WO2015099188A1 - Micro-arn de type à correspondance artificielle pour le contrôle de l'expression génétique et son utilisation - Google Patents

Micro-arn de type à correspondance artificielle pour le contrôle de l'expression génétique et son utilisation Download PDF

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WO2015099188A1
WO2015099188A1 PCT/JP2014/084725 JP2014084725W WO2015099188A1 WO 2015099188 A1 WO2015099188 A1 WO 2015099188A1 JP 2014084725 W JP2014084725 W JP 2014084725W WO 2015099188 A1 WO2015099188 A1 WO 2015099188A1
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雅彦 黒田
慎一郎 大野
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株式会社ボナック
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
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • 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.
    • C12N2310/141—MicroRNAs, miRNAs

Definitions

  • the present invention relates to an artificial match-type miRNA that suppresses gene expression and uses thereof.
  • Micro RNA is known as a nucleic acid molecule that suppresses gene expression, and has been reported to suppress transcription of a protein encoded by the gene through, for example, the following generation process. That is, first, a miRNA transcript (Pri-miRNA) having a cap structure at the 5 'end and poly (A) at the 3' end is generated in the nucleus. The Pri-miRNA is cleaved by RNase (Drosha) to generate a miRNA precursor (Pre-miRNA). The Pre-miRNA has a hairpin structure having a loop region and a stem region.
  • Pri-miRNA miRNA transcript having a cap structure at the 5 'end and poly (A) at the 3' end is generated in the nucleus.
  • the Pri-miRNA is cleaved by RNase (Drosha) to generate a miRNA precursor (Pre-miRNA).
  • the Pre-miRNA has a hairpin structure having a loop region and a stem region.
  • This Pre-miRNA moves out of the nucleus, and then is degraded by cytoplasmic RNase (Dicer) to cut out double-stranded miRNA (mature miRNA) having a 1 to 4 base overhang at the 3 'end.
  • double-stranded miRNAs one strand is called a guide strand, the other strand is called a passenger strand, and the guide strand binds to a complex similar to RNA-induced Silencing Complex (RISC).
  • RISC RNA-induced Silencing Complex
  • the miRNA / RISC complex binds to the 3 'untranslated region (3'UTR) of a specific mRNA, thereby suppressing protein translation from the mRNA.
  • the miRNA for example, there is a method using a double-stranded mature miRNA.
  • this method requires the annealing of two single-stranded nucleic acid molecules prior to use, and may generate autoimmunity by TLR3 or the like that recognizes the double strand.
  • an object of the present invention is to provide a new artificial match-type miRNA using miRNA.
  • the artificial match-type miRNA of the present invention comprises: A single-stranded nucleic acid having an X region and a Y region, The 3 ′ end of the X region and the 5 ′ end of the Y region are linked via a non-nucleotide structure containing at least one of a pyrrolidine skeleton and a piperidine skeleton,
  • the X region comprises a guide strand sequence of a mature miRNA of hsa-miR-34;
  • the Y region includes a sequence completely complementary to the X region.
  • composition of the present invention is a composition for suppressing gene expression, and includes the artificial match-type miRNA of the present invention.
  • composition of the present invention is a pharmaceutical composition, characterized in that it contains the artificial match-type miRNA of the present invention.
  • the expression suppression method of the present invention is a method of suppressing the expression of a target gene, characterized by using the artificial match-type miRNA of the present invention.
  • the method for treating a disease of the present invention comprises the step of administering the artificial match-type miRNA of the present invention to a patient, wherein the disease is a disease involving a target gene of a mature miRNA of hsa-miR-34.
  • the artificial match-type miRNA of the present invention can be easily synthesized at low cost, and can suppress the translation of the protein encoded by the target gene of the mature miRNA of hsa-miR-34.
  • FIG. 1 is a schematic view showing an example of the artificial match-type miRNA of the present invention.
