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WO2020204151A1 - Aptamère dirigé contre fgf9 et son utilisation - Google Patents

Aptamère dirigé contre fgf9 et son utilisation Download PDF

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WO2020204151A1
WO2020204151A1 PCT/JP2020/015263 JP2020015263W WO2020204151A1 WO 2020204151 A1 WO2020204151 A1 WO 2020204151A1 JP 2020015263 W JP2020015263 W JP 2020015263W WO 2020204151 A1 WO2020204151 A1 WO 2020204151A1
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aptamer
seq
nucleotide
nucleotide sequence
fgf9
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健朗 安達
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Ribomic Inc
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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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • 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
    • 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
    • A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • 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
    • 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
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers

Definitions

  • Fibroblast Growth Factor 9 is one of the FGF family proteins and is a protein involved in various biological processes such as embryogenesis, development, angiogenesis, and tumorigenesis (Fibroblast Growth Factor 9; FGF9).
  • FGF9 Fibroblast Growth Factor 9
  • Non-Patent Document 1 it has been reported that it is expressed in various cancers like other FGF family proteins, and it has also been reported that various organ-specific cancer models can be created by expressing FGF9 in a cell-specific manner.
  • Non-Patent Document 2 suggests that an anti-FGF9 antibody is effective for prostate cancer-induced osteoblastic bone metastasis.
  • Aptamers are nucleic acids that specifically bind to target molecules (proteins, sugar chains, hormones, etc.) and can bind to target molecules via the three-dimensional structure formed by single-stranded RNA (or DNA). it can.
  • a screening method called the SELEX method Systematic Evolution of Ligands by Exponential Enrichment
  • the chain length of the aptamer obtained by the SELEX method is about 80 nucleotides, and then the chain length is shortened using the physiological inhibitory activity of the target molecule as an index.
  • chemical modification is added for the purpose of improving stability in the living body, and optimization as a pharmaceutical product is achieved.
  • Aptamers are less susceptible to immune exclusion, and side effects such as antibody-specific antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cellular cytotoxicity (CDC) are less likely to occur.
  • ADCC antibody-specific antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cellular cytotoxicity
  • low-molecular-weight compounds which are the same molecular-targeted drugs, also have poorly soluble molecules, and optimization may be required for their formulation, but aptamers are highly water-soluble, which is also advantageous.
  • cost reduction can be achieved by mass production.
  • long-term storage stability, heat, and solvent resistance are also predominant features of aptamers.
  • aptamers generally have a shorter half-life in blood than antibodies. However, this point may also be advantageous from the viewpoint of toxicity.
  • RNA aptamer drugs have been developed, including Macugen (target disease: age-related macular degeneration), the first RNA aptamer drug approved in the United States in December 2004.
  • Macugen target disease: age-related macular degeneration
  • DNA aptamers that can be stably and inexpensively produced in vivo have been developed.
  • aptamers for purification and molecular targeting of target molecules by utilizing their high affinity for target molecules.
  • Aptamers often have a higher affinity than antibodies with similar functions. From the viewpoint of delivery, since the aptamer has a molecular size of about 1/10 of that of the antibody, tissue migration is likely to occur, and it is easier to deliver the drug to the target site.
  • Antibodies to FGF9 are already commercially available, but no aptamers to FGF9 have been reported so far.
  • An object of the present invention is to provide an aptamer for FGF9.
  • this aptamer has succeeded in producing an aptamer that binds to FGF9 as a result of diligent studies to solve the above problems. We also showed that this aptamer inhibits the activity of FGF9.
  • this aptamer was a novel aptamer having a characteristic potential secondary structure and a motif sequence of a loop portion, which is a characteristic of the secondary structure.
  • [6] The following (A), (B) or (C): (A) Nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-6, 10-15, 17, 18, 20, 21 and 23-35, (B) In a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 6, 10 to 15, 17, 18, 20, 21 and 23 to 35, one to several nucleotides are substituted, deleted, inserted or added.
  • nucleotide sequence (However, excluding the nucleotide sequence represented by the formula (I)), the nucleotide sequence, (C) Has 95% or more identity with the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-6, 10-15, 17, 18, 20, 21 and 23-35 (provided that it is in formula (I)).
  • Each pyrimidine nucleotide is deoxyribose
  • An aptamer in which each purine nucleotide is ribose.
  • FIG. 4-1 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 2 as expected by the MFOLD program. U or u represents deoxythymidine. In addition, the nucleotide sequence circled corresponds to a common sequence.
  • FIG. 4-2 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 3 as expected by the MFOLD program. U or u represents deoxythymidine. In addition, the nucleotide sequence circled corresponds to a common sequence.
  • FIG. 4-3 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 4 as expected by the MFOLD program.
  • FIG. 4-4 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 5 as expected by the MFOLD program.
  • U or u represents deoxythymidine.
  • the nucleotide sequence circled corresponds to a common sequence.
  • FIG. 4-5 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 6 as expected by the MFOLD program.
  • U or u represents deoxythymidine.
  • the nucleotide sequence circled corresponds to a common sequence.
  • FIG. 4-8 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 9 as expected by the MFOLD program.
  • U or u represents deoxythymidine.
  • the nucleotide sequence circled corresponds to a partial sequence of the common sequence.
  • FIG. 5-1 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 10 as expected by the MFOLD program.
  • U or u represents deoxythymidine.
  • the loop surrounded by a square corresponds to a loop formed by a common sequence.
  • FIG. 5-3 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 12 as expected by the MFOLD program.
  • U or u represents deoxythymidine.
  • the loop surrounded by a square corresponds to a loop formed by a common sequence.
  • FIG. 5-3 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 12 as expected by the MFOLD program.
  • U or u represents deoxythymidine.
  • the loop surrounded by a square corresponds to a loop formed by a common sequence.
  • FIG. 5-4 shows the secondary structure of the aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 13 as expected by the MFOLD program.
  • U or u represents deoxythymidine.
  • the loop surrounded by a square corresponds to a loop formed by a common sequence.
  • FIG. 5-5 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 14 as expected by the MFOLD program.
  • U or u represents deoxythymidine.
  • the loop surrounded by a square corresponds to a loop formed by a common sequence.
  • FIG. 5-6 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 15 as expected by the MFOLD program.
