WO2015093495A1 - Molécule d'acide nucléique simple brin pour la régulation de l'expression du gène tgf-β1 - Google Patents

Molécule d'acide nucléique simple brin pour la régulation de l'expression du gène tgf-β1 Download PDF

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WO2015093495A1
WO2015093495A1 PCT/JP2014/083312 JP2014083312W WO2015093495A1 WO 2015093495 A1 WO2015093495 A1 WO 2015093495A1 JP 2014083312 W JP2014083312 W JP 2014083312W WO 2015093495 A1 WO2015093495 A1 WO 2015093495A1
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region
nucleic acid
acid molecule
bases
expression
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PCT/JP2014/083312
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Japanese (ja)
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雅彦 黒田
エステバン ガバザ
小林　哲
忠明 大木
智洋 濱崎
志織 加藤
松本　貴博
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株式会社ボナック
国立大学法人三重大学
学校法人東京医科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • 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
    • C12N15/1136—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 against growth factors, growth regulators, cytokines, lymphokines or hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin

Definitions

  • the present invention relates to a nucleic acid molecule that suppresses the expression of TGF- ⁇ 1 gene, a composition containing the same, and use thereof.
  • Lung fibrosis is a disease in which lung tissue becomes fibrotic due to the accumulation of excess collagen and other cell matrices.
  • pulmonary fibrosis idiopathic pulmonary fibrosis is a chronic intractable disease with an average intermediate survival period of 3 years and a 5-year survival rate of 20 to 40%.
  • Acute lung injury is a kind of acute respiratory disorder and is acute respiratory failure showing lung infiltration shadow.
  • the pathology of acute lung injury is pulmonary edema due to increased permeability caused by lung capillary endothelial cells and alveolar wall cell damage. This interstitial pulmonary edema, alveolar pulmonary edema and pulmonary collapse leads to increased dead space ventilation and hypoxemia.
  • Patent Documents 1-5 Non-Patent Document 1
  • Patent Documents 1-7 Patent Documents 1-7).
  • RNA interference is known as a technique for suppressing gene expression. Inhibition of gene expression by RNA interference is generally performed, for example, by administering a short double-stranded RNA molecule to a cell or the like.
  • the double-stranded RNA molecule is usually referred to as siRNA (small interfering RNA).
  • siRNA small interfering RNA
  • an object of the present invention is to provide a new nucleic acid molecule that is effective for treating diseases involving the expression of TGF- ⁇ 1, such as pulmonary fibrosis and acute lung injury, and a medicine using the same. To do.
  • the TGF- ⁇ 1 gene expression suppressing nucleic acid molecule of the present invention is characterized by comprising the following nucleotide sequence as a TGF- ⁇ 1 gene expression suppressing sequence.
  • SEQ ID NO: 1 3'-CGUCUCAUGUGUGUCGUAU-5 '
  • composition of the present invention is a composition for suppressing the expression of the TGF- ⁇ 1 gene, and includes the nucleic acid molecule of the present invention.
  • composition of the present invention is a pharmaceutical composition and is characterized by containing the nucleic acid molecule of the present invention.
  • the expression suppression method of the present invention is a method of suppressing the expression of TGF- ⁇ 1 gene, characterized by using the nucleic acid molecule of the present invention.
  • the method for treating pulmonary fibrosis of the present invention is characterized by including a step of administering the nucleic acid molecule of the present invention to a patient.
  • the method for treating acute lung injury of the present invention is characterized by including a step of administering the nucleic acid molecule of the present invention to a patient.
  • the nucleic acid molecule of the present invention can suppress the expression of TGF- ⁇ 1 gene. Therefore, the present invention is effective for treating diseases caused by the expression of the TGF- ⁇ 1 gene, such as pulmonary fibrosis and acute lung injury.
  • FIG. 1 is a schematic diagram showing an example of the nucleic acid molecule of the present invention.
  • FIG. 2 is a schematic diagram showing another example of the nucleic acid molecule of the present invention.
  • FIG. 3 is a schematic diagram showing another example of the nucleic acid molecule of the present invention.
  • FIG. 4 is a schematic diagram showing another example of the nucleic acid molecule of the present invention.
  • FIG. 5 is a graph showing the relative value of TGF- ⁇ 1 amount in Example 1 of the present invention.
  • FIG. 6 is an electrophoretogram of serum stability in Example 2 of the present invention.
  • FIG. 7 is a photograph of electrophoresis for reactivity to Dicer protein in Example 3 of the present invention.
  • FIG. 8 is a graph showing the inhibitory effect of TGF- ⁇ expression in lung tissue by administration of the nucleic acid molecule of the present invention in pulmonary fibrosis spontaneous model mice.
  • FIG. 9 is a graph showing the effect of suppressing the amount of hydroxyproline in lung tissue by administration of the nucleic acid molecule of the present invention in a pulmonary fibrosis spontaneous model mouse.
  • FIG. 10 is a graph showing the inhibitory effect on the total number of cells in bronchoalveolar lavage fluid (BALF) by administration of the nucleic acid molecule of the present invention in bleomycin-induced pulmonary fibrosis model mice.
  • BALF bronchoalveolar lavage fluid
  • FIG. 11 is a graph showing the survival rate improving effect of the bleomycin-induced pulmonary fibrosis model mouse by the administration of the nucleic acid molecule of the present invention.
  • FIG. 12 is a graph showing the effect of suppressing the amount of hydroxyproline in lung tissue by administration of the nucleic acid molecule of the present invention in a bleomycin-induced pulmonary fibrosis model mouse.
  • FIG. 13 is a graph showing the inhibitory effect on the total number of cells in bronchoalveolar lavage fluid (BALF) by administration of the nucleic acid molecule of the present invention in LPS-induced pulmonary fibrosis / acute lung injury combined model mice.
  • FIG. 14 is a graph showing the effect of administration of the nucleic acid molecule of the present invention on the amount of IFN in the bronchoalveolar lavage fluid (BALF) of LPS-induced pulmonary fibrosis / acute lung injury combined model mice.
  • BALF bronchoalveolar lavage fluid
  • nucleic acid molecule for suppressing expression of TGF- ⁇ 1 gene (1) Expression suppressing sequence and complementary sequence As described above, the nucleic acid molecule of the present invention is for suppressing the expression of TGF- ⁇ 1 gene and suppresses the expression of TGF- ⁇ 1 gene.
  • the sequence includes the following nucleotide sequence. (SEQ ID NO: 1) 3'-CGUCUCAUGUGUGUCGUAU-5 '
  • the expression suppression sequence may be, for example, a sequence consisting of the nucleotide sequence or a sequence containing the nucleotide sequence.
  • the length of the expression suppression sequence is not particularly limited, and is, for example, 18 to 32 bases long, preferably 19 to 30 bases long, and more preferably 19, 20, or 21 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 single-stranded nucleic acid molecule of the present invention preferably further has, for example, a complementary sequence that can be annealed with the expression suppressing sequence.
  • the complementary sequence is, for example, in the same strand as the expression suppressing sequence and forms a single-stranded nucleic acid molecule composed of one single strand.
  • the complementary sequence only needs to be annealable with the expression suppression sequence, for example.
  • the complementary sequence may be, for example, a sequence exhibiting 100% complementarity with the expression suppression sequence, or a sequence exhibiting complementarity of less than 100% within a range that can be annealed.
  • the complementarity is not particularly limited, and examples thereof include 90% to 100%, 93% to 100%, 95% to 100%, 98% to 100%, and 99% to 100%.
  • Examples of the complementary sequence include those containing the following nucleotide sequences. (SEQ ID NO: 2) 5'-GCAGAGUACACACAGCAUA-3 '
  • nucleotide sequence of SEQ ID NO: 2 is referred to as s nucleotide sequence.
  • the complementary sequence may be, for example, a sequence composed of the s nucleotide sequence or a sequence containing the s nucleotide sequence.
  • the length of the complementary sequence is not particularly limited, and is, for example, 18 to 32 bases long, preferably 19 to 30 bases long, and more preferably 19, 20, or 21 bases long.
  • the expression suppression sequence and the complementary sequence may each be, for example, an RNA molecule consisting only of ribonucleotide residues, or an RNA molecule containing deoxyribonucleotide residues in addition to ribonucleotide residues.