  • FIG. 2 is a graph showing the number of cells per well in Example 1 of the present invention.
  • FIG. 3 is a graph showing the relative values of cell proliferation in Example 1 of the present invention.
  • FIG. 4 is a graph showing the percentage of apoptosis in Example 1 of the present invention.
  • FIG. 5 is a graph showing the relative values of the amount of AXL mRNA and the amount of MET mRNA in Example 1 of the present invention.
  • FIG. 6 is a graph showing the relative value of the amount of AXL mRNA in Example 2 of the present invention.
  • FIG. 7 is a graph showing the relative value of the amount of MET mRNA in Example 2 of the present invention.
  • the artificial match-type miRNA of the present invention as described above, A single-stranded nucleic acid having an X region and a Y region, The 3 ′ end of the X region and the 5 ′ end of the Y region are linked via a non-nucleotide structure containing at least one of a pyrrolidine skeleton and a piperidine skeleton,
  • the X region comprises a guide strand sequence of a mature miRNA of hsa-miR-34;
  • the Y region includes a sequence completely complementary to the X region.
  • the artificial match-type miRNA of the present invention can suppress the expression of the target gene of hsa-miR-34.
  • Expression suppression means, for example, suppression of translation of the target gene, that is, suppression of translation of a protein encoded by the target gene, and more specifically, suppression of translation of the protein from mRNA of the target gene.
  • the suppression of the expression of the target gene can be achieved, for example, by reducing the production amount of the transcription product from the target gene, reducing the activity of the transcription product, reducing the production amount of the translation product from the target gene, or activity of the translation product. It can be confirmed by decrease of Examples of the protein include a mature protein or a precursor protein before undergoing processing or post-translational modification.
  • the artificial match-type miRNA of the present invention is a single-stranded nucleic acid molecule, for example, it is not necessary to anneal two single-strands like mature miRNA, and can be produced at low cost. Furthermore, since the artificial match-type miRNA of the present invention is a single-stranded nucleic acid molecule, it can be prevented from being recognized by, for example, TLR3, RIG-I, MDA5, etc. involved in autoimmunity.
  • FIG. 1 shows an outline of the positional relationship between the X region and the Y region in the artificial match-type miRNA of the present invention.
  • FIG. 1 is schematic, and for example, the length, shape, and the like of each region are not limited.
  • the artificial match-type miRNA of the present invention has the X region disposed on the 5 ′ side, the Y region disposed on the 3 ′ side, and the 3 ′ end of the X region and the Y region.
  • the 5 ′ end is linked via a non-nucleotide structure (indicated as “P” in the figure) containing at least one of a pyrrolidine skeleton and a piperidine skeleton.
  • P non-nucleotide structure
  • the Y region includes a sequence that is completely complementary to the X region
  • the X region and the Y region are annealed intramolecularly, for example.
  • Intramolecular annealing is also referred to as self-annealing, for example.
  • the artificial match-type miRNA of the present invention is also said to form a double strand in the intramolecularly annealed region.
  • the artificial match-type miRNA of the present invention can also be referred to as a linear single-stranded nucleic acid molecule in which the 5 'end and 3' end are not linked.
  • the artificial match-type miRNA of the present invention preferably has a non-phosphate group at the 5 'end, for example, to maintain unbonded both ends.
  • the X region contains the guide strand sequence of the mature miRNA of hsa-miR-34 as described above.
  • the guide strand sequence of mature miRNA is registered in various databases (for example, http://www.mirbase.org/ etc.). Therefore, for example, the X region can be set based on information of these known mature miRNAs.
  • the guide strand of the mature miRNA is a strand that is incorporated into the RNA-induced silencing complex (RISC) Argonaute (Ago) protein and binds to the target mRNA.
  • RISC RNA-induced silencing complex
  • the X region may consist of, for example, only the guide strand sequence or may have an additional sequence.