  • U or u represents deoxythymidine.
  • the loop surrounded by a square corresponds to a loop formed by a common sequence.
  • FIG. 5-7 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 16 as expected by the MFOLD program.
  • U or u represents deoxythymidine.
  • FIG. 5-8 shows the secondary structure of the aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 17 as expected by the MFOLD program.
  • U or u represents deoxythymidine.
  • FIG. 5-9 shows the secondary structure of the aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 18 as expected by the MFOLD program. U or u represents deoxythymidine.
  • the loop surrounded by a square corresponds to a loop formed by a common sequence.
  • FIG. 5-10 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 19 as expected by the MFOLD program. U or u represents deoxythymidine.
  • FIG. 5-11 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 20 as expected by the MFOLD program.
  • U or u represents deoxythymidine.
  • the loop surrounded by a square corresponds to a loop formed by a common sequence.
  • FIG. 5-12 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 21 as expected by the MFOLD program.
  • U or u represents deoxythymidine.
  • the loop surrounded by a square corresponds to a loop formed by a common sequence.
  • FIG. 5-16 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 25, as expected by the MFOLD program. U or u represents deoxythymidine.
  • the loop surrounded by a square corresponds to a loop formed by a common sequence.
  • FIG. 5-17 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 26, as expected by the MFOLD program. U or u represents deoxythymidine.
  • the loop surrounded by a square corresponds to a loop formed by a common sequence.
  • FIG. 6-2 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 29, as expected by the MFOLD program.
  • U or u represents deoxythymidine.
  • the loop surrounded by a square corresponds to a loop formed by a common sequence.
  • FIG. 6-3 shows the secondary structure of the aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 30 as expected by the MFOLD program.
  • U or u represents deoxythymidine.
  • the loop surrounded by a square corresponds to a loop formed by a common sequence.
  • FIG. 6-4 shows the secondary structure of the aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 31 as expected by the MFOLD program.
  • U or u represents deoxythymidine.
  • the loop surrounded by a square corresponds to a loop formed by a common sequence.
  • FIG. 6-5 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 32, as expected by the MFOLD program.
  • U or u represents deoxythymidine.
  • the loop surrounded by a square corresponds to a loop formed by a common sequence.
  • FIG. 6-6 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 33, as expected by the MFOLD program.
  • U or u represents deoxythymidine.
  • the loop surrounded by a square corresponds to a loop formed by a common sequence.
  • FIG. 6-7 shows the secondary structure of an aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 34, as expected by the MFOLD program.
  • U or u represents deoxythymidine.
  • the loop surrounded by a square corresponds to a loop formed by a common sequence.
  • the present invention provides an aptamer having a binding activity to FGF9.
  • An aptamer is a nucleic acid molecule having a binding activity to a predetermined target molecule. Aptamers can inhibit the activity of a given target molecule by binding to the given target molecule.
  • the aptamers of the present invention can be RNA, DNA, modified nucleic acids or mixtures thereof, but preferably pyrimidines are DNA and purines are RNA.
  • the aptamers of the present invention may also be in linear, cyclic or stem-loop form.
  • the aptamer of the present invention binds to FGF9 in a physiological buffer solution.
  • the buffer solution is not particularly limited, but one having a pH of about 5.0 to 10.0 is preferably used, and examples of such a buffer solution include solution C (see Example 1) described later.
  • the aptamer of the present invention binds to FGF9 with a strength that can be detected by any of the following tests. Biacore T200 manufactured by GE Healthcare is used to measure the bond strength. One measurement method is to first immobilize the aptamer on the sensor chip. The amount of immobilization is about 370 RU.
  • the FGF9 solution for analite is prepared to 0.1 ⁇ M and injected to detect the binding of FGF9 to the aptamer.
  • the aptamer of the present invention can bind to FGF9 and inhibit the activity of FGF9. That is, the aptamer of the present invention may also have an inhibitory activity on FGF9.
  • the inhibitory activity on FGF9 means an inhibitory ability against any activity possessed by FGF9.
  • FGF9 acts on FGF receptor-expressing cells to activate signal transduction and induce the production of various cell growth factors and their receptors. Therefore, the inhibitory activity on FGF9 may be an activity that inhibits intracellular signal transduction via the FGF receptor.
  • the expression of these various cell growth factors and their receptors results in the enhancement of cell growth activity and migration activity, and thus the inhibitory activity of FGF9 means inhibition of those activities.
  • the aptamer of the present invention binds to FGF9 and inhibits the binding between FGF9 and the FGF receptor, the action associated with the activation of the intracellular signal transduction pathway mediated by the FGF receptor, for example, phosphorylation of ERK1 / 2 and The accompanying proliferation of fibroblasts and the like can be inhibited.
  • FGF9 is a protein that is strongly expressed during embryogenesis, development, angiogenesis, and tumorigenesis, and is, for example, a protein having the amino acid sequence represented by SEQ ID NO: 36.
  • FGF9 in the present invention can also be produced using mammalian cells such as mice, insect cells, and cultured cells such as Escherichia coli, and can also be produced by chemical synthesis. When prepared by cultured cells or chemical synthesis, a mutant can be easily prepared by a method known per se.
  • the "mutant" of FGF9 is one in which one to several amino acids are substituted, deleted, added, etc.
  • FGF9 in the present invention contains these variants.
  • the FGF9 receptor means a cell surface protein to which FGF9 binds.
  • FGFR3c is known as the FGF9 receptor.
  • the FGF9 receptor in the present invention may be a protein containing a natural amino acid sequence or a mutant thereof.
  • the "mutant" of the FGF9 receptor is one in which one to several amino acids are substituted, deleted, added, etc., or one consisting of a part of the amino acid sequence of a known FGF9 receptor.
  • the invention provides an aptamer that inhibits the binding of FGF9 to the FGF9 receptor.
  • the aptamer of the present invention is not particularly limited as long as it is an aptamer that binds to an arbitrary portion of FGF9. Further, the aptamer of the present invention is not particularly limited as long as it can bind to any portion of FGF9 and inhibit its activity.
  • the length of the aptamer of the present invention is not particularly limited and may be usually about 200 nucleotides or less, but for example, it is about 100 nucleotides or less, preferably about 50 nucleotides or less, and more preferably about 40 nucleotides or less. obtain.
