発現とが相関していることを確認した。 (Example 5)
Postoperative adhesion model mice were prepared, and it was confirmed that the formation of adhesions and periostin expression were correlated.
（１）癒着モデルマウスの作製
　１２週齢の雄性マウス（C57BL/6J、日本SLCより入手）を、イソフルラン（Isoflurane）で麻酔し、麻酔下で下腹部を正中切開した。盲腸を露出させ、点状出血が認められるまで盲腸の全周囲を綿棒で擦過した後、閉腹することにより癒着モデルマウスを作製した。 (1) Preparation of adhesion model mouse A 12-week-old male mouse (C57BL / 6J, obtained from Japan SLC) was anesthetized with Isoflurane, and a midline incision was made in the lower abdomen under anesthesia. An adhesion model mouse was prepared by exposing the cecum, rubbing the entire circumference of the cecum with a cotton swab until punctate bleeding was observed, and then closing the abdomen.
（２）癒着スコアの測定
　手術後１、３、５、７、１４日の時点で、マウスをイソフルランで麻酔した後、下腹部の縫合部位を切開し、盲腸を露出させ、盲腸の癒着スコアを測定した。癒着スコアは、０が癒着なし、１が剥離容易な癒着形成、２が局所的に剥離困難な癒着形成、３が全体的に剥離困難な癒着形成、４が全体的に剥離不可能な癒着形成とした。 (2) Measurement of adhesion score At 1, 3, 5, 7 and 14 days after surgery, the mouse was anesthetized with isoflurane, then the incision was made in the lower abdomen, the cecum was exposed, and the cecal adhesion score was determined. It was measured. As for the adhesion score, 0 is no adhesion, 1 is adhesion formation that is easy to peel, 2 is adhesion formation that is difficult to peel locally, 3 is adhesion formation that is difficult to peel entirely, 4 is adhesion formation that is totally impossible to peel It was.
　癒着スコアの結果を図８に示す。図８において、横軸は、手術後の日数を示し、縦軸は、癒着スコアを示す。図８に示すように、手術後１日から、スコア１程度の、フィルム状の弱い癒着形成が認められた。さらに、継時的に癒着スコアが高くなり、１４日では、平均スコア３以上の癒着形成が認められた。 The result of adhesion score is shown in FIG. In FIG. 8, the horizontal axis indicates the number of days after surgery, and the vertical axis indicates the adhesion score. As shown in FIG. 8, film-like weak adhesion formation with a score of about 1 was observed from 1 day after surgery. Furthermore, the adhesion score increased over time, and on the 14th, adhesion formation with an average score of 3 or more was observed.
（３）ペリオスチン遺伝子の発現量の測定
　癒着スコア測定後、盲腸先端部を採取後、常法によりＲＮＡを抽出し、前記ＲＮＡにおけるペリオスチン遺伝子の発現量および１８Ｓ　ｒＲＮＡの発現量をＲＴ－ＰＣＲにより定量した。ペリオスチン遺伝子の発現量は、１８Ｓ　ｒＲＮＡで補正した相対値とした。 (3) Measurement of periostin gene expression level After measurement of adhesion score, the cecal tip was collected, RNA was extracted by a conventional method, and periostin gene expression level and 18S rRNA expression level in the RNA were quantified by RT-PCR. did. The expression level of the periostin gene was a relative value corrected with 18S rRNA.
ペリオスチン遺伝子増幅用プライマーセット
　　　（配列番号２３８）5’-ATCAGGGGTCGGGATCAG-3’
　　　（配列番号２３９）5’-GGAGCTGAAGTATTTCTTTTTGGT-3’
１８ｓ　ｒＲＮＡ増幅用プライマーセット
　　　（配列番号２４０）5’-GCAATTATTCCCCATGAACG-3’
　　　（配列番号２４１）5’-GGGACTTAATCAACGCAAGC-3’ Primer set for periostin gene amplification (SEQ ID NO: 238) 5'-ATCAGGGGTCGGGATCAG-3 '
(SEQ ID NO: 239) 5'-GGAGCTGAAGTATTTCTTTTTGGT-3 '
18s rRNA amplification primer set (SEQ ID NO: 240) 5'-GCAATTATTCCCCATGAACG-3 '
(SEQ ID NO: 241) 5'-GGGACTTAATCAACGCAAGC-3 '
　ペリオスチン遺伝子の発現量の測定結果を図９に示す。図９において、横軸は、手術後の日数を示し、縦軸は、ペリオスチン遺伝子の発現量を示す。図９に示すように、ペリオスチンの発現は、手術後１日から１４日にかけて経時的に増加傾向を示した。 The measurement result of the expression level of periostin gene is shown in FIG. In FIG. 9, the horizontal axis indicates the number of days after surgery, and the vertical axis indicates the expression level of the periostin gene. As shown in FIG. 9, the expression of periostin tended to increase over time from day 1 to day 14 after surgery.
（４）病理組織解析
　前記採取した盲腸先端部から組織切片を作製し、マロリー・アゾカルミンＧ液（武藤化学社製）とマロリー・アニリン青オレンジＧ液（武藤化学社製）を用いて、常法によりアザン染色により組織染色を行った。また、一次抗体として抗ペリオスチン抗体（sc-49480, Santa Cruz Biotechnology, Inc.社製）用い、ＬＳＡＢ染色キット（DAKO社製）を用いて、ペリオスチンの免疫染色を行った。その後、顕微鏡下、２０倍または２００倍の倍率で撮影した。また、コントロールは、擦過処理を行わなかった以外は、同様にして撮影した。 (4) Histopathological analysis A tissue section was prepared from the collected cecal tip, and using a Mallory azocarmine G solution (Muto Kagaku Co.) and Mallory aniline blue orange G solution (Muto Kagaku Co.) Tissue staining was performed by Azan staining. Further, periostin was immunostained using an anti-periostin antibody (sc-49480, manufactured by Santa Cruz Biotechnology, Inc.) as a primary antibody and an LSAB staining kit (produced by DAKO). Then, it image | photographed by 20 times or 200 times magnification under the microscope. The control was photographed in the same manner except that the rubbing treatment was not performed.