  • nucleic acid molecule examples include a form in which the expression suppressing sequence and the complementary sequence are directly linked and a form in which they are indirectly linked.
  • Examples of the direct linking include linking by a phosphodiester bond.
  • Examples of the indirect linkage include linkage via a linker region.
  • the order in which the expression suppressing sequence and the complementary sequence are linked is not particularly limited, and for example, the 3 ′ end of the expression suppressing sequence and the 5 ′ end of the complementary sequence may be linked. The 5 ′ end and the 3 ′ end of the complementary sequence may be linked, preferably the former.
  • the linker region may be composed of, for example, nucleotide residues, may be composed of non-nucleotide residues, or may be composed of the nucleotide residues and non-nucleotide residues.
  • nucleotide residues include a ribonucleotide residue and a deoxyribonucleotide residue.
  • a molecule in which a 5′-side region and a 3′-side region are annealed with each other to form a double-stranded structure can be mentioned.
  • This can also be said to be a form of shRNA (small hairpin RNA or short hairpin RNA).
  • shRNA small hairpin RNA or short hairpin RNA.
  • the shRNA has a hairpin structure and generally has one stem region and one loop region.
  • the nucleic acid molecule of this embodiment includes, for example, a region (X), a linker region (Lx), and a region (Xc), and the linker region (Lx) is between the region (X) and the region (Xc). Takes a linked structure.
  • the region (Xc) preferably has a structure complementary to the region (X). Specifically, one of the region (X) and the region (Xc) has the expression suppressing sequence.
  • the other includes the complementary sequence. Since the region (X) and the region (Xc) each have one of the expression suppression sequence and the complementary sequence, for example, a stem structure can be formed by intramolecular annealing, and the linker region (Lx) It becomes a loop structure.
  • the nucleic acid molecule may have, for example, the region (Xc), the linker region (Lx), and the region (X) in the order from 5 ′ side to 3 ′ side, or from the 3 ′ side. You may have the said area
  • the expression suppressing sequence may be arranged, for example, in any of the region (X) and the region (Xc), and may be arranged upstream of the complementary sequence, that is, 5 ′ side of the complementary sequence. preferable.
  • FIG. 1A is a schematic diagram showing an outline of the order of each region
  • FIG. 1B is a schematic diagram showing a state in which the nucleic acid molecule forms a double strand in the molecule. is there.
  • the nucleic acid molecule forms a double strand between the region (Xc) and the region (X), and the Lx region loops according to its length.
  • FIG. 1 merely shows the linking order of the regions and the positional relationship of each region forming a duplex. For example, the length of each region, the shape of the linker region (Lx), etc. Not limited.
  • the number of bases in the region (Xc) and the region (X) is not particularly limited. Although the length of each area
  • the relationship between the number of bases (X) in the region (X) and the number of bases (Xc) in the region (Xc) satisfies, for example, the following (3) or (5) In this case, specifically, for example, the following condition (11) is satisfied.
  • X ⁇ Xc 1 to 10, preferably 1, 2 or 3, More preferably 1 or 2 (11)
  • X Xc (5)
  • the region may be a region composed of only the expression suppression sequence or a region including the expression suppression sequence, for example.
  • the number of bases of the expression suppression sequence is, for example, as described above.
  • the region containing the expression suppression sequence may further have an additional sequence on the 5 'side and / or 3' side of the expression suppression sequence, for example.
  • the number of bases of the additional sequence is, for example, 1 to 31 bases, preferably 1 to 21 bases, and more preferably 1 to 11 bases.
  • the number of bases in the region (Xc) is not particularly limited.
  • the lower limit is, for example, 19 bases.
  • the upper limit is, for example, 50 bases, preferably 30 bases, and more preferably 25 bases.
  • Specific examples of the number of bases in the region (X) are, for example, 19 to 50 bases, preferably 19 to 30 bases, more preferably 19 to 25 bases.
  • the number of bases in the region (X) is not particularly limited.
  • the lower limit is, for example, 19 bases, preferably 20 bases, and more preferably 21 bases.
  • the upper limit is 50 bases, for example, More preferably, it is 40 bases, More preferably, it is 30 bases.
  • the linker region (Lx) includes a nucleotide residue as described above, the length is not particularly limited.
  • the linker region (Lx) preferably has a length that allows the region (X) and the region (Xc) to form a double chain.
  • the lower limit of the number of bases in the linker region (Lx) is, for example, 1 base, preferably 2 bases, more preferably 3 bases, and the upper limit thereof is, for example, 100 bases, preferably 80 bases, more preferably 50 bases.
  • a second form of the single-stranded nucleic acid molecule is a molecule in which the 5 ′ region and the 3 ′ region are separately annealed in the molecule to form two double-stranded structures (stem structures).
  • the nucleic acid molecule of the present embodiment includes, for example, a 5 ′ side region (Xc), an internal region (Z), and a 3 ′ side region (Yc) from the 5 ′ side to the 3 ′ side in the order described above.
  • Z) is formed by connecting an inner 5 ′ side region (X) and an inner 3 ′ side region (Y), and the 5 ′ side region (Xc) is complementary to the inner 5 ′ side region (X).
  • the 3 ′ side region (Yc) is preferably complementary to the inner 3 ′ side region (Y).
  • the 5 ′ region (Xc) when the internal 5 ′ region (X) of the internal region (Z) has the expression suppressing sequence, the 5 ′ region (Xc) preferably has the complementary sequence, When the internal 3 ′ side region (Y) of the region (Z) has the expression suppressing sequence, the 3 ′ side region (Yc) preferably has the complementary sequence.
  • the inner 5 ′ region (X) of the inner region (Z) when the 5 ′ region (Xc) has the expression suppressing sequence, the inner 5 ′ region (X) of the inner region (Z) preferably has the complementary sequence, and the 3 ′ region When (Yc) has the expression suppression sequence, the internal 3 ′ side region (Y) of the internal region (Z) preferably has the complementary sequence.
  • the 5′-side region (Xc) is complementary to the inner 5′-side region (X), and the 3′-side region (Yc) is the inner 3′-side region (Y).
  • the region (Xc) is folded toward the region (X), and the region (Xc) and the region (X) can form a double chain by self-annealing.
  • the region (Yc) is folded toward the region (Y), and the region (Yc) and the region (Y) can form a double chain by self-annealing.
  • the inner region (Z) is connected to the inner 5 'region (X) and the inner 3' region (Y).
  • the region (X) and the region (Y) are directly connected, for example, and do not have an intervening sequence therebetween.
  • the inner region (Z) is defined as “the inner 5 ′ side region (X) and the inner 3 ′ side in order to indicate the arrangement relationship between the 5 ′ side region (Xc) and the 3 ′ side region (Yc)”.
  • the region (Y) is connected to each other ”, and in the inner region (Z), the 5 ′ side region (Xc) and the 3 ′ side region (Yc) are, for example,
  • the use of nucleic acid molecules is not limited to being a separate and independent region. That is, for example, when the internal region (Z) has the expression suppression sequence, the expression suppression sequence is arranged across the region (X) and the region (Y) in the internal region (Z). Also good.
  • the 5 'side region (Xc) and the inner 5' side region (X) may be directly connected or indirectly connected, for example.
  • direct linkage includes, for example, linkage by a phosphodiester bond.
  • a linker region (Lx) is provided between the region (Xc) and the region (X), and the region (Xc) and the region ( And X) are linked together.
  • the 3'-side region (Yc) and the internal 3'-side region (Y) may be directly connected or indirectly connected, for example.
  • direct linkage includes, for example, linkage by a phosphodiester bond.
  • a linker region (Ly) is provided between the region (Yc) and the region (Y), and the region (Yc) and the region ( And Y) are linked.
  • the nucleic acid molecule may have, for example, both the linker region (Lx) and the linker region (Ly), or one of them.
  • the linker region (Lx) is provided between the 5 ′ side region (Xc) and the inner 5 ′ side region (X), and the 3 ′ side region (Yc) and the inner 3 'The linker region (Ly) is not present between the side region (Y), that is, the region (Yc) and the region (Y) are directly linked.
  • the linker region (Ly) is provided between the 3 ′ side region (Yc) and the inner 3 ′ side region (Y), and the 5 ′ side region (Xc) and the The linker region (Lx) is not provided between the internal 5′-side region (X), that is, the region (Xc) and the region (X) are directly linked.