  • the total number of nucleotides is small, chemical synthesis and mass production are easier, and there is a great cost advantage.
  • the lower limit of the aptamer length of the present invention is not particularly limited as long as it contains the common sequence represented by SEQ ID NO: 37 (CAAGGHTGCCG; H represents A, C or T), but the aptamer length is preferably 15 nucleotides or more, for example. Can be 20 nucleotides or more, more preferably 25 nucleotides or more. In a particularly preferred embodiment, the aptamer length of the present invention is 25-40 nucleotides.
  • the aptamer of the present invention is formulated (I): CAAGGHTGCCG (SEQ ID NO: 37) (I) (In the formula, H represents A, C or T) It is an aptamer that binds to FGF9 (hereinafter, also referred to as the aptamer (I) of the present invention) containing the nucleotide sequence represented by.
  • Each nucleotide constituting the aptamer (I) of the present invention may be ribose or deoxyribose independently.
  • thymine (T) shall be read as uracil (U).
  • the aptamer of the present invention is formulated (I): CAAGGHTGCCG (SEQ ID NO: 37) (I) (In the formula, H represents A, C or T) An aptamer that binds to FGF9 and contains the nucleotide sequence represented by (a) or (b): (a) In the nucleotide sequence contained in the aptamer, (i) Each pyrimidine nucleotide is deoxyribose, (ii) Each purine nucleotide is ribose; (b) In the nucleotide sequence contained in the aptamer (i) Each pyrimidine nucleotide is deoxyribose, and the hydrogen atom at the 2'position of the deoxyribose is independently unsubstituted or substituted with a methoxy group.
  • Each purine nucleotide is ribose, and the hydroxy group at the 2'position of the ribose is independently unsubstituted or substituted with a methoxy group; It is an aptamer (hereinafter, also referred to as an aptamer (II) of the present invention) which is one of the above.
  • the aptamer (II) of the present invention comprises the following (A) to (C): (A) Aptamer containing a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16, 19 and 22; (B) In the nucleotide sequence selected from the group consisting of SEQ ID NOs: 16, 19 and 22, one to several nucleotides were substituted, deleted, inserted or added (however, CAAGGHTGCCG (SEQ ID NO: 37) (in the formula).
  • H represents A, C or T
  • C has 95% or more identity with the nucleotide sequence selected from the group consisting of SEQ ID NOs: 16, 19 and 22 (where CAAGGHTGCCG (SEQ ID NO: 37) (wherein H stands for A, C or T).
  • the nucleotide sequence represented by) is the same.) An aptamer that binds to FGF9 and contains a nucleotide sequence; In any of the aptamers, in the nucleotide sequence contained in the aptamer, (i) Each pyrimidine nucleotide is deoxyribose, (ii) Each purine nucleotide is an aptamer that is ribose (hereinafter, also referred to as an aptamer (III) of the present invention).
  • the aptamers of (B) and (C) above include the sequence represented by the formula (I): CAAGGHTGCCG (SEQ ID NO: 37) (where H represents A, C or T) as a common sequence.
  • the number of nucleotides to be substituted, deleted, inserted or added is not particularly limited as long as the aptamer can bind to FGF9 and inhibit the activity of FGF9, but is, for example, 10 or less, preferably 5 or less. , More preferably 4, 3, 2, or 1.
  • NCBI BLAST-2 National Center for Biotechnology Information Basic Local Alignment Search Tool
  • the aptamer (III) of the present invention also (D) can be one or more of the above (A) and / or one or more of the above (B) and / or one or more of the above (C).
  • the connection can be performed by a tandem bond.
  • a linker may be used for the connection.
  • Linkers include nucleotide chains (eg, 1 to about 20 nucleotides), non-nucleotide chains (eg,-(CH 2 ) n-linker,-(CH 2 CH 2 O) n-linker, hexaethylene glycol linker, TEG linker.
  • the plurality of the above-mentioned plurality of connected products is not particularly limited as long as it is two or more, but may be, for example, two, three, or four.
  • the aptamer (II) of the present invention comprises a nucleotide sequence represented by formula (I), adjacent to nucleotides X1, X2, X3 and 3'ends adjacent to the 5'end of the nucleotide sequence.
  • Nucleotides X4, X5, X6 are expressed in formula (II):
  • H represents A, C or T
  • X1 to X6 are nucleotides selected from the group consisting of A, G, C and T, which are the same or different, respectively
  • X1 and X6, X2 and X5, X3 and X4 and the 5'end C and 3'end G of the nucleotide sequence represented by formula (I) form Watson-Crick base pairs, respectively
  • the stem structure is formed by Watson-Crick base pairing of C at the 5'end and G at the 3'end of the nucleotide sequence represented by X1 and X6, X2 and X5, X3 and X4, and the formula (I). Is formed. Having a stem structure stabilizes the aptamer and gives it a strong activity.
  • the number of base pairs forming the stem structure is not particularly limited. Preferably, the stem length is 4 base pairs or more.
  • the upper limit is not particularly limited, but is, for example, 10 base pairs or less, preferably 7 base pairs or less. Further, in the stem structure, as long as the stem structure is formed as a whole, the aptamer activity is maintained even if a part of the stem structure does not form a base pair.
  • the aptamer (IV) of the present invention further has a nucleotide number 4th G and a nucleotide number 10th C, a nucleotide number 5th G and a nucleotide number in the nucleotide sequence represented by the formula (I).
  • the ninth C may each potentially form a Watson click base pair. Therefore, the aptamer (IV) of the present invention has the following formula (II').
  • the aptamer (V) of the present invention According to the secondary structure prediction by Mfold, when there are multiple energetically stable candidates, multiple candidates may be presented.
  • the nucleotide sequence of the aptamer of the present invention is applied to Mfold and the secondary structure is predicted, both the structure of the formula (II) and the structure of the formula (II') may be obtained. Therefore, the aptamer (IV) of the present invention may adopt the structure of the formula (II) or the structure of the formula (II').