　これらの結果を図１０に示す。図１０は、盲腸先端部の組織のアザン染色およびペリオスチン免疫染色の結果であり、図１０Ａ～Ｆは、それぞれ、コントロールの染色結果（Ｎｏｒｍａｌ）、および、手術後１、３、５、７、１４日における染色結果を示す。図１０Ａ～Ｆにおいて、上段の写真は、アザン染色の染色結果、下段の写真は、ペリオスチン免疫染色の染色結果を示す。また、図１０Ａ～Ｆにおいて、左列の写真は、倍率２０倍、右列の写真は、左列の写真の四角で囲い、矢印Ｘで示す領域の倍率２００倍の結果を示す。図１０の上段に示すように、アザン染色で染色された矢印Ｙの部分は線維化部位を示す。手術後１日後から１４日後のいずれにおいても、矢印Ｙで示すように癒着部位の線維化形成が確認できた。また図１０Ａの下段に示すように、ペリオスチン免疫染色の結果、擦過処理を行っていない盲腸組織では、基底部（矢印Ｚで示す実線で囲んだ領域）においてペリオスチンの発現が認められた。他方、図１０Ｂ～Ｆの下段に示すように、擦過処理を行った組織では、基底部に加え、癒着部位（矢印Ｐで示す実線で囲んだ領域）においても、ペリオスチンの強い発現が確認された。 These results are shown in FIG. FIG. 10 shows the results of Azan staining and periostin immunostaining of the cecal tip tissue, and FIGS. 10A to 10F show the control staining results (Normal) and 1, 3, 5, 7, 14 after the operation, respectively. The dyeing result in day is shown. 10A to F, the upper photograph shows the staining result of Azan staining, and the lower photograph shows the staining result of periostin immunostaining. In FIGS. 10A to 10F, the left column photo shows the result of 20 times magnification, and the right column photo shows the result of enclosing the rectangle in the left column photo with the magnification of 200 times in the region indicated by the arrow X. As shown in the upper part of FIG. 10, a portion indicated by an arrow Y stained with Azan staining indicates a fibrosis site. As shown by arrow Y, fibrosis formation at the adhesion site was confirmed from 1 day to 14 days after the operation. Further, as shown in the lower part of FIG. 10A, as a result of periostin immunostaining, expression of periostin was observed in the cecum tissue (the area surrounded by the solid line indicated by the arrow Z) in the cecal tissue that was not subjected to scratching. On the other hand, as shown in the lower part of FIGS. 10B to 10F, in the tissue subjected to the abrasion treatment, strong expression of periostin was confirmed not only at the base but also at the adhesion site (the region surrounded by the solid line indicated by the arrow P). .
　以上の結果から、癒着スコアの上昇と相関するようにペリオスチンの遺伝子発現が増加すること、および、癒着部位においてペリオスチンが強く発現していることがわかった。これらの結果から、癒着の形成とペリオスチンの発現とが相関していることがわかった。 From the above results, it was found that periostin gene expression increased in correlation with an increase in adhesion score, and that periostin was strongly expressed at the adhesion site. From these results, it was found that the formation of adhesions was correlated with the expression of periostin.
（実施例６）
　術後癒着モデルマウスを用いて、本発明の発現抑制核酸分子の投与により、癒着の形成が抑制されることを確認した。 (Example 6)
It was confirmed that the formation of adhesions was suppressed by administration of the expression-suppressing nucleic acid molecule of the present invention using postoperative adhesion model mice.
　ペリオスチンの発現抑制核酸分子として、配列番号２１７の核酸分子（Ｘ１７７５２）を使用した。 The nucleic acid molecule of SEQ ID NO: 217 (X17752) was used as the periostin expression-suppressing nucleic acid molecule.
　１０週齢の雄性マウス（C57BL/6J、日本SLCより入手）を用い、実施例５と同様にして、盲腸を擦過した。その後、０．２ｍＬの前記核酸溶液を腹腔内に投与し、閉腹した。手術から１週後、再度、０．２ｍＬの前記核酸溶液を腹腔内に投与した。なお、各マウスに対する核酸分子の投与量は、４０ｍｇ／ｋｇ／回とした。 Using a 10-week-old male mouse (C57BL / 6J, obtained from Japan SLC), the cecum was scraped in the same manner as in Example 5. Thereafter, 0.2 mL of the nucleic acid solution was administered intraperitoneally, and the abdomen was closed. One week after the operation, 0.2 mL of the nucleic acid solution was again administered intraperitoneally. The dose of the nucleic acid molecule for each mouse was 40 mg / kg / dose.
　手術から１４日後、前記各群のマウスに対し、ペリオスチン遺伝子の発現量の測定、癒着スコアの測定、および病理組織解析を、実施例５と同様にして行った（実施例６）。また、コントロール１は、ペリオスチンアンチセンス核酸に代えて、前記コントロールの核酸分子（配列番号２３３）を使用した以外は、同様にして実施した。さらに、コントロール２は、前記核酸溶液に代えてＰＢＳを使用した以外は、同様にして実施した。なお、各群のサンプル数は、ｎ＝６とした。 14 days after the operation, measurement of the expression level of the periostin gene, measurement of adhesion score, and histopathological analysis were performed on the mice of each group in the same manner as in Example 5 (Example 6). Control 1 was carried out in the same manner except that the control nucleic acid molecule (SEQ ID NO: 233) was used in place of the periostin antisense nucleic acid. Furthermore, Control 2 was carried out in the same manner except that PBS was used instead of the nucleic acid solution. The number of samples in each group was n = 6.