  • the linker region (Lx) and the linker region (Ly) each preferably have a structure that does not cause self-annealing within its own region.
  • FIG. 2 (A) is a schematic diagram showing an outline of the order of each region from the 5 ′ side to the 3 ′ side of the nucleic acid molecule
  • FIG. 2 (B) shows that the nucleic acid molecule is the molecule. It is a schematic diagram which shows the state which forms the double chain
  • FIG. 2 merely shows the connection order of the regions and the positional relationship of the regions forming the double chain.
  • the length of each region is not limited to this.
  • the number of bases in the 5 ′ region (Xc), the internal 5 ′ region (X), the internal 3 ′ region (Y) and the 3 ′ region (Yc) is particularly limited. For example, it is as follows.
  • the 5′-side region (Xc) may be complementary to the entire region of the inner 5′-side region (X), for example.
  • the region (Xc) has the same base length as the region (X), and is composed of a base sequence complementary to the entire region from the 5 ′ end to the 3 ′ end of the region (X).
  • the region (Xc) has the same base length as the region (X), and all bases in the region (Xc) are complementary to all bases in the region (X). That is, for example, it is preferably completely complementary.
  • the present invention is not limited to this.
  • 1 to several (2, 3, 4 or 5) bases may be non-complementary.
  • the 5′-side region (Xc) may be complementary to a partial region of the inner 5′-side region (X), for example.
  • the region (Xc) has, for example, the same base length as the partial region of the region (X), that is, consists of a base sequence having a base length shorter by one base or more than the region (X). preferable. More preferably, the region (Xc) has the same base length as the partial region of the region (X), and all the bases of the region (Xc) are included in the partial region of the region (X). It is preferred that it is complementary to all bases, that is, for example, completely complementary.
  • the partial region of the region (X) is preferably, for example, a region (segment) having a base sequence continuous from the 5 ′ terminal base (first base) in the region (X).
  • the 3′-side region (Yc) may be complementary to the entire region of the inner 3′-side region (Y), for example.
  • the region (Yc) has, for example, the same base length as the region (Y) and is composed of a base sequence complementary to the entire region from the 5 ′ end to the 3 ′ end of the region (Y).
  • the region (Yc) has the same base length as the region (Y), and all bases in the region (Yc) are complementary to all bases in the region (Y). That is, for example, it is preferable to be completely complementary.
  • the present invention is not limited to this.
  • 1 to several (2, 3, 4 or 5) bases may be non-complementary.
  • the 3′-side region (Yc) may be complementary to a partial region of the inner 3′-side region (Y), for example.
  • the region (Yc) has, for example, the same base length as the partial region of the region (Y), that is, consists of a base sequence having a base length shorter by one base or more than the region (Y). preferable. More preferably, the region (Yc) has the same base length as the partial region of the region (Y), and all the bases of the region (Yc) are included in the partial region of the region (Y). It is preferred that it is complementary to all bases, that is, for example, completely complementary.
  • the partial region of the region (Y) is preferably, for example, a region (segment) having a base sequence continuous from the base at the 3 'end (first base) in the region (Y).
  • the number of bases (Z) in the internal region (Z), the number of bases (X) in the internal 5 ′ side region (X), and the number of bases (Y) in the internal 3 ′ side region (Y) Relationship between the number of bases (Z) in the internal region (Z), the number of bases (Xc) in the 5′-side region (Xc), and the number of bases (Yc) in the 3′-side region (Yc) Satisfies, for example, the conditions of the following formulas (1) and (2).
  • Z X + Y (1)
  • the relationship between the number of bases (X) in the inner 5 ′ region (X) and the number of bases (Y) in the inner 3 ′ region (Y) is not particularly limited, For example, any condition of the following formula may be satisfied.
  • X Y (19) X ⁇ Y (20) X> Y (21)
  • the number of bases (X) in the inner 5 ′ side region (X), the number of bases (Xc) in the 5 ′ side region (Xc), the number of bases in the inner 3 ′ side region (Y) (Y ) And the number of bases (Yc) in the 3′-side region (Yc) satisfy, for example, the following conditions (a) to (d).
  • Y Yc (4)
  • X Xc (5) Y> Yc (6) (C)
  • the conditions of the following formulas (7) and (8) are satisfied.
  • X> Xc (7) Y> Yc (8) (D)
  • the conditions of the following formulas (9) and (10) are satisfied.
  • X Xc (9)
  • Y Yc (10)
  • the difference between the number of bases (X) in the inner 5 ′ side region (X) and the number of bases (Xc) in the 5 ′ side region (Xc), the inner 3 ′ side region ( The difference between the number of bases (Y) of Y) and the number of bases (Yc) of the 3 ′ side region (Yc) preferably satisfies the following condition, for example.
  • A The conditions of the following formulas (11) and (12) are satisfied.
  • FIG. 4 has the number of bases (X) in the inner 5 ′ side region (X) and the number of bases (Y) in the inner 3 ′ side region (Y) as “X ⁇ Y” in the formula (20).
  • FIG. 4 is merely a relationship between the inner 5 ′ side region (X) and the 5 ′ side region (Xc), and the relationship between the inner 3 ′ side region (Y) and the 3 ′ side region (Yc).
  • the length and shape of each region are not limited thereto, and the presence or absence of the linker region (Lx) and the linker region (Ly) is not limited thereto.
  • the nucleic acid molecules (a) to (c) include, for example, the 5 ′ side region (Xc) and the internal 5 ′ side region (X), and the 3 ′ side region (Yc) and the internal 3 ′ side.
  • the region (Y) has a base that cannot be aligned with any of the 5 ′ side region (Xc) and the 3 ′ side region (Yc) in the internal region (Z) by forming a double chain, respectively. It can be said that the structure has a base that does not form a double chain.
  • the base that cannot be aligned also referred to as a base that does not form a double chain
  • free base In FIG.
  • the free base region is indicated by “F”.
  • the number of bases in the region (F) is not particularly limited.
  • the number of bases (F) in the region (F) is, for example, the number of bases “X-Xc” in the case of the nucleic acid molecule (a), and “Y—Yc” in the case of the nucleic acid molecule (b). In the case of the nucleic acid molecule (c), it is the total number of bases “X—Xc” and “Y—Yc”.
  • the nucleic acid molecule of (d) has a structure in which, for example, the entire region of the internal region (Z) is aligned with the 5 ′ side region (Xc) and the 3 ′ side region (Yc), It can also be said that the entire region (Z) forms a double chain.
  • the 5 'end of the 5' side region (Xc) and the 3 'end of the 3' side region (Yc) are unlinked.
  • each region is exemplified below for the nucleic acid molecule, but the present invention is not limited to this.
  • the total number of bases of the free base (F) in the 5 ′ side region (Xc), the 3 ′ side region (Yc), and the internal region (Z) is the number of bases in the internal region (Z). .
  • the lengths of the 5 ′ side region (Xc) and the 3 ′ side region (Yc) depend on, for example, the length of the internal region (Z), the number of free bases (F), and the position thereof. Can be determined as appropriate.
  • the number of bases in the internal region (Z) is, for example, 19 bases or more.
  • the lower limit of the number of bases is, for example, 19 bases, preferably 20 bases, and more preferably 21 bases.
  • the upper limit of the number of bases is, for example, 50 bases, preferably 40 bases, and more preferably 30 bases.
  • Specific examples of the number of bases in the internal region (Z) include, for example, 19 bases, 20 bases, 21 bases, 22 bases, 23 bases, 24 bases, 25 bases, 26 bases, 27 bases, 28 bases, 29 bases, or , 30 bases.
  • the internal region (Z) may be, for example, a region composed only of the expression suppression sequence or a region including the expression suppression sequence.
  • the number of bases of the expression suppression sequence is, for example, as described above.
  • the internal region (Z) contains the expression suppression sequence it may further have an additional sequence on the 5 'side and / or 3' side of the expression suppression sequence.
  • the number of bases of the additional sequence is, for example, 1 to 31 bases, preferably 1 to 21 bases, more preferably 1 to 11 bases, and further preferably 1 to 7 bases.
  • the number of bases in the 5 ′ side region (Xc) is, for example, 1 to 29 bases, preferably 1 to 11 bases, more preferably 1 to 7 bases, and further preferably 1 to 4 bases. Particularly preferred are 1 base, 2 bases and 3 bases.