  • aptamer that binds to FGF9 which comprises a nucleotide sequence (in the formula, H represents A, C or T) has the same nucleotide sequence;
  • H represents A, C or T
  • aptamers in the nucleotide sequence contained in the aptamer, (i) Each pyrimidine nucleotide is deoxyribose, (ii) Each purine nucleotide is an aptamer that is ribose (hereinafter, also referred to as an aptamer (VI) of the present invention).
  • the aptamer (VII) of the present invention is, in a preferred embodiment, an aptamer that binds to FGF9, which comprises the nucleotide sequence represented by SEQ ID NO: 30, in the nucleotide sequence contained in the aptamer.
  • the hydrogen atom at the 2'position of deoxyribose of each pyrimidine nucleotide is independently unsubstituted or substituted with a methoxy group.
  • sugar residues can be carried out by a method known per se (eg, Sproat et al., (1991) Nucle. Acid. Res. 19, 733-738; Cotton et al., (1991)). Nucl. Acid. Res. 19, 2629-2635; Hobbs et al., (1973) Biochemistry 12, 5138-5145).
  • the sugar residue can also be BNA: Bridged nucleic acid (LNA: Linked nucleic acid) having a crosslinked structure formed at the 2'and 4'positions.
  • Such modification of sugar residues can also be performed by a method known per se (eg, Tetrahedron Lett., 38, 8735-8738 (1997); Tetrahedron, 59, 5123-5128 (2003), Rahman SMA, Seki. See S., Obika S., Yoshikawa H., Miyashita K., Imanishi T., J. Am. Chem. Soc., 130, 4886-4896 (2008)).
  • the aptamer of the present invention may also have a nucleobase (eg, purine, pyrimidine) modified (eg, chemically substituted) in order to enhance the binding activity to FGF9 and the like.
  • a nucleobase eg, purine, pyrimidine
  • modifications include, for example, 5-position pyrimidine modification, 6- and / or 8-position purine modification, modification with extracyclic amine, substitution with 4-thiouridine, substitution with 5-bromo or 5-iodo-uracil.
  • the phosphate group contained in the aptamer of the present invention may be modified so as to be resistant to nucleases and hydrolysis, or for the purpose of enhancing the binding activity to FGF9.
  • the aptamer of the present invention can be synthesized by the methods disclosed in the present specification and known in the art.
  • One of the synthetic methods is a method using RNA polymerase.
  • the target RNA can be obtained by chemically synthesizing the DNA having the target sequence and the promoter sequence of RNA polymerase and transcribing this as a template by a method already known. It can also be synthesized by using DNA polymerase. DNA having the desired sequence is chemically synthesized, and this is used as a template for amplification by a known method, the polymerase chain reaction (PCR). This is made into a single strand by a already known method such as polyacrylamide gel electrophoresis or an enzyme treatment method.
  • PCR polymerase chain reaction
  • the ionic bond utilizing the negative charge of the phosphate group existing as many as the number of constituent nucleotides is strong and binds to the positive charge of lysine and arginine existing on the surface of the protein. Therefore, nucleobases that are not directly involved in binding to the target substance can be replaced.
  • the stem structure part since the stem structure part has already been base paired and faces the inside of the double helix structure, it is difficult for the nucleobase to directly bind to the target substance. Therefore, replacing a base pair with another base pair often does not reduce the activity of the aptamer. Even in structures such as loop structures that do not form base pairs, base substitution is possible when the nucleobase is not involved in direct binding to the target molecule.
  • Aptamers are prepared by using the SELEX method and its improved methods (for example, Ellington et al., (1990) Nature, 346, 818-822; Tuerk et al., (1990) Science, 249, 505-510). can do.
  • SELEX method aptamers with stronger binding force to the target substance are concentrated and sorted by increasing the number of rounds or using competing substances to tighten the sorting conditions. Therefore, by adjusting the number of rounds of SELEX and / or changing the race condition, aptamers with different binding forces, aptamers with different binding forms, and aptamers with the same binding force and binding form but different base sequences. You may be able to get an aptamer.
  • the SELEX method includes an amplification process by PCR, and by inserting mutations such as using manganese ions in the process, it is possible to perform SELEX with a greater variety.
  • SELEX can be performed by further changing the primer in order to obtain an aptamer having higher activity.
  • the specific method is to prepare a template in which a part of the aptamer having a certain sequence is made into a random sequence or a template in which a random sequence of about 10 to 30% is doped, and perform SELEX again.
  • the aptamer obtained by SELEX has a length of about 80 nucleotides, and it is difficult to use this as it is as a medicine. Therefore, it is necessary to repeat trial and error to shorten the length to about 50 nucleotides or less, which can be easily chemically synthesized.
  • the aptamer obtained by SELEX depends on its primer design, and the ease of subsequent minimization work changes. If the primers are not designed properly, even if SELEX can select active aptamers, further development will be impossible. In the present invention, it was possible to obtain an aptamer that retains inhibitory activity even at 29 nucleotides.
  • the important part of the obtained aptamer for binding to the target substance can be identified by repeating the above trial and error, the activity often changes even if a new sequence is added to both ends of the sequence. do not do.
  • the length of the new sequence is not particularly limited.
  • the aptamer of the present invention or the complex of the present invention can be used, for example, as a medicine (hereinafter, also referred to as the medicine of the present invention).
  • Glue gelatin, gum arabic, polyethylene glycol, sucrose, starch and other binders, starch, carboxymethyl cellulose, hydroxypropyl starch, sodium-glycol-starch, sodium hydrogen carbonate, calcium phosphate, calcium citrate and other disintegrants, magnesium stearate , Lubricants such as aerodil, starch, sodium lauryl sulfate, fragrances such as citric acid, menthol, glycyrrhizin / ammonium salt, glycine, orange powder, preservatives such as sodium benzoate, sodium hydrogen sulfite, methylparaben, propylparaben, citric acid , Stabilizers such as sodium citrate, acetic acid, suspending agents such as methyl cellulose, polyvinylpyrrolidone, aluminum stearate, dispersants such as surfactants, diluents such as water, physiological saline, orange juice, cacao butter, polyethylene. Examples thereof include
  • the medicament of the present invention can be coated by a method known per se for the purpose of masking taste, enteric acidity or persistence, if necessary.
  • the coating agent used for coating include hydroxypropylmethylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, polyoxyethylene glycol, Tween 80, Pluronic F68, cellulose acetate phthalate, hydroxypropylmethylcellulose phthalate, hydroxymethylcellulose acetate succinate, and the like.