　ペリオスチン遺伝子の発現量の測定結果を図１１に示す。図１１において、横軸は、サンプル名を示し、縦軸は、ペリオスチン遺伝子の発現量を示す。図１１に示すように、実施例６は、コントロール１およびコントロール２と比較して、ペリオスチン遺伝子の発現量が有意に減少していた。これらの結果から、術後癒着モデルマウスにおいて、ペリオスチンの発現抑制核酸分子の投与により、ペリオスチン遺伝子の発現を抑制できることがわかった。 The measurement result of the expression level of the periostin gene is shown in FIG. In FIG. 11, the horizontal axis represents the sample name, and the vertical axis represents the expression level of the periostin gene. As shown in FIG. 11, in Example 6, the expression level of the periostin gene was significantly reduced as compared with Control 1 and Control 2. From these results, it was found that periostin gene expression can be suppressed by administration of periostin expression-suppressing nucleic acid molecules in postoperative adhesion model mice.
　つぎに、癒着スコアの結果を図１２に示す。図１２において、横軸は、サンプル名を示し、縦軸は、癒着スコアを示す。図１２に示すように、実施例６は、コントロール１およびコントロール２と比較して、癒着スコアが有意に低下していた。これらの結果から、術後癒着モデルマウスにおいて、ペリオスチンの発現抑制核酸分子の投与により、癒着の形成が抑制されることが示された。 Next, the result of the adhesion score is shown in FIG. In FIG. 12, the horizontal axis indicates the sample name, and the vertical axis indicates the adhesion score. As shown in FIG. 12, in Example 6, the adhesion score was significantly reduced as compared with Control 1 and Control 2. From these results, it was shown that the formation of adhesions is suppressed by administration of periostin expression-inhibiting nucleic acid molecules in postoperative adhesion model mice.
　つぎに、病理組織解析の結果を図１３ＡおよびＢに示す。図１３Ａは、コントロール２の染色結果を示し、図１３Ｂは、実施例６の染色結果を示す。図１３ＡおよびＢにおいて、左列は、アザン染色の染色結果、右列は、ペリオスチン免疫染色の染色結果を示す。また、図１３において、上段は、倍率２０倍、下段は、上段の写真の四角で囲い、矢印Ｘで示す領域の倍率２００倍の結果を示す。図１３Ａに示すように、コントロール２では、矢印Ｙで示すように線維化形成が認められ、また、癒着部位（矢印Ｐで示す実線で囲んだ領域）において、ペリオスチンの強い発現が確認された。図１３Ｂに示すように、ペリオスチンアンチセンス核酸の投与により、実施例６では、矢印Ｙで示すように癒着部位の線維化形成の低減が認められ、また癒着部位（矢印Ｐで示す実線で囲んだ領域）において、ペリオスチンの発現が抑制されていることが確認された。 Next, the results of histopathological analysis are shown in FIGS. 13A and 13B. FIG. 13A shows the staining result of Control 2, and FIG. 13B shows the staining result of Example 6. 13A and 13B, the left column shows the staining result of Azan staining, and the right column shows the staining result of periostin immunostaining. In FIG. 13, the upper part shows the result of 20 times magnification, and the lower part shows the result of enlarging the area shown by the arrow X by 200 times with the square of the upper photograph. As shown in FIG. 13A, fibrosis formation was observed in control 2 as indicated by arrow Y, and strong expression of periostin was confirmed at the adhesion site (region surrounded by a solid line indicated by arrow P). As shown in FIG. 13B, administration of periostin antisense nucleic acid showed a decrease in fibrosis formation at the adhesion site as indicated by arrow Y in Example 6, and the adhesion site (enclosed by a solid line indicated by arrow P). In the region), it was confirmed that the expression of periostin was suppressed.
　以上の結果から、術後癒着モデルマウスにおいて、本発明の発現抑制核酸分子の投与により、ペリオスチンの発現が抑制され、それと相関するように、癒着の形成が抑制されることがわかった。したがって、本発明の発現抑制核酸分子の投与により、癒着の形成が抑制されることがわかった。 From the above results, it was found that, in the postoperative adhesion model mouse, the expression of periostin was suppressed by the administration of the expression-suppressing nucleic acid molecule of the present invention, and the formation of adhesion was suppressed so as to correlate with it. Therefore, it was found that the formation of adhesions is suppressed by administration of the expression-suppressing nucleic acid molecule of the present invention.
（実施例７）
　異なる長さを有するペリオスチンの発現抑制核酸分子および異なる修飾ヌクレオチド残基を含むペリオスチンの発現抑制核酸分子を合成し、ｉｎ　ｖｉｔｒｏにおけるヒトペリオスチン遺伝子の発現抑制を確認した。 (Example 7)
Periostin expression-suppressing nucleic acid molecules having different lengths and periostin expression-suppressing nucleic acid molecules containing different modified nucleotide residues were synthesized, and human periostin gene expression suppression was confirmed in vitro .