  • the internal region (Z) or the 3 'side region (Yc) includes the expression suppression sequence, for example, such a base number is preferable.
  • the number of bases in the internal region (Z) is 19 to 30 bases (for example, 19 bases)
  • the number of bases in the 5 ′ side region (Xc) is, for example, 1 to 11 bases
  • the number is preferably 1 to 7 bases, more preferably 1 to 4 bases, and still more preferably 1 base, 2 bases, and 3 bases.
  • the 5′-side region (Xc) may be, for example, a region composed only of the expression suppression sequence, or a region including the expression suppression sequence But you can.
  • the length of the expression suppression sequence is, for example, as described above.
  • the 5 'region (Xc) contains the expression suppression sequence, it may further have an additional sequence on the 5' side and / or 3 'side of the expression suppression sequence.
  • the number of bases of the additional sequence is, for example, 1 to 11 bases, and preferably 1 to 7 bases.
  • the number of bases in the 3 ′ side region (Yc) is, for example, 1 to 29 bases, preferably 1 to 11 bases, more preferably 1 to 7 bases, and further preferably 1 to 4 bases. Particularly preferred are 1 base, 2 bases and 3 bases.
  • the internal region (Z) or the 5 'side region (Xc) includes the expression suppression sequence, for example, such a base number is preferable.
  • the number of bases in the internal region (Z) is 19 to 30 bases (for example, 19 bases)
  • the number of bases in the 3 ′ side region (Yc) is, for example, 1 to 11 bases
  • the number is preferably 1 to 7 bases, more preferably 1 to 4 bases, and still more preferably 1 base, 2 bases, and 3 bases.
  • the 3 ′ side region (Yc) may be, for example, a region composed only of the expression suppression sequence, or a region including the expression suppression sequence But you can.
  • the length of the expression suppression sequence is, for example, as described above.
  • the 3 'side region (Yc) includes the expression suppression sequence, it may further have an additional sequence on the 5' side and / or 3 'side of the expression suppression sequence.
  • the number of bases of the additional sequence is, for example, 1 to 11 bases, and preferably 1 to 7 bases.
  • the number of bases in the internal region (Z), the 5′-side region (Xc), and the 3′-side region (Yc) is expressed by, for example, “Z ⁇ Xc + Yc” in the formula (2). Can do.
  • the number of bases “Xc + Yc” is, for example, the same as or smaller than the inner region (Z).
  • “Z ⁇ (Xc + Yc)” is, for example, 1 to 10, preferably 1 to 4, more preferably 1, 2 or 3.
  • the “Z ⁇ (Xc + Yc)” corresponds to the number of bases (F) in the free base region (F) in the internal region (Z).
  • the linker region (Lx) preferably has, for example, a length that allows the internal 5 ′ side region (X) and the 5 ′ side region (Xc) to form a double chain, and the linker region (Ly) ) Is, for example, preferably a length such that the inner 3 ′ side region (Y) and the 3 ′ side region (Yc) can form a double chain.
  • the lengths of the linker region (Lx) and the linker region (Ly) may be the same or different, and the base sequences thereof may be the same or different.
  • the lower limit of the number of bases in the linker region (Lx) and the linker region (Ly) is, for example, 1 base, preferably 2 bases, more preferably 3 bases, and the upper limit thereof is, for example, 100 bases, preferably 80 bases, more preferably 50 bases.
  • Specific examples of the number of bases in each linker region include 1 to 50 bases, 1 to 30 bases, 1 to 20 bases, 1 to 10 bases, 1 to 7 bases, and 1 to 4 bases. This is not a limitation.
  • the total length of the nucleic acid molecule is not particularly limited.
  • the lower limit of the total number of bases is, for example, 38 bases, preferably 42 bases, more preferably 50 bases, and even more preferably 51 bases.
  • the upper limit is, for example, 52 bases, and the upper limit thereof is, for example, 300 bases, preferably 200 bases, more preferably 150 bases, still more preferably 100 bases, and particularly preferably 80 bases. .
  • the lower limit of the total number of bases excluding the linker region (Lx) and the linker region (Ly) is, for example, 38 bases, preferably 42 bases, more preferably 50 bases, More preferably, it is 51 bases, particularly preferably 52 bases, and the upper limit is, for example, 300 bases, preferably 200 bases, more preferably 150 bases, still more preferably 100 bases, Preferably, it is 80 bases.
  • the 5 'end and the 3' end may be bound or unbound.
  • the nucleic acid molecule of this form is a circular single-stranded nucleic acid molecule.
  • the nucleic acid molecule of the present embodiment is preferably a non-phosphate group at the 5 'end, for example, since it can maintain unbonded at both ends.
  • a third form of the single-stranded nucleic acid molecule is a molecule in which the linker region has a non-nucleotide structure.
  • This embodiment can use the above description except that the linker region (Lx) and / or the linker region (Ly) has a non-nucleotide structure in the nucleic acid molecules of the first and second forms.
  • the non-nucleotide structure is not particularly limited, and examples thereof include polyalkylene glycol, pyrrolidine skeleton and piperidine skeleton.
  • examples of the polyalkylene glycol include polyethylene glycol.
  • the pyrrolidine skeleton may be, for example, a skeleton of a pyrrolidine derivative in which one or more carbons constituting the 5-membered ring of pyrrolidine are substituted, and when substituted, for example, a carbon atom other than C-2 carbon. It is preferable.
  • the carbon may be substituted with, for example, nitrogen, oxygen or sulfur.
  • the pyrrolidine skeleton may contain, for example, a carbon-carbon double bond or a carbon-nitrogen double bond in the 5-membered ring of pyrrolidine.
  • the carbon and nitrogen constituting the 5-membered ring of pyrrolidine may be bonded, for example, to hydrogen or a substituent as described below.
  • the linker region (Lx) is the region (X) and the region (Xc), and the linker region (Ly) is the region (Y) and the region (Yc), for example, any of the pyrrolidine skeleton. It may be bonded via a group, preferably any one carbon atom of the five-membered ring and nitrogen, preferably two-position carbon (C-2) and nitrogen of the five-membered ring. is there.
  • the pyrrolidine skeleton include a proline skeleton and a prolinol skeleton.
  • the proline skeleton, prolinol skeleton, and the like are excellent in safety because they are, for example, in-vivo substances and their reduced forms.
  • the piperidine skeleton may be, for example, a skeleton of a piperidine derivative in which one or more carbons constituting the six-membered ring of piperidine are substituted, and when substituted, for example, a carbon atom other than C-2 carbon. It is preferable.
  • the carbon may be substituted with, for example, nitrogen, oxygen or sulfur.
  • the piperidine skeleton may contain, for example, a carbon-carbon double bond or a carbon-nitrogen double bond in the 6-membered ring of piperidine.
  • the carbon and nitrogen constituting the piperidine 6-membered ring may be bonded to, for example, a hydrogen group or a substituent as described later.
  • the linker region (Lx) includes the region (X) and the region (Xc), the linker region (Ly) includes the region (Y) and the region (Yc), and any one of the piperidine skeleton, for example. It may be bonded via a group, preferably any one carbon atom of the six-membered ring and nitrogen, more preferably carbon (C-2) at the 2-position of the six-membered ring and nitrogen. It is.
  • the linker region may include, for example, only a non-nucleotide residue having the non-nucleotide structure, or may include a non-nucleotide residue having the non-nucleotide structure and a nucleotide residue.
  • the linker region is represented by the following formula (I), for example.
  • X 1 and X 2 are each independently H 2 , O, S or NH; Y 1 and Y 2 are each independently a single bond, CH 2 , NH, O or S; R 3 is a hydrogen atom or substituent bonded to C-3, C-4, C-5 or C-6 on ring A; L 1 is an alkylene chain consisting of n atoms, wherein the hydrogen atom on the alkylene carbon atom is replaced with OH, OR a , NH 2 , NHR a , NR a R b , SH, or SR a May or may not be substituted, or L 1 is a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with an oxygen atom,
  • L 2 is an alkylene chain consisting of n atoms, wherein the hydrogen atom on the alkylene carbon atom is replaced with OH, OR a , NH 2 , NHR a , NR a R b , SH, or
  • the ring A may contain a carbon-carbon double bond or a carbon-nitrogen double bond
  • the region (Xc) and the region (X) are each bonded to the linker region (Lx) via —OR 1 — or —OR 2 —;
  • the region (Yc) and the region (Y) are each bonded to the linker region (Ly) via —OR 1 — or —OR 2 —,
  • R 1 and R 2 may be present or absent, and when present, R 1 and R 2 are each independently a nucleotide residue or the structure (I).