  • Eudragit manufactured by Roam, Germany, acrylate copolymer of methacrylic acid
  • dyes eg, red iron oxide, titanium dioxide, etc.
  • the drug may be either a rapid-release preparation or a sustained-release preparation.
  • the sustained-release substrate include liposomes, atelocollagen, gelatin, hydroxyapatite, PLGA and the like.
  • Suitable formulations for parenteral administration are aqueous and non-aqueous isotonic.
  • parenteral administration eg, intravenous, subcutaneous, intramuscular, topical, intraperitoneal, nasal, pulmonary, etc.
  • aqueous and non-aqueous isotonic are aqueous and non-aqueous isotonic.
  • sterile injection solutions which may contain antioxidants, buffers, antibacterial agents, isotonic agents and the like.
  • aqueous and non-aqueous sterile suspensions may be mentioned, which may include suspending agents, solubilizing agents, thickeners, stabilizers, preservatives and the like.
  • the formulation can be encapsulated in single doses or multiple doses, such as ampoules and vials.
  • sustained-release preparations can also be mentioned as suitable preparations.
  • Sustained-release preparations include artificial bone, biodegradable or non-degradable sponges, bags, drug pumps, osmotic pumps, and other sustained-release forms from carriers or containers embedded in the body, or continuous or intermittent from outside the body. Examples include devices that are delivered internally or locally.
  • examples of the surfactant include oleic acid, lecithin, diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glyceryl trioleate, glyceryl monolaurate, glyceryl monooleate, and glyceryl monosteer.
  • Span, Tween, Epicron, Brij, Genapol and Synperonic are trademarks.
  • the oil include corn oil, olive oil, cottonseed oil, sunflower oil and the like.
  • appropriate pharmaceutically acceptable bases yellow petrolatum, white petrolatum, paraffin, plastic base, silicone, white ointment, beeswax, pig oil, vegetable oil, hydrophilic ointment, hydrophilic petrolatum, purified lanolin, hydrous lanolin , Water-absorbing ointment, hydrophilic plastic base, macrogol ointment, etc.
  • the inhalant can be manufactured according to the conventional method. That is, it can be produced by powdering or liquidating the aptamer of the present invention or the composite of the present invention, blending it in an inhalation propellant and / or a carrier, and filling it in an appropriate inhalation container. Further, when the aptamer of the present invention or the complex of the present invention is powder, a normal mechanical powder inhaler can be used, and when the complex is liquid, an inhaler such as a nebulizer can be used.
  • propellants can be widely used, and CFC-11, CFC-12, CFC-21, CFC-22, CFC-113, CFC-114, CFC-123, CFC-142c, CFC-134a , Freon-227, Freon-C318, Freon compounds such as 1,1,1,2-tetrafluoroethane, hydrocarbons such as propane, isobutane, n-butane, ethers such as diethyl ether, nitrogen gas, carbon dioxide A compressed gas such as a gas can be exemplified.
  • the dose of the medicament of the present invention varies depending on the type of active ingredient, activity, severity of disease, animal species to be administered, drug acceptability to be administered, body weight, age, etc., but is usually per adult per day.
  • the amount of active ingredient can be from about 0.0001 to about 100 mg / kg, such as from about 0.0001 to about 10 mg / kg, preferably from about 0.005 to about 1 mg / kg.
  • the aptamer of the present invention or the complex of the present invention can specifically bind to FGF9. Therefore, the aptamer of the present invention or the complex of the present invention is useful as a detection reagent for FGF9 (hereinafter, also referred to as a detection reagent of the present invention).
  • the detection reagent is useful for in vivo imaging of FGF9, blood concentration measurement, tissue staining, ELISA and the like.
  • the aptamer of the present invention or the complex of the present invention can be used as a ligand for separation and purification of FGF9 (hereinafter, also referred to as a ligand for separation and purification of the present invention).
  • the present invention also provides a solid phase carrier on which the aptamer of the present invention or the complex of the present invention is immobilized (hereinafter, also referred to as the solid phase carrier of the present invention).
  • the solid phase carrier include substrates, resins, plates (eg, multi-well plates), filters, cartridges, columns, and porous materials.
  • the substrate may be one used for a DNA chip, a protein chip, or the like.
  • the resin examples include agarose particles, silica particles, a copolymer of acrylamide and N, N'-methylenebisacrylamide, polystyrene-crosslinked divinylbenzene particles, particles obtained by cross-linking dextran with epichlorohydrin, cellulose fibers, and allyl dextran.
  • examples thereof include crosslinked polymers of N, N'-methylenebisacrylamide, monodisperse synthetic polymers, monodisperse hydrophilic polymers, sepharose, toyopearl, and resins in which various functional groups are bonded to these resins. ..
  • the solid phase carrier of the present invention can be useful, for example, for purification of FGF9 and detection and quantification of FGF9.
  • the aptamer of the present invention or the complex of the present invention can be immobilized on a solid phase carrier by a method known per se.
  • an affinity substance eg, as described above
  • a predetermined functional group is introduced into an aptamer of the present invention or a complex of the present invention, and then the affinity substance or a predetermined functional group is used as a solid phase carrier.
  • a method of immobilization can be mentioned.
  • the present invention also provides such a method.
  • the predetermined functional group can be a functional group that can be subjected to a coupling reaction, and examples thereof include an amino group, a thiol group, a hydroxyl group, and a carboxyl group.
  • the present invention also provides an aptamer into which such a functional group has been introduced.
  • the present invention also provides a method for purifying and concentrating FGF9 (hereinafter, also referred to as the method for purifying and concentrating the present invention).
  • the present invention is capable of separating FGF9 from other family proteins.
  • the purification and concentration method of the present invention may include adsorbing FGF9 on the solid phase carrier of the present invention and eluting the adsorbed FGF9 with an eluate.
  • Adsorption of FGF9 onto the solid phase carrier of the present invention can be carried out by a method known per se. For example, a sample containing FGF9 (eg, bacterial or cell culture or culture supernatant, blood) is introduced into the solid phase carrier of the invention or its content.
  • FGF9 eg, bacterial or cell culture or culture supernatant, blood
  • the elution of autotaxine can be performed using an eluate such as a neutral solution.