（１）核酸分子
　実施例の核酸分子として、下記表７の核酸分子Ｘ２２８１６、Ｘ１６２５６、Ｘ２２８１７、Ｘ２２８１８、Ｘ２２８２２、およびＸ２２８２３を取得した。具体的には、前記実施例１（１）と同様にして、取得し、また、精製を確認した。なお、実施例の核酸分子は、下記表７の各核酸分子の塩基配列において、「a、u、ｇ、ｃ」を、２’－Ｏ－メチルリボースおよびホスホロチオエートを含む修飾リボヌクレオチド残基とし、「Ａ、Ｔ、Ｇ、Ｃ」を、ホスホロチオエートを含む修飾デオキシリボヌクレオチド残基とした。また、下記表７の各核酸分子の塩基配列において、「ｃ」および「Ｃ」は、５－メチルシトシンとした。なお、各核酸分子は、Ｘ１３７９２における修飾ヌクレオチド残基のみを変更した核酸分子、または修飾ヌクレオチド残基および長さを短縮化した核酸分子である。また、下記表７において、Ｗ－Ｇ－Ｗは、それぞれ、各核酸分子における５’側領域、内部領域および３’側領域の長さを示す。 (1) Nucleic acid molecule Nucleic acid molecules X22816, X16256, X22817, X22818, X22822, and X22823 shown in Table 7 below were obtained as the nucleic acid molecules of Examples. Specifically, it was obtained in the same manner as in Example 1 (1), and purification was confirmed. In the nucleic acid molecules of the examples, in the base sequences of the nucleic acid molecules in Table 7 below, “a, u, g, c” is a modified ribonucleotide residue containing 2′-O-methylribose and phosphorothioate, “A, T, G, C” were modified deoxyribonucleotide residues including phosphorothioate. In the base sequence of each nucleic acid molecule in Table 7 below, “c” and “C” were 5-methylcytosine. Each nucleic acid molecule is a nucleic acid molecule in which only the modified nucleotide residue in X13792 is changed, or a nucleic acid molecule in which the modified nucleotide residue and the length are shortened. In Table 7 below, WGW represents the lengths of the 5 ′ region, the internal region, and the 3 ′ region in each nucleic acid molecule.
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
　また、修飾ヌクレオチド残基として、ＬＮＡを用いた実施例の核酸分子として、下記表８の核酸分子Ｌ－５１０５、Ｌ－４１２４、Ｌ－３１４３、Ｌ－４１０４、Ｌ－３１２３、Ｌ－２１４２、Ｌ－４８４、Ｌ－３１０３、およびＬ－３７３を取得した。なお、実施例の核酸分子は、下記表８の各核酸分子の塩基配列において、「a、ｔ、ｇ、ｃ」を、リボース環の２’位の酸素原子と４’位の炭素原子とを、メチレンを介して架橋し、且つホスホロチオエートを含む修飾リボヌクレオチド残基、すなわち、ホスホロチオエートを含むＬＮＡとし、「Ａ、Ｔ、Ｇ、Ｃ」を、ホスホロチオエートを含む修飾デオキシリボヌクレオチド残基とした。また、下記表８の各核酸分子の塩基配列において、「ｃ」は、５－メチルシトシンとした。なお、各核酸分子は、Ｘ１３７９２における修飾ヌクレオチド残基のみを変更した核酸分子、または修飾ヌクレオチド残基および長さを短縮化した核酸分子である。また、下記表８において、Ｗ－Ｇ－Ｗは、それぞれ、各核酸分子における５’側領域、内部領域および３’側領域の長さを示す。 Further, as nucleic acid molecules of Examples using LNA as modified nucleotide residues, nucleic acid molecules L-5105, L-4124, L-3143, L-4104, L-3123, L-2142, L in Table 8 below are used. -484, L-3103, and L-373 were obtained. In addition, the nucleic acid molecule of an Example WHEREIN: In the base sequence of each nucleic acid molecule of following Table 8, "a, t, g, c" are the oxygen atom of 2'-position of a ribose ring, and the carbon atom of 4'-position. , A modified ribonucleotide residue cross-linked via methylene and containing phosphorothioate, ie LNA containing phosphorothioate, and “A, T, G, C” were modified deoxyribonucleotide residues containing phosphorothioate. In the base sequence of each nucleic acid molecule in Table 8 below, “c” was 5-methylcytosine. Each nucleic acid molecule is a nucleic acid molecule in which only the modified nucleotide residue in X13792 is changed, or a nucleic acid molecule in which the modified nucleotide residue and the length are shortened. In Table 8, WGW represents the lengths of the 5 'region, the internal region, and the 3' region in each nucleic acid molecule, respectively.
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
（２）ペリオスチン遺伝子の発現量の測定
　トランスフェクション試薬Lipofectamine（登録商標）2000を用いて、添付プロトコールに従って、前記核酸分子をトランスフェクションするためのトランスフェクション液をそれぞれ調製した。つぎに、各トランスフェクション液を、９６穴プレートに０．２μＬ／ウェルとなるように添加した。さらに、各ウェルに、１．２×１０4細胞／ウェルとなるように、前記Ａ１７２細胞を播種した。前記ウェルあたり、前記核酸分子の最終濃度は、２５または７５ｎｍｏｌ／Ｌとした。前記Ａ１７２細胞の培地および培養条件は、前記実施例１（２）と同様とした。これらの点を除き、前記実施例１（２）と同様にして、前記ヒトペリオスチン遺伝子の発現量の相対値を算出した。 (2) Measurement of expression level of periostin gene Transfection solutions for transfection of the nucleic acid molecules were prepared using the transfection reagent Lipofectamine (registered trademark) 2000 according to the attached protocol. Next, each transfection solution was added to a 96-well plate at 0.2 μL / well. Furthermore, the A172 cells were seeded at 1.2 × 10 4 cells / well in each well. The final concentration of the nucleic acid molecule per well was 25 or 75 nmol / L. The medium and culture conditions for the A172 cells were the same as in Example 1 (2). Except for these points, the relative value of the expression level of the human periostin gene was calculated in the same manner as in Example 1 (2).