  • the X region is composed of, for example, the guide strand sequence and the additional sequence, and the additional sequence is linked to the 3 ′ end of the guide strand sequence, for example.
  • the Y region includes a sequence that is completely complementary to the X region when the X region and the Y region are aligned.
  • the Y region may consist of, for example, a sequence that is completely complementary to the X region, or may have an overhang in addition to the complementary sequence. That is, in the artificial match-type miRNA of the present invention, for example, when the Y region and the X region are aligned, the Y region may have an overhang at the 3 ′ end.
  • the overhang of the Y region is, for example, a terminal base that the Y region has more than the X region when the Y region and the X region are aligned.
  • the length of each region is not particularly limited.
  • the conditions are exemplified below, but the artificial match-type miRNA of the present invention is not limited to these descriptions.
  • the numerical range of bases discloses all positive integers belonging to the range.
  • the description “1 to 4 bases” means “1, 2, 3, 4 bases”. (Hereinafter the same).
  • the length of the guide strand sequence is not particularly limited, and for example, the length of the guide strand sequence in the reported mature miRNA can be exemplified.
  • the lower limit is, for example, 19 base lengths and 20 base lengths
  • the upper limit is, for example, 25 base lengths, 24 base lengths
  • the range is, for example, 19-25 base lengths, 20-24 base lengths It is.
  • the length of the additional sequence is not particularly limited, and the lower limit is, for example, 0 base length, 1 base length, 2 base lengths, and the upper limit is, for example, 5 bases Length, 4 base length, 3 base length, and the range is, for example, 0 to 5 base length, 1 to 4 base length, 2 to 3 base length.
  • the length of the X region is not particularly limited, and the lower limit is, for example, 19 base length, 21 base length, 23 base length, and the upper limit is, for example, 33 base length, 30 base length, 28 base length, 26
  • the range is, for example, 19-33 base length, 19-30 base length, 21-28 base length, 23-26 base length.
  • the length of the overhang is not particularly limited, and the lower limit is, for example, 0 base length, 1 base length, and the upper limit is, for example, 4 base length, 3 bases
  • the range is, for example, 0 to 4 bases long, 1 to 3 bases long, 2 bases long.
  • the arrangement of the overhang is not particularly limited, and examples thereof include UU, CU, GC, UA, AA, CC, UG, CG, AU, and TT from the 3 'side.
  • the overhang is TT, resistance to RNase can be added.
  • the length of the Y region is not particularly limited, and the lower limit is, for example, 19 base length, 21 base length, 23 base length, and the upper limit is, for example, 35 base length, 32 base length, 30 base length, 28
  • the range is, for example, 19-35 base length, 21-35 base length, 19-32 base length, 21-30 base length, 23-28 base length.
  • the total length (T) of the artificial match-type miRNA of the present invention is not particularly limited, and the lower limit is, for example, 38 base length, 42 base length, 46 base length, and the upper limit is, for example, 62 base length, 58 base length. 54 base length, and the range is, for example, 38 to 62 base length, 42 to 58 base length, 46 to 54 base length.
  • the mature miRNA may be, for example, a mature miRNA of hsa-miR-34a (SEQ ID NO: 1) as hsa-miR-34.
  • hsa-miR-34a (SEQ ID NO: 1) UGGCAGUGUCUUAGCUGGUUGU
  • the guide chain of miR-34a targets, for example, AXL, MET, CDK4, CDK6, SIRT1, CCND1, SIRT1, BCL-2 and the like. By suppressing the expression of these target genes, for example, cancer (lung cancer, colon) Diseases such as cancer, stomach cancer, liver cancer, breast cancer, etc.).
  • examples of the polynucleotide of the artificial match-type miRNA include at least one base sequence selected from the group consisting of SEQ ID NOs: 5 and 13-23.
  • the structural unit of the artificial match-type miRNA 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 is preferably, for example, a ribonucleotide residue.