  • the neutral eluate is not particularly limited, but may have, for example, a pH of about 6 to about 9, preferably about 6.5 to about 8.5, and more preferably about 7 to about 8.
  • Neutral solutions also include, for example, urea, chelating agents (eg, EDTA), sodium salts (eg, NaCl), potassium salts (eg, KCl), magnesium salts (eg, MgCl 2 ), surfactants (eg, Tween20). , Triton, NP40), glycerin.
  • the purification and concentration method of the present invention may further include washing the solid phase carrier with a washing solution after adsorption of autotaxin.
  • the cleaning solution examples include urea, a chelating agent (eg, EDTA), Tris, an acid, an alkali, a surface active agent such as Tranfer RNA, DNA, Tween 20, and a salt such as NaCl.
  • the purification and concentration methods of the present invention may further include heat treating the solid phase carrier. By this step, the solid phase carrier can be regenerated and sterilized.
  • the present invention also provides a method for detecting and quantifying FGF9 (hereinafter, also referred to as a method for detecting and quantifying FGF9 of the present invention).
  • the present invention can be detected and quantified separately from FGF9 and other family proteins.
  • the detection and quantification methods of the present invention may include measuring FGF9 using the aptamers of the present invention (eg, by using the complex and solid phase carriers of the present invention).
  • the method for detecting and quantifying FGF9 can be carried out by the same method as the immunological method except that the aptamer of the present invention is used instead of the antibody.
  • EIA enzyme-linked immunosorbent assay
  • ELISA direct-competitive ELISA, indirect-competitive ELISA, sandwich ELISA
  • RIA radioimmunoassay
  • FIA Western blot method
  • immunohistochemical staining method cell sorting method and the like. It can also be used as a molecular probe for PET and the like.
  • RNA aptamers that bind to FGF9 were prepared using the SELEX method. SELEX was performed with reference to the method of Ellington et al. (Ellington and Szostak, Nature 346, 818-822, 1990) and the method of Tuerk et al. (Tuerk and Gold, Science 249, 505-510, 1990).
  • FGF9 manufactured by R & D systems
  • immobilized on a carrier of NHS-activated Sepharose 4 Fast Flow manufactured by GE Healthcare
  • the method for immobilizing FGF9 on the carrier was performed according to the specifications of GE Healthcare Japan.
  • the amount of immobilization was confirmed by examining the FGF9 solution before immobilization and the supernatant immediately after immobilization by SDS-PAGE. As a result of SDS-PAGE, no band of FGF9 was detected from the supernatant, and it was confirmed that almost all of the FGF9 used was coupled. About 217 pmol of FGF9 was immobilized on about 3 ⁇ L of resin.
  • RNA (35N) used in the first round was obtained by transcribing the DNA obtained by chemical synthesis using mutant T7 RNA polymerase (Sousa and Padilla, EMBO J. 14,4609-4621, 1995).
  • the substrate for this transcription reaction was selected so that the purine nucleotide of the transcript was RNA and the pyrimidine nucleotide was DNA.
  • As the DNA template a 71-nucleotide long DNA having primer sequences at both ends of the 35-nucleotide random sequence shown below was used. DNA templates and primers were made by chemical synthesis.
  • DNA template 5'-AGGAGCTACGCAGGCGTANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGCTCGACGGAGCTTCCC-3'(SEQ ID NO: 39)
  • Primer Fwd 5'-TAATACGACTCACTATAGGGAAGCTCCGTCGAGCT-3'(SEQ ID NO: 40)
  • Primer Rev 5'-AGGAGCTACGCAGGCGTA-3' (SEQ ID NO: 41)
  • N in the DNA template is any combination of 35 nucleotides (35N: each N is A, C, G or T) and the resulting aptamer-specific sequence region. Occurs.
  • Primer Fwd contains the promoter sequence of T7 RNA polymerase. The variation of the RNA pool used in the first round was theoretically 10 14 .
  • RNA bound to FGF9 was recovered from the supernatant after adding solution B as an eluate and heat-treating at 85 ° C. for 3 minutes.
  • solution B is a mixed solution of 7M Urea, 5mM EDTA, and 20mM tris (pH 6.6).
  • the recovered RNA was amplified by reverse transcription PCR, transcribed with mutant T7 RNA polymerase, and used as a pool for the next round. The above was regarded as one round, and the same work was repeated multiple times.
  • the base sequence was analyzed using a next-generation sequencer.
  • the Ion PGM TM system manufactured by Thermo
  • was used for the next-generation sequencer was used for the next-generation sequencer, and the analysis was performed according to the specifications of Thermo.
  • the nucleotide sequence represented by SEQ ID NO: 1 is one of the representative sequences of those clonal sequences, and there were 65 copies in the clonal sequence.
  • the nucleotide sequence represented by SEQ ID NO: 1 contained common sequence 1.
  • Common sequence 1 was present in at least 28 of the 25,205 sequences. When the secondary structure of these 28 types of sequences was predicted using the MFOLD program (Zukker, Nucleic Acids Res. 31, 3406-3415, 2003), the common sequence 1 formed a similar loop structure.
  • the secondary structure of the aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 1 is shown in FIG.
  • nucleotide sequence represented by SEQ ID NO: 1 and the common sequence 1 are shown below. Unless otherwise stated, the individual sequences listed below shall be represented in the 5'to 3'direction, with purine nucleotides (A and G) being 2'-OH (ie, ribonucleotides) and pyrimidine nucleotides (ie, ribonucleotides). T and C) are 2'-H (ie, deoxyribonucleotides). In addition, H in the sequence indicates T, C or A.
  • SEQ ID NO: 1 GGGAAGCTCCGTCGAGCTGAGGGCACCCAAGGCTGCCGGGTGCCATGCACACATACGCCTGCGTAGCTCCT
  • CAAGGHTGCCG SEQ ID NO: 37
  • the binding activity of the aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 1 to FGF9 was evaluated by the surface plasmon resonance method.
  • a Biacore T200 manufactured by GE Healthcare was used for the measurement.
  • As the sensor chip an SA chip on which streptavidin was immobilized was used.
  • About 740 RU of 16 nucleotides of PolydT to which biotin was bound at the 5'end was bound to this.
