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WO2017026496A1 - Molécule d'acide nucléique supprimant l'expression du gène de la périostine et application associée - Google Patents

Molécule d'acide nucléique supprimant l'expression du gène de la périostine et application associée 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)
Inventor
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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AQUA THERAPEUTICS Co Ltd
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AQUA THERAPEUTICS Co Ltd
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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

Definitions

  • 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

L'invention concerne une nouvelle molécule permettant de supprimer l'expression du gène de la périostine. L'invention concerne une molécule d'acide nucléique permettant de supprimer l'expression du gène de la périostine, la molécule d'acide nucléique supprimant l'expression étant caractérisé en ce qu'il contient le nucléotide (as4) suivant comme séquence de suppression de l'expression du gène de la périostine. (as4) Nucléotide ayant la capacité à supprimer l'expression du gène de la périostine, ledit nucléotide comprenant une séquence de bases qui s'hybride dans des conditions stringentes avec un polynucléotide comprenant l'une quelconque des séquences de base de la SEQ ID n° 121-127. Etant donné que cette molécule d'acide nucléique supprimant l'expression peut supprimer l'expression du gène de la périostine, ladite molécule peut être utilisée, par exemple, dans le traitement de maladies résultant de l'expression génique de la périostine.
PCT/JP2016/073500 2015-08-10 2016-08-09 Molécule d'acide nucléique supprimant l'expression du gène de la périostine et application associée Ceased WO2017026496A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013147140A1 (fr) * 2012-03-29 2013-10-03 国立大学法人九州大学 Molécule d'acide nucléique apte à inhiber l'expression du gène périostine, procédé d'inhibition de l'expression du gène périostine et utilisation de ladite molécule d'acide nucléique
WO2015046451A1 (fr) * 2013-09-27 2015-04-02 株式会社アクアセラピューティクス Médicament pour maladie causée par l'expression de la périostine à l'exception d'une maladie oculaire et son utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013147140A1 (fr) * 2012-03-29 2013-10-03 国立大学法人九州大学 Molécule d'acide nucléique apte à inhiber l'expression du gène périostine, procédé d'inhibition de l'expression du gène périostine et utilisation de ladite molécule d'acide nucléique
WO2015046451A1 (fr) * 2013-09-27 2015-04-02 株式会社アクアセラピューティクス Médicament pour maladie causée par l'expression de la périostine à l'exception d'une maladie oculaire et son utilisation

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Title
DATABASE DDBJ/EMBL/GenBank [O] 26 August 2016 (2016-08-26), KIKUCHI, Y. ET AL.: "Homo sapiens periostin, osteoblast specific factor (POSTN", XP055364815, Database accession no. NM_001135935 *
DATABASE DDBJ/EMBL/GenBank 21 October 2008 (2008-10-21), KIKUCHI, Y. ET AL.: "Definition: Homo sapiens periostin, osteoblast specific factor (POSTN), transcript variant 1, mRNA", XP003034204, Database accession no. NM_006475 *
DATABASE DDBJ/EMBL/GenBank 21 October 2008 (2008-10-21), KIKUCHI, Y. ET AL.: "Homo sapiens periostin, osteoblast specific factor (POSTN", XP003034204, Database accession no. NM_001135934 *
DATABASE GenBank [O] 26 August 2016 (2016-08-26), KANEMITSU, Y. ET AL.: "Homo sapiens periostin, osteoblast specific factor (POSTN", XP055364827, Database accession no. NM_001286667 *
DATABASE GenBank 26 August 2016 (2016-08-26), KANEMITSU, Y. ET AL.: "Homo sapiens periostin, osteoblast specific factor (POSTN", XP055364824, Database accession no. NM_001286665 *
DATABASE GenBank 26 August 2016 (2016-08-26), KANEMITSU, Y. ET AL.: "Homo sapiens periostin, osteoblast specific factor (POSTN", XP055364825, Database accession no. NM_001286666 *
DATABASE GenBank 26 August 2016 (2016-08-26), KIKUCHI, Y. ET AL.: "Homo sapiens periostin, osteoblast specific factor (POSTN", XP055364820, Database accession no. NM_001135936 *

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