  • X 1 and X 2 are each independently, for example, H 2 , O, S or NH.
  • X 1 being H 2 means that X 1 together with the carbon atom to which X 1 is bonded forms CH 2 (methylene group). The same is true for X 2.
  • Y 1 and Y 2 are each independently a single bond, CH 2 , NH, O or S.
  • l 1 or 2.
  • ring A is a 5-membered ring, for example, the pyrrolidine skeleton.
  • the pyrrolidine skeleton include a proline skeleton and a prolinol skeleton, and examples thereof include a bivalent structure.
  • ring A is a 6-membered ring, for example, the piperidine skeleton.
  • one carbon atom other than C-2 on ring A may be substituted with nitrogen, oxygen or sulfur.
  • Ring A may contain a carbon-carbon double bond or a carbon-nitrogen double bond in ring A.
  • Ring A may be, for example, either L-type or D-type.
  • R 3 is a hydrogen atom or a substituent bonded to C-3, C-4, C-5 or C-6 on the ring A.
  • R 3 is the above-described substituent, the substituent R 3 may be one, plural, or absent, and when plural, it may be the same or different.
  • the substituent R 3 is, for example, halogen, OH, OR 4 , NH 2 , NHR 4 , NR 4 R 5 , SH, SR 4 or an oxo group ( ⁇ O).
  • R 4 and R 5 are, for example, each independently a substituent or a protecting group, and may be the same or different.
  • substituents include halogen, alkyl, alkenyl, alkynyl, haloalkyl, aryl, heteroaryl, arylalkyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, cyclylalkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, heterocyclylalkenyl. , Heterocyclylalkyl, heteroarylalkyl, silyl, silyloxyalkyl and the like. The same applies hereinafter.
  • the substituent R 3 may be any of these listed substituents.
  • the protecting group is, for example, a functional group that converts a highly reactive functional group to inert, and examples thereof include known protecting groups.
  • the description of the literature J. F. W. McOmie, “Protecting Groups in Organic Chemistry” Prenum Press, London and New York, 1973) can be used for the protecting group.
  • the protective group is not particularly limited, and examples thereof include tert-butyldimethylsilyl group (TBDMS), bis (2-acetoxyethyloxy) methyl group (ACE), triisopropylsilyloxymethyl group (TOM), 1- (2 -Cyanoethoxy) ethyl group (CEE), 2-cyanoethoxymethyl group (CEM), tolylsulfonylethoxymethyl group (TEM), dimethoxytrityl group (DMTr) and the like.
  • R 3 is OR 4
  • the protecting group is not particularly limited, and examples thereof include a TBDMS group, an ACE group, a TOM group, a CEE group, a CEM group, and a TEM group.
  • the silyl-containing group of [Chemical Formula 5] described later is also included. The same applies hereinafter.
  • L 1 is an alkylene chain composed of n atoms.
  • the hydrogen atom on the alkylene carbon atom may be substituted with, for example, OH, OR a , NH 2 , NHR a , NR a R b , SH, or SR a , or may not be substituted.
  • L 1 may be a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with an oxygen atom.
  • the polyether chain is, for example, polyethylene glycol.
  • L 2 is an alkylene chain composed of m atoms.
  • the hydrogen atom on the alkylene carbon atom may be substituted with, for example, OH, OR c , NH 2 , NHR c , NR c R d , SH or SR c , or may not be substituted.
  • L 2 may be a polyether chain in which one or more carbon atoms of the alkylene chain are substituted with an oxygen atom.
  • Y 2 is NH, O or S
  • the L 2 atom bonded to Y 2 is carbon
  • the L 2 atom bonded to OR 2 is carbon
  • oxygen atoms are not adjacent to each other. That is, for example, when Y 2 is O, the oxygen atom and the oxygen atom of L 2 are not adjacent, and the oxygen atom of OR 2 and the oxygen atom of L 2 are not adjacent.
  • N in L 1 and m in L 2 are not particularly limited, and the lower limit is, for example, 0, and the upper limit is not particularly limited.
  • n and m can be appropriately set according to the desired length of the linker region (Lx) or (Ly), for example.
  • n and m are each preferably 0 to 30, more preferably 0 to 20, and still more preferably 0 to 15 from the viewpoint of production cost and yield.
  • n + m is, for example, 0 to 30, preferably 0 to 20, and more preferably 0 to 15.
  • R a , R b , R c and R d are, for example, each independently a substituent or a protecting group.
  • the substituent and the protecting group are the same as described above, for example.
  • hydrogen atoms may be independently substituted with halogens such as Cl, Br, F and I, for example.
  • the region (Xc) and the region (X) are in the linker region (Lx), the region (Yc) and the region (Y) are in the linker region (Ly), for example, —OR 1 — Alternatively, the bonds are made through —OR 2 —.
  • R 1 and R 2 may or may not exist.
  • R 1 and R 2 are each independently a nucleotide residue or the structure of formula (I) above.
  • the linker region (Lx) and the linker region (Ly) are, for example, of the formula (I) except for the nucleotide residue R 1 and / or R 2 It is formed from the non-nucleotide residue consisting of a structure and the nucleotide residue.
  • the linker region (Lx) and the linker region (Ly) are, for example, the non-nucleotide residue having the structure of the formula (I) Two or more structures are connected.
  • the structure of the formula (I) may include 1, 2, 3, or 4, for example.
  • the structure of (I) may be directly linked or may be bonded via the nucleotide residue, for example.
  • R 1 and R 2 are not present, the linker region (Lx) and the linker region (Ly) are formed only from the non-nucleotide residue having the structure of the formula (I), for example.
  • the combination of the region (Xc) and the region (X), the region (Yc) and the region (Y), and the —OR 1 — and —OR 2 — is not particularly limited.
  • One of the following conditions can be given.
  • Condition (1) The region (Xc) is bonded to the structure of the formula (I) through —OR 2 —, and the region (X) is bonded through —OR 1 —.
  • the region (Yc) is bonded to the structure of the formula (I) through —OR 1 —, and the region (Y) is bonded through —OR 2 —.
  • Condition (2) The region (Xc) is bonded to the structure of the formula (I) through —OR 2 —, and the region (X) is bonded through —OR 1 —.
  • the region (Yc) is bonded to the structure of the formula (I) through —OR 2 —, and the region (Y) is bonded through —OR 1 —.
  • Condition (3) The region (Xc) is bonded to the structure of the formula (I) through —OR 1 —, and the region (X) is bonded through —OR 2 —.
  • the region (Yc) is bonded to the structure of the formula (I) through —OR 1 —, and the region (Y) is bonded through —OR 2 —.
  • Condition (4) The region (Xc) is bonded to the structure of the formula (I) through —OR 1 —, and the region (X) is bonded through —OR 2 —.
  • the region (Yc) is bonded to the structure of the formula (I) through —OR 2 —, and the region (Y) is bonded through —OR 1 —.
  • Examples of the structure of the formula (I) include the following formulas (I-1) to (I-9), in which n and m are the same as those in the formula (I).
  • q is an integer of 0 to 10.
  • n, m and q are not particularly limited and are as described above.
  • 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 can improve nuclease resistance and stability, for example, by including the modified nucleotide residue.
  • the nucleic acid molecule of the present invention may further contain a non-nucleotide residue in addition to the nucleotide residue, for example.
  • each of the constituent units in the region other than the linker is preferably the nucleotide residue.
  • Each region is composed of the following residues (1) to (3), for example. (1) Unmodified nucleotide residue (2) Modified nucleotide residue (3) Unmodified nucleotide residue and modified nucleotide residue
  • the structural unit of the linker region is not particularly limited, and examples thereof include the nucleotide residue and the non-nucleotide residue.
  • the linker region may be composed of, for example, only the nucleotide residue, may be composed of only the non-nucleotide residue, or may be composed of the nucleotide residue and the non-nucleotide residue.
  • the linker region is composed of the following residues (1) to (7), for example.
  • both structural units may be the same or different.
  • Specific examples include, for example, a form in which the constituent units of both linker regions are the nucleotide residues, a form in which the constituent units of both linker regions are the non-nucleotide residues, and the constituent units of one region are the nucleotide residues.