（３）結果
　これらの結果を、図１４および１５に示す。図１４は、前記表７の核酸分子の結果を示し、図１５は、前記表８の核酸分子の結果を示す。図１４および１５は、発現量の相対値を示すグラフであり、縦軸は、発現量の相対値であり、図中の黒色のバーが２５ｎｍｏｌ／Ｌの結果を示し、白色のバーが７５ｎｍｏｌ／Ｌの結果を示す。図１４および１５に示すように、前記実施例の核酸分子は、発現量の相対値が１よりも低い値を示したことから、いずれも発現抑制活性を有することが確認できた。これらの結果から、異なる長さを有するペリオスチンの発現抑制核酸分子および異なる修飾ヌクレオチド残基を含むペリオスチンの発現抑制核酸分子においても、優れた発現抑制活性を有することがわかった。 (3) Results These results are shown in FIGS. FIG. 14 shows the results for the nucleic acid molecules in Table 7, and FIG. 15 shows the results for the nucleic acid molecules in Table 8. 14 and 15 are graphs showing the relative value of the expression level, the vertical axis is the relative value of the expression level, the black bar in the figure shows the result of 25 nmol / L, and the white bar is 75 nmol / L The result of L is shown. As shown in FIGS. 14 and 15, the nucleic acid molecules of the above examples were confirmed to have expression suppression activity since the relative value of the expression level was lower than 1. From these results, it was found that periostin expression-suppressing nucleic acid molecules having different lengths and periostin expression-suppressing nucleic acid molecules containing different modified nucleotide residues also have excellent expression-suppressing activity.
　以上、実施形態および実施例を参照して本発明を説明したが、本発明は、上記実施形態および実施例に限定されるものではない。本発明の構成や詳細には、本発明のスコープ内で当業者が理解しうる様々な変更をすることができる。 As mentioned above, although this invention was demonstrated with reference to embodiment and an Example, this invention is not limited to the said embodiment and Example. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.
　この出願は、２０１５年８月１０日に出願された国際特許出願ＰＣＴ／ＪＰ２０１５／０７２６９６を基礎とする優先権を主張し、その開示の全てをここに取り込む。 This application claims priority based on international patent application PCT / JP2015 / 072696 filed on August 10, 2015, the entire disclosure of which is incorporated herein.
　本発明の核酸分子によれば、ペリオスチン遺伝子の発現またはペリオスチンタンパク質の機能を抑制できる。このため、本発明は、ペリオスチン遺伝子の発現またはペリオスチンタンパク質が原因となる疾患の治療に有効である。 According to the nucleic acid molecule of the present invention, the expression of periostin gene or the function of periostin protein can be suppressed. Therefore, the present invention is effective for treatment of diseases caused by periostin gene expression or periostin protein.

Claims (37)

  1. ペリオスチン遺伝子の発現抑制核酸分子であり、
    前記発現抑制核酸分子が、前記ペリオスチン遺伝子の発現抑制配列として、下記（ａｓ４）のヌクレオチドを含むことを特徴とする、ペリオスチン遺伝子の発現抑制核酸分子。
    （ａｓ４）配列番号１２１～１２７のいずれか１つの塩基配列からなるポリヌクレオチドに対して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からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチド     Periostin gene expression-suppressing nucleic acid molecule,
    The expression-inhibiting nucleic acid molecule of the periostin gene, wherein the expression-inhibiting nucleic acid molecule comprises the following (as4) nucleotide as an expression-inhibiting sequence of the periostin gene.
    (As4) a nucleotide comprising a base sequence that hybridizes under stringent conditions to a polynucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 121 to 127, and having a function of suppressing expression of a periostin gene
  2. 前記（ａｓ４）のヌクレオチドが、配列番号１２１～１２７のいずれか１つの塩基配列からなるポリヌクレオチドのコーディング領域および３’非翻訳領域の少なくとも一方に対して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からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチドである、請求項１記載の発現抑制核酸分子。 A nucleotide sequence in which the nucleotide (as4) hybridizes under stringent conditions to at least one of the coding region and 3 ′ untranslated region of a polynucleotide comprising any one nucleotide sequence of SEQ ID NOS: 121 to 127 The expression-suppressing nucleic acid molecule according to claim 1, which is a nucleotide comprising a periostin gene expression-suppressing function.
  3. 前記（ａｓ４）のヌクレオチドが、配列番号１２１の塩基配列における１２２９～３１３２番目の塩基配列からなるポリヌクレオチドに対して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からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチドである、請求項１または２記載の発現抑制核酸分子。 The nucleotide (as4) comprises a base sequence that hybridizes under stringent conditions to a polynucleotide comprising the 1229 to 3132st base sequence in the base sequence of SEQ ID NO: 121, and has a function to suppress the expression of the periostin gene. The expression-suppressing nucleic acid molecule according to claim 1 or 2, which is a nucleotide having a nucleotide.
  4. 前記（ａｓ４）のヌクレオチドが、配列番号１２１の塩基配列における１２２９～１２５７番目の塩基配列からなるポリヌクレオチドに対して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からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチドである、請求項３記載の発現抑制核酸分子。 The nucleotide (as4) comprises a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide comprising the 1229 to 1257th nucleotide sequence in the nucleotide sequence of SEQ ID NO: 121, and has a function to suppress the expression of the periostin gene. The expression-suppressing nucleic acid molecule according to claim 3, which is a nucleotide having a nucleotide.