  • 16 nucleotides of PolyA was added to the 3'end and immobilized on the SA chip by annealing T and A.
  • Nucleic acid was injected for 30 seconds at a flow rate of 10 ⁇ L / min to immobilize approximately 370 RU of nucleic acid.
  • FGF9 for analysis was prepared to 0.1 ⁇ M and injected at a flow rate of 30 ⁇ L / min for 60 seconds.
  • Solution C was used as the running buffer.
  • the solution C is a mixed solution of 300 mM sodium chloride, 5.4 mM potassium chloride, 1.8 mM calcium chloride, 0.8 mM magnesium chloride, 20 mM tris (pH 7.6), and 0.05% Tween 20.
  • aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 1 binds to FGF9 (Fig. 2).
  • Nucleic acid pool (35N) used in the first round containing a random sequence of 35 nucleotides used as a negative control did not show binding to FGF9.
  • Whether or not the aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 1 inhibits the activity of FGF9 was evaluated by the following method.
  • a fusion protein of FGFR3c and the Fc portion of IgG (manufactured by R & D systems) was selected as the receptor for FGF9.
  • Biacore T200 manufactured by GE Healthcare was used.
  • the sensor chip used was a protein A immobilized on CM5.
  • FGFR3c was immobilized there at about 500 RU.
  • a mixture of FGF9 (0.1 ⁇ M), heparin (0.1 ⁇ M) (manufactured by Calbiochem) and aptamer (0.5 ⁇ M) was flowed as an analyzer.
  • Solution D was used as the running buffer.
  • solution D is a mixed solution of 295 mM sodium chloride, 5.4 mM potassium chloride, 1.8 mM calcium chloride, 0.8 mM magnesium chloride, 20 mM tris (pH 7.6), and 0.05% Tween 20.
  • a mixture of FGF9 and heparin binds to FGFR3c.
  • the aptamer represented by SEQ ID NO: 1 showed strong inhibitory activity.
  • FIG. 3 shows a sensorgram showing that the aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 1 inhibits the binding between FGF9 and FGFR3c.
  • the inhibition rate of the test substance was calculated using the following formula.
  • Inhibition rate (%) (R Max -R apt ) / R Max x100
  • R Max and R apt are the RU values indicated by the mixture of FGF9 and heparin with and without the addition of the test substance.
  • the inhibition rate of the aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 1 was 95.7%.
  • 35N which is a negative control, showed no inhibitory activity (inhibition rate 0%). From the above results, it can be said that the aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 1 exhibits an excellent inhibitory effect on FGF9.
  • Example 2 Shortening the aptamer (1) The aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 1 was shortened to obtain an aptamer consisting of the nucleotide sequence represented by SEQ ID NOs: 2-9.
  • FIG. 4 shows a prediction of the secondary structure of the aptamer consisting of the nucleotide sequences represented by SEQ ID NOs: 2 to 9.
  • nucleotide sequences represented by SEQ ID NOs: 2 to 9 are shown below. Unless otherwise stated, the individual sequences listed below shall be represented in the 5'to 3'direction, with purine nucleotides (A and G) being 2'-OH (ie, ribonucleotides) and pyrimidine nucleotides (ie, ribonucleotides). T and C) are 2'-H (ie, deoxyribonucleotides).
  • SEQ ID NO: 2 (Sequence in which the nucleotide sequence represented by SEQ ID NO: 1 is shortened to 25 nucleotides including a common sequence) GGGCACCCAAGGCTGCCGGGTGCCA SEQ ID NO: 3: (Sequence in which the nucleotide sequence represented by SEQ ID NO: 1 is shortened to 59 nucleotides including a common sequence) GGAGCTGAGGGCACCCAAGGCTGCCGGGTGCCATGCACACATACGCCTGCGTAGCTCCT SEQ ID NO: 4: (Sequence in which the nucleotide sequence represented by SEQ ID NO: 1 is shortened to 43 nucleotides including a common sequence) GGGCACCCAAGGCTGCCGGGTGCCATGCACACATACGCCTGCG SEQ ID NO: 5: (Sequence obtained by shortening the nucleotide sequence represented by SEQ ID NO: 1 to 57 nucleotides including a common sequence) GGGAAGCTCCGTCGAGCT
  • GGGCTGCCGGGTGCCATGCACACATACGCCTGCGTAGCC SEQ ID NO: 9: (Sequence obtained by shortening the nucleotide sequence represented by SEQ ID NO: 1 to 31 nucleotides including a partial sequence of the common sequence) GGCCGGGTGCCATGCACACATACGCCTGCGT
  • Example 3 Preparation of RNA aptamer that binds to FGF9 (2)
  • the total length of the aptamer obtained was about 70 bases, and then the work of shortening the chain was required. However, the shortened form was often significantly reduced in activity. Therefore, SELEX was performed with reference to the Tailored-SELEX method developed by NOXXON (Vater et al. Nucleic Acids Res. 31, 2003, el30; Jarosch et al. Nucleic Acids Res. 34, 2006, e86).
  • the target immobilization and washing method was substantially the same as in Example 1, and FGF9 (manufactured by R & D systems) immobilized on a carrier of NHS-activated Sepharose 4 Fast Flow (manufactured by GE Healthcare) was used.
  • DNA templates and primers were prepared by chemical synthesis.
  • the substrate for the transcription reaction was selected so that the purine nucleotide was RNA and the pyrimidine nucleotide was DNA with reference to Example 1.
  • Two SELEXs were performed with different molds and primer sets. The sequences of the templates and primers used are shown below.