  • the other linker region is a non-nucleotide residue.
  • nucleic acid molecule of the present invention examples include a molecule composed only of the nucleotide residue, a molecule containing the non-nucleotide residue in addition to the nucleotide residue, and the like.
  • the nucleotide residue may be, for example, only the unmodified nucleotide residue, only the modified nucleotide residue, or the unmodified nucleotide residue and the modification. Both nucleotide residues may be used.
  • the number of the modified nucleotide residue is not particularly limited, and is, for example, “one or several”, specifically For example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and most preferably 1 or 2.
  • the number of the non-nucleotide residue is not particularly limited, and is, for example, “one or several”, specifically, for example, 1 to Eight, one to six, one to four, one, two or three.
  • the number of the modified ribonucleotide residue is not particularly limited, and for example, “1 or several” Specifically, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and most preferably 1 or 2.
  • the modified ribonucleotide residue relative to the unmodified ribonucleotide residue may be, for example, the deoxyribonucleotide residue in which a ribose residue is replaced with a deoxyribose residue.
  • the number of the deoxyribonucleotide residue is not particularly limited, and is, for example, “one or several” Specifically, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and most preferably 1 or 2.
  • 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, Cy3 dye, and Cy5 dye, and examples of the dye include Alexa dye such as Alexa488.
  • 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 can suppress the expression of the TGF- ⁇ 1 gene. Therefore, the nucleic acid molecule of the present invention can be used, for example, as a therapeutic agent for diseases caused by the TGF- ⁇ 1 gene.
  • “treatment” includes, for example, the meanings of preventing the disease, improving the disease, and improving the prognosis. Specific examples of the disease include acute lung injury; pulmonary fibrosis.
  • the method of using the nucleic acid molecule of the present invention is not particularly limited, and for example, the nucleic acid molecule may be administered to an administration subject having the TGF- ⁇ 1 gene.
  • Examples of the administration target include cells, tissues or organs.
  • Examples of the administration subject include non-human animals such as humans and non-human mammals other than humans.
  • the administration may be, for example, in vivo or in vitro.
  • the cells are not particularly limited, and examples thereof include various cultured cells such as A549, HeLa, 293, and COS7, pluripotent stem cells such as ES cells and iPS cells, somatic stem cells such as hematopoietic stem cells, the pluripotent stem cells or Examples thereof include various cultured cells derived from somatic stem cells, cells isolated from living bodies such as primary cultured cells, and the like.
  • nucleic acid molecule of the present invention refers to the description of the composition of the present invention, expression suppression method, treatment method and the like described later.
  • nucleic acid molecule of the present invention can suppress the expression of the TGF- ⁇ 1 gene as described above, it is useful, for example, as a research tool for pharmaceuticals, diagnostic agents, agricultural chemicals, agricultural chemicals, medicine, life sciences and the like. is there.
  • nucleotide residues include, for example, sugars, bases and phosphates 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 U (uracil) as bases
  • the deoxyribose 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, and 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. For example, Limbac et al.
  • modified nucleosides of RNA Nucleic Acids Res. 22: 2183-2196
  • the modified nucleotide residue may be, for example, a residue of the nucleotide substitute.
  • ribophosphate skeleton examples include modification of a ribose-phosphate skeleton (hereinafter referred to as ribophosphate skeleton).
  • a ribose residue can be modified.
  • 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 can be replaced with deoxyribose.
  • the ribose residue can be substituted with, for example, a stereoisomer, and can be substituted with, for example, an arabinose residue.
  • the ribophosphate skeleton may be substituted with a non-ribophosphate skeleton having a non-ribose residue and / or non-phosphate, for example.
  • the non-ribophosphate skeleton include uncharged ribophosphate skeletons.
  • the substitute for the nucleotide substituted with the non-ribophosphate skeleton include morpholino, cyclobutyl, pyrrolidine and the like.
  • Other examples of the substitute include artificial nucleic acid monomer residues. Specific examples include PNA (peptide nucleic acid), LNA (Locked Nucleic Acid), ENA (2'-O, 4'-C-Ethylenebridged Nucleic Acid), and PNA is preferred.
  • 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, phosphorodithioate in which the two non-bonded oxygens are both substituted with S is 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 above.
  • 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 to be added to the terminal includes, for example, a dye, an intercalating agent (for example, acridine), a crosslinking agent (for example, psoralen, mitomycin C), a porphyrin (TPPC4, texaphyrin, suffirin), a 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- (oleoy
  • 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, thymine, xanthine, hypoxanthine, nubalarine, isoguanisine, and tubercidine.
  • 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 ribophosphate skeleton.
  • the modified nucleotide residues are, for example, US provisional application 60 / 465,665 (filing date: April 25, 2003), and international application 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.
  • Method for synthesizing nucleic acid molecule of the present invention is not particularly limited, and conventionally known methods can be adopted.
  • Examples of the synthesis method include a synthesis method using a genetic engineering technique, a chemical synthesis method, and the like.
  • Examples of genetic engineering techniques include in vitro transcription synthesis, a method using a vector, and a method using a PCR cassette.
  • the vector is not particularly limited, and examples thereof include non-viral vectors such as plasmids and viral vectors.
  • the chemical synthesis method is not particularly limited, and examples thereof include a phosphoramidite method and an H-phosphonate method.
  • a commercially available automatic nucleic acid synthesizer can be used.
  • amidite is generally used.
  • the amidite is not particularly limited, and commercially available amidites include, for example, RNA Phosphoramidates (2′-O-TBDMSi, trade name, Michisato Pharmaceutical), ACE amidite, TOM amidite, CEE amidite, CEM amidite, TEM amidite, etc. Can be given.
  • the expression vector of the present invention is characterized by comprising DNA encoding the nucleic acid molecule of the present invention.
  • the expression vector of the present invention is characterized by containing the DNA, and other configurations are not limited at all.
  • the DNA is inserted so that the vector can be expressed.
  • the vector into which the DNA is inserted is not particularly limited, and for example, a general vector can be used, and examples thereof include viral vectors and non-viral vectors. Examples of the non-viral vector include a plasmid vector.
  • composition for suppressing expression of the present invention is a composition for suppressing the expression of the TGF- ⁇ 1 gene, and includes the nucleic acid molecule of the present invention.
  • the composition of the present invention is characterized by including the nucleic acid molecule of the present invention, and other configurations are not limited at all.
  • the expression suppressing composition of the present invention can also be referred to as an expression suppressing reagent, for example.
  • the expression of the TGF- ⁇ 1 gene can be suppressed by administration to a subject in which the TGF- ⁇ 1 gene is present.
  • the pharmaceutical composition of the present invention is characterized by containing the nucleic acid molecule of the present invention.
  • the composition of the present invention is characterized by containing the nucleic acid molecule of the present invention, and other configurations are not limited at all.
  • the pharmaceutical composition of the present invention can also be referred to as a pharmaceutical product, for example.
  • administration to a patient with a disease caused by the TGF- ⁇ 1 gene can suppress the expression of the gene and treat the disease.
  • diseases are as described above, and include acute lung injury, interstitial pneumonia / pulmonary fibrosis and the like.
  • treatment includes, for example, the meanings of prevention of the above-mentioned diseases, improvement of the diseases, and improvement of the prognosis.
  • composition for suppressing expression and the pharmaceutical composition (hereinafter referred to as composition) of the present invention
  • the nucleic acid molecule is administered to an administration subject having the TGF- ⁇ 1 gene. do it.
  • Examples of the administration target include cells, tissues or organs.
  • Examples of the administration subject include non-human animals such as humans and non-human mammals other than humans.
  • the administration may be, for example, in vivo or in vitro.
  • the cells are not particularly limited, and examples thereof include the cells described above.
  • the administration method is not particularly limited, and can be appropriately determined according to the administration subject, for example.
  • the administration subject is a cultured cell
  • examples thereof include a method using a transfection reagent and an electroporation method.
  • composition of the present invention may contain, for example, only the nucleic acid molecule of the present invention or may contain other additives.
  • 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 nucleic acid molecule may form a complex with the additive, for example.
  • the additive can also be referred to as a complexing agent, for example.
  • the complex formation for example, the nucleic acid molecule can be efficiently delivered.
  • the binding between the 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.