  • the nucleotide residue include an unmodified unmodified nucleotide residue and a modified modified nucleotide residue.
  • the artificial match-type miRNA of the present invention can improve nuclease resistance and stability, for example, by including the modified nucleotide residue.
  • the artificial match-type miRNA of the present invention may further include a non-nucleotide residue in addition to the nucleotide residue, for example.
  • the number of the modified ribonucleotide residue is not particularly limited.
  • “1 Specifically, the number is, 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 artificial match-type miRNA of the present invention includes, for example, the deoxyribonucleotide residue in addition to the unmodified ribonucleotide residue
  • the number of the deoxyribonucleotide residue is not particularly limited. Specifically, for example, 1 to 5, preferably 1 to 4, more preferably 1 to 3, and most preferably 1 or 2.
  • the nucleotide residue includes, for example, a sugar, a base and a phosphate as constituent elements.
  • the ribonucleotide residue has, for example, a ribose residue as a sugar, and has adenine (A), guanine (G), cytosine (C), and uracil (U) as bases
  • the 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.
  • each component is the same or substantially the same as, for example, naturally occurring, specifically, for example, the same or substantially the same as that naturally occurring in the human body. Are identical.
  • the modified nucleotide residue may be modified, for example, with any component of the unmodified nucleotide residue.
  • Examples of the modified nucleotide residue include naturally occurring nucleotide residues, artificially modified nucleotide residues, and the like.
  • the modified nucleotide residue may be, for example, a residue of a substitute for the unmodified nucleotide.
  • 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 the like.
  • 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.
  • the non-nucleotide structure is represented, for example, by the following formula (I).
  • 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 X region and the Y region are each bonded to the non-nucleotide structure 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 as 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.
  • TBDMS tert-butyldimethylsilyl group
  • ACE (2-acetoxyethyloxy) methyl group
  • TOM triisopropylsilyloxymethyl group
  • CEE 2-Cyanoethoxymethyl group
  • CEM 2-cyanoethoxymethyl group
  • TEM dimethoxytrityl group
  • DMTr dimethoxytrityl group
  • R 3 is OR 4
  • the protecting group is not particularly
  • 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 non-nucleotide structure, 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 X region and the Y region are bonded to the non-nucleotide structure via, for example, —OR 1 — or —OR 2 —, respectively.
  • 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 non-nucleotide structure is, for example, the non-nucleotide residue consisting of the structure of the formula (I) except the nucleotide residue R 1 and / or R 2 , And the nucleotide residues.
  • the non-nucleotide structure is, for example, a structure in which two or more of the non-nucleotide residues having the structure of the formula (I) are linked. Become.
  • 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 non-nucleotide structure is formed only from the non-nucleotide residue consisting of the structure of the formula (I), for example.
  • the combination of the bond between the X region and the Y region and —OR 1 — and —OR 2 — is not particularly limited, and examples thereof include any of the following conditions.
  • Condition (1) The X region is bonded to the structure of the formula (I) through —OR 2 —, and the Y region is bonded through —OR 1 —.
  • Condition (2) The X region is bonded to the structure of the formula (I) through —OR 1 — and the Y region is bonded through —OR 2 —.
  • 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 artificial match-type miRNA of the present invention may contain, for example, a labeling substance and may 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 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 artificial match-type miRNA of the present invention can suppress the expression of the target gene. Therefore, the artificial match-type miRNA of the present invention can be used as a therapeutic agent for diseases caused by genes, for example.
  • the disease can be treated, for example, by suppressing the expression of the target gene.
  • “treatment” includes, for example, the meanings of preventing the disease, improving the disease, and improving the prognosis.
  • the disease is not particularly limited, and for example, the expression suppression sequence can be appropriately set according to the target disease. Examples of the disease include cancer (breast cancer, lung cancer, stomach cancer, colon cancer, liver cancer).
  • the method for using the artificial match-type miRNA of the present invention is not particularly limited, and for example, the artificial match-type miRNA may be administered to an administration subject having the target gene.