  • DNA template 1 5'-TCGAGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGCCCTATAGTGAGTCGTATTA-3' (SEQ ID NO: 42) Forward ligate 1: 5'-UAAUACGACUCACUAUA-3' (SEQ ID NO: 43) Forward primer 1: 5'-TAGACCATTCGACTATAATACGACTCACTATAGGGC-3' (SEQ ID NO: 44) Forward bridge 1: 5'-GCCCTATAGTGAGTCGTATT-NH 2-3 ' (SEQ ID NO: 45) Reverse bridge 1: 5'-TTACGTCTTGTTTTTCTCGAG-3' (SEQ ID NO: 46) Reverse ligate 1: 5'-p-GAAAAACAAGACGTAA-NH 2-3 ' (SEQ ID NO: 47) DNA template 2: 5'-TCGAGNNNNNNNNNNNNNNNNNNNNNNNNTCCCTATAGTGAGTCGTATTA-3' (SEQ ID NO: 48) Forward ligate 2: (Sam
  • sequence analysis was performed using the next-generation sequencer in the same manner as in Example 1. From SELEX using DNA template 1, 12,737 types of clone sequences were identified, and it was confirmed that they converged to 353 types of sequences. From SELEX using DNA template 2, 28,646 types of clone sequences were identified, and it was confirmed that they converged to 3,852 types of sequences. There were clone sequences having a common sequence in these clone sequences. The nucleotide sequences represented by SEQ ID NOs: 10 to 27 are representative sequences of those clonal sequences. FIG. 5 shows a secondary structure prediction of an aptamer consisting of the nucleotide sequences represented by SEQ ID NOs: 10 to 27.
  • nucleotide sequences represented by SEQ ID NOs: 10 to 27 are shown below. Unless otherwise stated, the individual sequences listed below shall be represented in the 5'to 3'direction, with purine nucleotides (A and G) being 2'-OH (ie, ribonucleotides) and pyrimidine nucleotides (ie, ribonucleotides). T and C) are 2'-H (ie, deoxyribonucleotides).
  • the stem to be produced was found to be a 4-base pair stem.
  • the result of SEQ ID NO: 11 showed that the nucleotide sequence adjacent to the 5'side of the stem can be deleted.
  • Example 4 Shortening the aptamer (2) The aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 27 was shortened to obtain an aptamer consisting of the nucleotide sequence represented by SEQ ID NOs: 28 to 35.
  • FIG. 6 shows a secondary structure prediction of an aptamer consisting of the nucleotide sequences represented by SEQ ID NOs: 28 to 35.
  • nucleotide sequences represented by SEQ ID NOs: 28 to 35 are shown below. Unless otherwise stated, the individual sequences listed below shall be represented in the 5'to 3'direction, with purine nucleotides (A and G) being 2'-OH (ie, siribonucleotides) and pyrimidine nucleotides. (T and C) are 2'-H (ie, deoxyribonucleotides).
  • SEQ ID NO: 28 (A sequence in which the 3'end of the nucleotide sequence represented by SEQ ID NO: 27 is shortened by 2 nucleotides) GGGAGGTGCCAAGGTTGCCGGTACCAATAATGTGCTC
  • SEQ ID NO: 29 (A sequence in which the 3'end of the nucleotide sequence represented by SEQ ID NO: 27 is shortened by 4 nucleotides) GGGAGGTGCCAAGGTTGCCGGTACCAATAATGTGC
  • SEQ ID NO: 30 (A sequence in which the 3'end of the nucleotide sequence represented by SEQ ID NO: 27 is shortened by 5 nucleotides) GGGAGGTGCCAAGGTTGCCGGTACCAATAATGTG
  • SEQ ID NO: 31 (A sequence in which the 3'end of the nucleotide sequence represented by SEQ ID NO: 27 is shortened by 6 nucleotides) GGGAGGTGCCAAGGTTGCCGGTACCAATAATGT
  • the aptamers of the invention can be shortened to at least 29 mer while maintaining a 6 base pair stem formed by a nucleotide sequence containing nucleotides adjacent to the 3'side.
  • Example 5 Modification of aptamer Chemical modification of the aptamer consisting of the nucleotide sequence represented by SEQ ID NO: 30 was examined, and an aptamer consisting of the nucleotide sequence represented by SEQ ID NOs: 30 (1) to 30 (22) was obtained. ..
  • nucleotide sequences represented by SEQ ID NOs: 30 (1) to 30 (22) are shown below.
  • the individual sequences listed below shall be represented in the 5'to 3'direction, and unless otherwise noted, purine nucleotides (A and G) are 2'-OH (ie, ribonucleotides) and pyrimidine nucleotides (ie, ribonucleotides).
  • T and C) are 2'-H (ie, deoxyribonucleotides).
  • a (M), G (M), C (M), and U (M) indicate that the nucleotides 2'of A, G, C, and U are methoxylated, respectively.
  • U is uridine (ribonucleotide).
  • SEQ ID NO: 30 (11): (2'of the 29th and 30th nucleotides of the nucleotide sequence represented by SEQ ID NO: 30 are methoxylated, and the 31st nucleotide is replaced with uridine in which the carbon at the 2'position is methoxylated.
  • SEQ ID NO: 30 (12): (2'of the 32nd and 34th nucleotides of the nucleotide sequence represented by SEQ ID NO: 30 are methoxylated, and the 33rd nucleotide is replaced with uridine in which the carbon at the 2'position is methoxylated.
  • SEQ ID NO: 30 (13) (includes all modifications of SEQ ID NO: 30 (1), 30 (2), 30 (3), 30 (4), 30 (10), 30 (11), 30 (12)' Array) G (M) G (M) G (M) A (M) G (M) G (M) U (M) G (M) C (M) C (M) AAGGTTTGCCG (M) G (M) U ( M) A (M) C (M) C (M) A (M) A (M) C (M) C (M) A (M) A (M) U (M) A (M) U (M) G (M) U (M) G (M) _ (T) SEQ ID NO: 30 (14): (sequence in which 2'of the nucleotides 11, 12, 13, 14, and 17 of the nucleotide sequence represented by SEQ ID NO: 30 (13) is methoxylated) G (M) G
  • the aptamers or complexes of the present invention may also be useful for purification and enrichment of FGF9, labeling of FGF9, and detection and quantification of FGF9.
  • This application is based on Japanese Patent Application No. 2019-072337 filed in Japan (Filing date: April 4, 2019), the contents of which are all incorporated herein by reference.

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Abstract

La présente invention concerne un aptamère se liant à FGF9, qui contient une séquence nucléotidique représentée par la formule (I) : CAAGGHTGCCG (SEQ ID NO: 37) (I) (où H représente A, C ou T), etc.
PCT/JP2020/015263 2019-04-04 2020-04-03 Aptamère dirigé contre fgf9 et son utilisation Ceased WO2020204151A1 (fr)

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JP2019-072337 2019-04-04

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