  • Examples of the additive include a carrier, a binding substance to a target cell, a condensing agent, a fusing agent, an excipient, and the like.
  • the expression suppression method of the present invention is a method of suppressing the expression of the TGF- ⁇ 1 gene, characterized by using the nucleic acid molecule of the present invention.
  • the expression suppression method of the present invention is characterized by using the nucleic acid molecule of the present invention, and other steps and conditions are not limited at all.
  • the expression suppression method of the present invention includes, for example, a step of administering the nucleic acid molecule to a subject in which the TGF- ⁇ 1 gene is present.
  • the administration step for example, the nucleic acid molecule is brought into contact with the administration subject.
  • the administration subject include cells, tissues, and organs.
  • the administration subject include non-human animals such as humans and non-human mammals other than humans.
  • the administration may be, for example, in vivo or in vitro.
  • the nucleic acid molecule may be administered alone, or the composition of the present invention containing the 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.
  • the therapeutic method of the disease of this invention is characterized by including the process of administering the nucleic acid molecule of the said this invention to a patient as mentioned above.
  • the therapeutic method of the present invention is characterized by using the nucleic acid molecule of the present invention, and other steps and conditions are not limited at all.
  • the diseases targeted by the present invention are, for example, as described above, and include acute lung injury, interstitial pneumonia / pulmonary fibrosis and the like.
  • the expression suppression method of the present invention can be used.
  • the administration method is not particularly limited, and may be, for example, oral administration or parenteral administration.
  • the dosage of the nucleic acid molecule of the present invention in the treatment method of the present invention is not particularly limited as long as it is a therapeutically effective amount for the above-mentioned disease, and the type, severity, species of animal to be administered, age, weight, drug Usually, it is about 0.0001 to about 100 mg / kg per adult, for example about 0.001 to about 10 mg / kg, preferably about 0.005 to about 5 mg / kg, although it varies depending on the acceptability, administration route, etc. obtain.
  • the amount can be administered, for example, at intervals of 3 times a day to once every 2 weeks, preferably once a day to once a week.
  • nucleic acid molecule The use of the present invention is the use of the nucleic acid molecule of the present invention for suppressing the expression of the TGF- ⁇ 1 gene.
  • the present invention also provides a nucleic acid molecule of the present invention for use in suppressing expression of the TGF- ⁇ 1 gene or treating pulmonary fibrosis or acute lung injury.
  • the present invention also provides use of the nucleic acid molecule of the present invention for the manufacture of an inhibitor of TGF- ⁇ 1 gene expression or a therapeutic agent for pulmonary fibrosis or acute lung injury.
  • TGF- ⁇ 1 gene expression inhibitory effect of single-stranded nucleic acid molecules in A549 cells (1) Synthesis of single-stranded nucleic acid molecules The following single-stranded nucleic acid molecules were synthesized based on the phosphoramidite method. The product was synthesized by a synthesizer (trade name: ABI Expedite (registered trademark) 8909 Nucleic Acid Synthesis System, Applied Biosystems). For the synthesis, RNA Phosphoramidites (2′-O-TBDMSi, trade name, Michisato Pharmaceutical) was used as an RNA amidite (hereinafter the same). The deprotection of the amidite followed a conventional method. The synthesized RNA was purified by HPLC. Each purified RNA was lyophilized.
  • PH-0009 is used as a single-stranded nucleic acid molecule in Examples, and single-stranded nucleic acid molecules nkRNA (hereinafter also referred to as NK-0133) and PnkRNA (hereinafter referred to as PKRNA) used by Hamasaki et al. (Also referred to as -0051) (PLoS ONE, 7 (8), e42655, doi: 10.1371, 2012, Table S5) were synthesized as described above.
  • Lx and Ly are linker regions Lx and Ly, respectively, and each has the following structural formula using L-proline diamide amidite.
  • the underlined portion is a human TGF- ⁇ 1 gene expression suppression sequence.
  • PH-0009 SEQ ID NO: 3
  • nkRNA SEQ ID NO: 4
  • PnkRNA SEQ ID NO: 5'-AGCAGAGUACACACAGCAUAUACC-Lx-GGUA UAUGCUGUGUGUACUCUGC UUC-Ly-G-3 '
  • RNA was dissolved in distilled water for injection (Otsuka Pharmaceutical Co., Ltd.) so as to be 20 ⁇ mol / L to prepare an RNA solution.
  • A549 cells (DS Pharma Biomedical) were used.
  • DMEM Invitrogen
  • the culture conditions were 37 ° C. and 5% CO 2 .
  • the cells were cultured in the medium, and the culture solution was dispensed into a 24-well plate in 400 ⁇ L portions at 5 ⁇ 10 4 cells / well. Furthermore, after culturing the cells in the well for 24 hours, the RNA was transfected using the transfection reagent Lipofectamine 2000 (Invitrogen) according to the protocol attached to the transfection reagent. Specifically, the composition per well was set as follows, and transfection was performed. In the following composition, (B) is Opti-MEM (Invitrogen), (C) is 20 ⁇ mol / L of the RNA solution, and 98.5 ⁇ L of both were added. In the well, the final concentration of RNA was 1 nmol / L.
  • PCR was performed using the synthesized cDNA as a template, and the expression level of the TGF- ⁇ 1 gene and the expression level of the ⁇ -actin gene as an internal standard were measured.
  • PCR primer set for TGF- ⁇ 1 gene (SEQ ID NO: 6) 5'-TTGTGCGGCAGTGGTTGAGCCG-3 ' (SEQ ID NO: 7) 5'-GAAGCAGGAAAGGCCGGTTCATGC-3 '
  • Primer set for ⁇ -actin gene (SEQ ID NO: 8) 5'-GCCACGGCTGCTTCCAGCTCCTC-3 ' (SEQ ID NO: 9) 5'-AGGTCTTTGCGGATGTCCACGTCAC-3 '
  • the gene expression level was also measured for cells in which only 100 ⁇ L of the solution (B) was added to the culture solution ( ⁇ ).
  • the RNA solution was not added, and the cells treated in the same manner except that (A) 1.5 ⁇ L and (B) were added in total 100 ⁇ L were also used for gene expression. The amount was measured (mock).
  • the expression level in the cells of the control ( ⁇ ) was taken as 1, and the relative value of the expression level in the cells into which each RNA was introduced was determined.
  • FIG. 5 is a graph showing the relative value of the TGF- ⁇ 1 gene expression level, and the vertical axis represents the relative gene expression level.
  • all single-stranded nucleic acid molecules showed strong gene expression suppression activity.
  • PH-0009 of the present invention exhibits a stronger gene expression suppressing activity than the other two single-stranded nucleic acid molecules.
  • Example 2 Stability of single-stranded nucleic acid molecule in human serum PH-0009 of Example 1 is used as the nucleic acid molecule of Example, and NK- The stability in human serum was examined using 0133 and PK-0051.
  • FIG. 6 (A) shows the result of electrophoresis showing the stability of PH-0009 against serum
  • FIG. 6 (B) shows the result of electrophoresis showing the stability of NK-0133 against serum.
  • lane “M” is a molecular weight marker
  • (min) indicates the incubation time.
  • NK-0133 and PK-0051 of the comparative example already started a rapid degradation reaction after 0.5 minutes of incubation, and as a result, all of 0.5 to 240 minutes In the result, it was confirmed that the size of the nucleic acid molecule was smaller than the result of 0 minute.
  • the change in the mobility with the passage of the incubation time that is, the decrease in the molecular weight due to the degradation was hardly confirmed. From this result, it was shown that PH-0009, which is a nucleic acid molecule of the present invention, has high stability in human serum.
  • Example 3 Reactivity of Single-Stranded Nucleic Acid Molecules to Dicer Protein PH-0009 of Example 1 is used as the nucleic acid molecule of Example, and NK-0133 shown in Example 1 is used as the nucleic acid molecule of Comparative Example. Using PK-0051, reactivity to Dicer protein was examined.
  • a reaction solution containing the Dicer protein and the nucleic acid molecule was prepared according to the attached protocol, and incubated at 37 ° C. Incubation times were 0, 0.5, 1, 2, 6, and 24 hours.
  • the reaction stop solution of the reagent was added to the reaction solution after incubation for a predetermined time, and 7M urea-20% polyacrylamide gel electrophoresis was performed. Thereafter, the polyacrylamide gel was stained with SYBR Green II (trade name, Lonza) and analyzed using ChemiDoc (trade name, Bio-Rad).