  5. 前記（ａｓ４）のヌクレオチドが、配列番号１２１の塩基配列における１２７０～１３０１番目の塩基配列からなるポリヌクレオチドに対して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からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチドである、請求項３記載の発現抑制核酸分子。 The nucleotide (as4) comprises a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide comprising the nucleotide sequence 1270 to 1301 in the nucleotide sequence of SEQ ID NO: 121, and has a function to suppress the expression of the periostin gene. The expression-suppressing nucleic acid molecule according to claim 3, which is a nucleotide having a nucleotide.
  6. 前記（ａｓ４）のヌクレオチドが、配列番号１２１の塩基配列における１４２６～１４４５番目の塩基配列からなるポリヌクレオチドに対して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からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチドである、請求項３記載の発現抑制核酸分子。 The nucleotide (as4) comprises a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide comprising the 1426th to 1445th nucleotide sequences in the nucleotide sequence of SEQ ID NO: 121, and has a function to suppress the expression of the periostin gene. The expression-suppressing nucleic acid molecule according to claim 3, which is a nucleotide having a nucleotide.
  7. 前記（ａｓ４）のヌクレオチドが、配列番号１２１の塩基配列における１４８８～１５０９番目の塩基配列からなるポリヌクレオチドに対して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からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチドである、請求項３記載の発現抑制核酸分子。 The nucleotide (as4) comprises a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide comprising the 1488th to 1509th nucleotide sequences in the nucleotide sequence of SEQ ID NO: 121, and has a function to suppress the expression of the periostin gene. The expression-suppressing nucleic acid molecule according to claim 3, which is a nucleotide having a nucleotide.
  8. 前記（ａｓ４）のヌクレオチドが、配列番号１２１の塩基配列における１７０６～１９６５番目の塩基配列からなるポリヌクレオチドに対して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からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチドである、請求項３記載の発現抑制核酸分子。 The nucleotide (as4) comprises a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide comprising the 1706th to 1965th nucleotide sequences in the nucleotide sequence of SEQ ID NO: 121, and has a function to suppress the expression of the periostin gene. The expression-suppressing nucleic acid molecule according to claim 3, which is a nucleotide having a nucleotide.
  9. 前記（ａｓ４）のヌクレオチドが、配列番号１２１の塩基配列における２０７８～２１０３番目の塩基配列からなるポリヌクレオチドに対して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からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチドである、請求項３記載の発現抑制核酸分子。 The nucleotide (as4) comprises a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide comprising the 2078 to 2103th nucleotide sequence in the nucleotide sequence of SEQ ID NO: 121, and has a function of suppressing the expression of the periostin gene. The expression-suppressing nucleic acid molecule according to claim 3, which is a nucleotide having a nucleotide.
  10. 前記（ａｓ４）のヌクレオチドが、配列番号１２１の塩基配列における２６３０～３３９０番目の塩基配列からなるポリヌクレオチドに対して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からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチドである、請求項３記載の発現抑制核酸分子。 The nucleotide (as4) comprises a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide comprising the nucleotide sequence from 2630 to 3390 in the nucleotide sequence of SEQ ID NO: 121, and has a function to suppress the expression of the periostin gene. The expression-suppressing nucleic acid molecule according to claim 3, which is a nucleotide having a nucleotide.
  11. 前記（ａｓ４）のヌクレオチドが、配列番号１２１の塩基配列における２９２１～３１３２番目の塩基配列からなるポリヌクレオチドに対して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からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチドである、請求項３記載の発現抑制核酸分子。 The nucleotide (as4) comprises a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide comprising the 2921st to 3132rd nucleotide sequence in the nucleotide sequence of SEQ ID NO: 121, and has a function to suppress the expression of the periostin gene. The expression-suppressing nucleic acid molecule according to claim 3, which is a nucleotide having a nucleotide.
  12. 前記（ａｓ４）のヌクレオチドが、配列番号１２１の塩基配列における２９２２～３０７８番目の塩基配列からなるポリヌクレオチドに対して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からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチドである、請求項１１記載の発現抑制核酸分子。 The nucleotide (as4) comprises a nucleotide sequence that hybridizes under stringent conditions to the polynucleotide comprising the nucleotide sequence 2922-3078 in the nucleotide sequence of SEQ ID NO: 121, and has a function to suppress the expression of the periostin gene. The expression-suppressing nucleic acid molecule according to claim 11, which is a nucleotide having a nucleotide.
  13. 前記（ａｓ４）のヌクレオチドが、配列番号１２１の塩基配列における８１５～１１２７番目の塩基配列からなるポリヌクレオチドに対して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からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチドである、請求項１または２記載の発現抑制核酸分子。 The nucleotide (as4) comprises a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide comprising the nucleotide sequence of positions 815 to 1127 in the nucleotide sequence of SEQ ID NO: 121, and has a function of suppressing the expression of the periostin gene. The expression-suppressing nucleic acid molecule according to claim 1 or 2, which is a nucleotide having a nucleotide.
  14. 前記（ａｓ４）のヌクレオチドが、配列番号１２１の塩基配列における９６５～９８８番目の塩基配列からなるポリヌクレオチドに対して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からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチドである、請求項１３記載の発現抑制核酸分子。 The nucleotide (as4) comprises a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide comprising the 965th to 988th nucleotide sequences in the nucleotide sequence of SEQ ID NO: 121, and has a function to suppress the expression of the periostin gene. The expression-suppressing nucleic acid molecule according to claim 13, which is a nucleotide having the same.