  • FIG. 7 shows the results of electrophoresis showing reactivity to Dicer protein
  • lane “M” shows molecular weight markers (20, 30, 40, 50 and 100 base).
  • Example 4 Inhibition of pulmonary fibrosis in spontaneously occurring pulmonary fibrosis model mice Using pulmonary fibrosis spontaneously occurring model mice (human TGF- ⁇ 1 transgenic mice described in Non-Patent Documents 6 and 7, hereinafter referred to as TG mice)
  • TG mice human TGF- ⁇ 1 transgenic mice described in Non-Patent Documents 6 and 7, hereinafter referred to as TG mice
  • nucleic acid molecule solution PH-0009 synthesized in Example 1 was used as the nucleic acid molecule of the example.
  • a nucleic acid molecule solution was prepared by dissolving PH-0009 in sterile physiological saline so as to be 2 ⁇ g / 75 ⁇ L.
  • nucleic acid molecule (2) Administration of nucleic acid molecule to spontaneously developing pulmonary fibrosis model mouse Using the above-mentioned nucleic acid molecule solution, MycroSprayer® (MSA-250-M: manufactured by PENNCENTURY), once / week, 4 times in total Administered intratracheally. As a negative control for the nucleic acid molecule solution, 75 ⁇ L of sterile physiological saline was used.
  • Each administration group is shown below. In each treatment group, 5 male mice were used. ⁇ Administration group 1 Wild type mice were administered 75 ⁇ L of sterile physiological saline and administered group 2 TG mice were administered 75 ⁇ L of sterile saline and administered group 3 Administration of 75 ⁇ L of 2 ⁇ g / 75 ⁇ L nucleic acid molecule solution (0.1 mg / kg mouse body weight) to TG mice
  • mice were anesthetized by intraperitoneal administration of pentobarbital (1.62 mg / 200 ⁇ L / head), and heparin-containing was obtained from the right ventricle of the mice after blood collection. After slow reflux with physiological saline, the right lung was extracted.
  • FIG. 8 is a graph showing the amount of TGF- ⁇ 1 in administration groups 2 and 3, and the vertical axis shows the amount of TGF- ⁇ 1 in lung tissue.
  • FIG. 9 is a graph showing the amount of hydroxyproline in each administration group, and the vertical axis shows the amount of hydroxyproline in the lung tissue.
  • TG mouse / nucleic acid molecule solution 0.1 mg / kg
  • administration group 3 the amount of TGF- ⁇ 1 and hydroxyproline in lung tissue was significantly higher than that in TG mouse / sterile saline administration group 2. Suppressed. From this, it was shown that PH-0009, which is a nucleic acid molecule of the present invention, suppresses the expression of target TGF- ⁇ 1 and suppresses lung fibrosis even in vivo.
  • Non-patent Document 7 Lung fibrosis inhibitory effect in bleomycin-induced pulmonary fibrosis model mouse
  • BALF bronchoalveolar lavage fluid
  • PH-0009 or PH-0000 was dissolved in sterile physiological saline so as to be 100 ⁇ g / 50 ⁇ L to prepare a nucleic acid molecule solution.
  • nucleic acid molecule solution was intratracheally administered to mice.
  • a wild-type mouse administered with 50 ⁇ L of sterile physiological saline was used as a normal control.
  • Each administration group is shown below.
  • Bleomycin-administered TG mice were administered 100 ⁇ g / 50 ⁇ L PH-0000 solution 50 ⁇ L and administered group 3 Bleomycin-administered TG mice were administered 100 ⁇ g / 50 ⁇ L PH-0009 solution 50 ⁇ L. Survival of mice in each administration group was monitored for 21 days from the start of bleomycin administration.
  • the total number of cells in the collected BALF was counted with a nucleocounter (ChemoMetec, Allerod, Denmark) on the day of bronchoalveolar lavage, and a cell specimen was prepared by cytospin (Labsystems Japan), and Giemsa staining (Merck Japan) was performed. The cell fraction was calculated. The amount of hydroxyproline in the lung tissue was measured in the same manner as in Example 4.
  • FIG. 10 is a graph showing the degree of inflammation associated with pulmonary fibrosis in each administration group, and the horizontal axis shows the total number of cells in BALF.
  • FIG. 11 is a graph showing a survival curve in each administration group, and the vertical axis shows the cumulative survival rate.
  • FIG. 12 is a graph showing the degree of fibrosis of lung tissue, and the horizontal axis shows the amount of hydroxyproline in lung tissue.
  • the survival rate of the mice was remarkably improved as compared with the TG mouse / PH-0000 administration group 2, and the BALF cell count and hydroxyproline in lung tissue The amount was significantly suppressed.
  • PH-0009 which is a nucleic acid molecule of the present invention, suppresses inflammation and lung fibrosis associated with pulmonary fibrosis and exhibits a therapeutic effect on pulmonary fibrosis in vivo.
  • Example 6 Inflammation inhibitory effect and safety in LPS-induced pulmonary fibrosis / acute lung injury combined model mice
  • Treatment with the nucleic acid molecule of the present invention using mice that have both pulmonary fibrosis and acute lung injury caused by LPS administration The effects and side effects were investigated.
  • the method described in Non-Patent Document 7 is used to confirm the therapeutic effect using the total number of cells in BALF as an index, and to confirm the presence or absence of side effects using the expression levels of interferon (IFN) - ⁇ and - ⁇ in BALF as an index. Went according to.
  • IFN interferon
  • Example 1 (1) Preparation of Nucleic Acid Molecule Solution PH-0009 synthesized in Example 1 was used as the nucleic acid molecule of Example, and PH-0000 was used as a negative control. Further, PK-0051 synthesized in Example 1 was used as a reference example, and PK-0000 having the following sequence was used as a negative control.
  • Lx and Ly represent a linker represented by the following structural formula.
  • Each nucleic acid molecule was dissolved in distilled water so as to be 50 ⁇ g / 50 ⁇ L to prepare a nucleic acid molecule solution.
  • Each administration group is shown below.
  • FIG. 13 is a graph showing the degree of inflammation associated with pulmonary fibrosis in each administration group, and the horizontal axis shows the total number of cells in BALF.
  • FIG. 14 is a graph showing the amount of IFN in BALF (upper: IFN- ⁇ , lower: IFN- ⁇ ) in each administration group, and the vertical axis shows the IFN concentration in BALF.
  • group 6 administered with TG mouse / PH-0009 solution, the number of BALF cells was significantly suppressed as compared with group 5 administered with TG mouse / PH-0000. Even when compared with PK-0051, the number of BALF cells tended to decrease, although there was no significant difference.
  • the amount of IFN in BALF was not significantly different from the control group in any of the nucleic acid molecule administration groups. From this, it was confirmed that PH-0009, which is a nucleic acid molecule of the present invention, suppresses inflammation and exhibits a therapeutic effect in vivo even in patients with pulmonary fibrosis and acute lung injury. Further, since no side effects were observed due to the administration of the nucleic acid molecule, it was suggested that PH-0009 can be safely administered to animals including humans in a therapeutically effective amount.
  • the single-stranded nucleic acid molecule of the present invention can suppress the expression of the TGF- ⁇ 1 gene. Therefore, the present invention is effective for treating diseases caused by the expression of the TGF- ⁇ 1 gene, such as pulmonary fibrosis and acute lung injury.

Abstract

　La présente invention porte sur une molécule d'acide nucléique simple brin pour l'inhibition de l'expression du gène TGF-β1, ladite molécule étant caractérisée en ce qu'elle comprend seulement une région (X), une région lieur (Lx) et une région (Xc), la région lieur (Lx) ayant une structure non nucléotidique contenant un squelette pyrrolidine et/ou un squelette qui pipéridine et la région (X) et/ou la région (Xc) comprenant une séquence d'inhibition d'expression qui inhibe l'expression du gène TGF-β1. La présente invention porte en outre sur une composition pharmaceutique thérapeutique pour la fibrose pulmonaire ou une lésion pulmonaire aiguë, qui contient la molécule d'acide nucléique simple brin.
PCT/JP2014/083312 2013-12-16 2014-12-16 Molécule d'acide nucléique simple brin pour la régulation de l'expression du gène tgf-β1 WO2015093495A1 (fr)

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