  15. 前記（ａｓ４）のヌクレオチドが、配列番号１２１の塩基配列における１１０５～１１２７番目の塩基配列からなるポリヌクレオチドに対して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からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチドである、請求項１３記載の発現抑制核酸分子。 The nucleotide (as4) comprises a nucleotide sequence that hybridizes under stringent conditions to a polynucleotide comprising the 1105th to 1127th nucleotide sequences in the nucleotide sequence of SEQ ID NO: 121, and has a function to suppress the expression of the periostin gene. The expression-suppressing nucleic acid molecule according to claim 13, which is a nucleotide having the same.
  16. 前記ペリオスチン遺伝子の発現抑制核酸分子が、前記ペリオスチン遺伝子の発現抑制配列として、下記（ａｓ１）、（ａｓ２）または（ａｓ３）のヌクレオチドを含む、請求項１から１５のいずれか一項に記載の発現抑制核酸分子。
    （ａｓ１）配列番号１～１２０および２１７～２３２のいずれか一つの塩基配列からなるヌクレオチド
    （ａｓ２）前記（ａｓ１）の塩基配列において、１もしくは数個の塩基が欠失、置換および／または付加された塩基配列からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチド
    （ａｓ３）前記（ａｓ１）の塩基配列と８０％以上の同一性を有する塩基配列からなり、ペリオスチン遺伝子の発現抑制機能を有するヌクレオチド     The expression according to any one of claims 1 to 15, wherein the periostin gene expression-suppressing nucleic acid molecule comprises the following (as1), (as2), or (as3) nucleotides as an expression-suppressing sequence for the periostin gene. Inhibitory nucleic acid molecule.
    (As1) Nucleotide comprising any one of the nucleotide sequences of SEQ ID NOS: 1-120 and 217-232 (as2) In the nucleotide sequence of (as1), one or several bases are deleted, substituted and / or added. A nucleotide having a function to suppress expression of a periostin gene (as3) A nucleotide having a base sequence having 80% or more identity with the base sequence of (as1) and having a function to suppress expression of a periostin gene
  17. 前記発現抑制核酸分子が、一本鎖核酸分子であり、
    ５’側から３’側にかけて、５’側領域、内部領域、および３’側領域を、前記順序で含み、
    前記内部領域を構成するヌクレオチド残基が、デオキシリボヌクレオチド残基である、請求項１から１６のいずれか一項に記載の発現抑制核酸分子。     The expression-suppressing nucleic acid molecule is a single-stranded nucleic acid molecule,
    From the 5 ′ side to the 3 ′ side, the 5 ′ side region, the inner region, and the 3 ′ side region are included in the order,
    The nucleic acid molecule for suppressing expression according to any one of claims 1 to 16, wherein the nucleotide residue constituting the internal region is a deoxyribonucleotide residue.
  18. 前記５’側領域および前記３’側領域の少なくとも一方を構成するヌクレオチド残基が、リボヌクレオチド残基である、請求項１から１７のいずれか一項に記載の発現抑制核酸分子。 The expression-suppressing nucleic acid molecule according to any one of claims 1 to 17, wherein the nucleotide residue constituting at least one of the 5'-side region and the 3'-side region is a ribonucleotide residue.
  19. 前記発現抑制核酸分子が、一本鎖核酸分子であり、
    ５’側から３’側にかけて、５’側領域、内部領域、および３’側領域を、前記順序で含み、
    前記５’側領域および前記３’側領域の少なくとも一方を構成するヌクレオチド残基が、修飾ヌクレオチド残基である、請求項１から１８のいずれか一項に記載の発現抑制核酸分子。     The expression-suppressing nucleic acid molecule is a single-stranded nucleic acid molecule,
    From the 5 ′ side to the 3 ′ side, the 5 ′ side region, the inner region, and the 3 ′ side region are included in the order,
    The expression-suppressing nucleic acid molecule according to any one of claims 1 to 18, wherein the nucleotide residue constituting at least one of the 5'-side region and the 3'-side region is a modified nucleotide residue.
  20. 前記修飾ヌクレオチド残基が、糖残基が修飾された修飾ヌクレオチド残基である、請求項１９記載の発現抑制核酸分子。 The expression-suppressing nucleic acid molecule according to claim 19, wherein the modified nucleotide residue is a modified nucleotide residue in which a sugar residue is modified.
  21. 前記糖残基が修飾された修飾ヌクレオチド残基が、２’－Ｏ－メチルリボースを含む修飾ヌクレオチド残基またはＬＮＡ（Locked Nucleic Acid）である、請求項２０記載の発現抑制核酸分子。 21. The expression-suppressing nucleic acid molecule according to claim 20, wherein the modified nucleotide residue in which the sugar residue is modified is a modified nucleotide residue containing 2'-O-methylribose or LNA (LockeducNucleic Acid).
  22. 前記５’側領域、前記内部領域、および前記３’側領域を構成するヌクレオチド残基が、リン酸基が修飾された修飾ヌクレオチド残基である、請求項１７から２１のいずれか一項に記載の発現抑制核酸分子。 The nucleotide residue constituting the 5 'region, the internal region, and the 3' region is a modified nucleotide residue in which a phosphate group is modified. Expression-suppressing nucleic acid molecule.
  23. 前記リン酸基が修飾された修飾ヌクレオチド残基が、ホスホロチオエートを含む修飾オリゴヌクレオチドである、請求項２２記載の発現抑制核酸分子。 The expression-suppressing nucleic acid molecule according to claim 22, wherein the modified nucleotide residue in which the phosphate group is modified is a modified oligonucleotide containing phosphorothioate.
  24. 前記５’側領域の塩基数が、１～１４個である、請求項１７から２３のいずれか一項に記載の発現抑制核酸分子。 The expression-suppressing nucleic acid molecule according to any one of claims 17 to 23, wherein the number of bases in the 5'-side region is 1 to 14.
  25. 前記３’側領域の塩基数が、１～１４個である、請求項１７から２４のいずれか一項に記載の発現抑制核酸分子。 The expression-suppressing nucleic acid molecule according to any one of claims 17 to 24, wherein the number of bases in the 3 'side region is 1 to 14.
  26. 前記内部領域の塩基数が、５～１６個である、請求項１７から２５のいずれか一項に記載の発現抑制核酸分子。 The expression-suppressing nucleic acid molecule according to any one of claims 17 to 25, wherein the number of bases in the internal region is 5 to 16.
  27. 前記ペリオスチン遺伝子の発現抑制核酸分子が、保護基を有する核酸分子である、請求項１から２６のいずれか一項に記載の発現抑制核酸分子。 27. The expression-suppressing nucleic acid molecule according to any one of claims 1 to 26, wherein the periostin gene expression-suppressing nucleic acid molecule is a nucleic acid molecule having a protecting group.
  28. 請求項１から２７のいずれか一項に記載の発現抑制核酸分子を含むことを特徴とする、ペリオスチン遺伝子の発現抑制試薬。 A periostin gene expression suppression reagent comprising the expression suppression nucleic acid molecule according to any one of claims 1 to 27.
  29. 請求項１から２７のいずれか一項に記載の発現抑制核酸分子を含むことを特徴とする、ペリオスチン遺伝子関連疾患用医薬品。 A medicine for a periostin gene-related disease comprising the expression-suppressing nucleic acid molecule according to any one of claims 1 to 27.
  30. 前記ペリオスチン遺伝子関連疾患が、眼疾患、皮膚疾患、呼吸器疾患、腎疾患、肝臓疾患、消化器疾患、耳鼻咽喉科疾患、循環器疾患、血液疾患、骨関節疾患、がん、炎症性疾患、および線維化疾患からなる群から選択された少なくとも１つである、請求項２９記載の医薬品。 The periostin gene-related diseases include eye diseases, skin diseases, respiratory diseases, kidney diseases, liver diseases, gastrointestinal diseases, otolaryngology diseases, cardiovascular diseases, blood diseases, bone and joint diseases, cancer, inflammatory diseases, 30. The pharmaceutical product of claim 29, wherein the pharmaceutical product is at least one selected from the group consisting of fibrotic diseases.
  31. ペリオスチン遺伝子の発現を抑制する方法であって、
    請求項１から２７のいずれか一項に記載の発現抑制核酸分子を使用することを特徴とする抑制方法。     A method of suppressing the expression of the periostin gene,
    An expression suppression method comprising using the expression-suppressing nucleic acid molecule according to any one of claims 1 to 27.
  32. 前記発現抑制核酸分子を、細胞、組織または器官に投与する工程を含む、請求項３１記載の抑制方法。 32. The suppression method according to claim 31, comprising a step of administering the expression-suppressing nucleic acid molecule to a cell, tissue or organ.
  33. 前記発現抑制核酸分子を、ｉｎ　ｖｉｖｏまたはｉｎ　ｖｉｔｒｏで投与する、請求項３１または３２記載の抑制方法。 The suppression method according to claim 31 or 32, wherein the expression suppressing nucleic acid molecule is administered in vivo or in vitro .
  34. 請求項１から２７のいずれか一項に記載の発現抑制核酸分子を、患者に投与する工程を含むことを特徴とする、ペリオスチン遺伝子関連疾患の治療方法。 A method for treating a periostin gene-related disease, comprising a step of administering the expression-suppressing nucleic acid molecule according to any one of claims 1 to 27 to a patient.
  35. 前記ペリオスチン遺伝子関連疾患が、眼疾患、皮膚疾患、呼吸器疾患、腎疾患、肝臓疾患、消化器疾患、耳鼻咽喉科疾患、循環器疾患、血液疾患、骨関節疾患、がん、炎症性疾患、および線維化疾患からなる群から選択された少なくとも１つである、請求項３４記載の治療方法。 The periostin gene-related diseases include eye diseases, skin diseases, respiratory diseases, kidney diseases, liver diseases, gastrointestinal diseases, otolaryngology diseases, cardiovascular diseases, blood diseases, bone and joint diseases, cancer, inflammatory diseases, 35. The treatment method according to claim 34, wherein the treatment method is at least one selected from the group consisting of fibrotic diseases.
  36. ペリオスチン遺伝子関連疾患の治療のために使用する請求項１から２７のいずれか一項に記載の発現抑制核酸分子。 The expression-suppressing nucleic acid molecule according to any one of claims 1 to 27, which is used for treatment of a periostin gene-related disease.
  37. 前記ペリオスチン遺伝子関連疾患が、眼疾患、皮膚疾患、呼吸器疾患、腎疾患、肝臓疾患、消化器疾患、耳鼻咽喉科疾患、循環器疾患、血液疾患、骨関節疾患、がん、炎症性疾患、および線維化疾患からなる群から選択された少なくとも１つである、請求項３６記載の発現抑制核酸分子。 The periostin gene-related diseases include eye diseases, skin diseases, respiratory diseases, kidney diseases, liver diseases, gastrointestinal diseases, otolaryngology diseases, cardiovascular diseases, blood diseases, bone and joint diseases, cancer, inflammatory diseases, 37. The expression-suppressing nucleic acid molecule according to claim 36, wherein the nucleic acid molecule is at least one selected from the group consisting of a fibrotic disease.
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