  • Examples of the administration subject include cells, tissues, and 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. For example, various cells such as NCI-H1299 cells, HeLa cells, 293 cells, NIH3T3 cells, and COS cells, stem cells such as ES cells and hematopoietic stem cells, and single cells from a living body such as primary culture cells. For example, separated cells.
  • the target gene to be subject to expression suppression is not particularly limited, and a desired gene can be set.
  • composition of the present invention expression suppression method, treatment method and the like described later can be referred to.
  • the artificial match-type miRNA of the present invention can suppress the expression of a target gene as described above, it is useful, for example, as a research tool for pharmaceuticals, diagnostic agents, agricultural chemicals, agriculture, medicine, life sciences and the like. is there.
  • the method for synthesizing the artificial match-type miRNA of the present invention is not particularly limited, and conventionally known nucleic acid production methods can be employed.
  • the synthesis method include a synthesis method using a genetic engineering technique, a chemical synthesis method, and the like.
  • 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. In the chemical synthesis method, for example, a commercially available automatic nucleic acid synthesizer can be used.
  • amidite is generally used.
  • the amidite is not particularly limited, and examples of commercially available amidites include RNA Phosphoramidates (2′-O-TBDMSi, trade name, Michisato Pharmaceutical), ACE amidite, TOM amidite, CEE amidite, CEM amidite, TEM amidite, and the like. Can be given.
  • the composition for suppressing expression of the present invention is a composition for suppressing the expression of a target gene, and includes the artificial match-type miRNA of the present invention. .
  • the composition of the present invention is characterized by including the artificial match-type miRNA 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.
  • expression of the target gene can be suppressed by administration to a subject in which the target gene exists.
  • the pharmaceutical composition of the present invention is characterized by containing the artificial match-type miRNA of the present invention.
  • the composition of the present invention is characterized by containing the artificial match-type miRNA 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.
  • treatment includes, for example, the meanings of prevention of the above-mentioned diseases, improvement of the diseases, and improvement of the prognosis.
  • a disease to be treated is not particularly limited, and examples thereof include diseases caused by expression of a target gene of a mature miRNA of hsa-miR-34.
  • the gene causing the disease is set as the target gene, and further, the guide strand sequence of the mature miRNA of hsa-miR-34 may be selected according to the target gene. .
  • composition for suppressing expression and the pharmaceutical composition (hereinafter referred to as composition) of the present invention
  • the method for using the composition for suppressing expression and the pharmaceutical composition (hereinafter referred to as composition) of the present invention is not particularly limited.
  • the artificial match-type miRNA is administered to an administration subject having the target gene. do it.
  • Examples of the administration subject include cells, tissues, and 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. For example, various cells such as NCI-H1299 cells, HeLa cells, 293 cells, NIH3T3 cells, and COS cells, stem cells such as ES cells and hematopoietic stem cells, and single cells from a living body such as primary culture cells. For example, separated cells.
  • 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 artificial match-type miRNA of the present invention, or may further 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 artificial match miRNA 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 artificial match miRNA can be efficiently delivered.
  • 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 a target gene, characterized by using the artificial match-type miRNA of the present invention.
  • the expression suppression method of the present invention is characterized by using the artificial match-type miRNA of the present invention, and other steps and conditions are not limited at all.
  • the mechanism for suppressing the expression of the target gene is not particularly limited, and examples thereof include expression suppression by mature miRNA.
  • the expression suppression method of the present invention includes, for example, a step of administering the artificial match-type miRNA to a subject in which the target gene is present.
  • the artificial match type miRNA 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 artificial match-type miRNA may be administered alone, or the composition of the present invention containing the artificial match-type miRNA 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 treatment method of the disease of the present invention comprises the step of administering the artificial match-type miRNA of the present invention to a patient, wherein the disease comprises the mature miRNA of hsa-miR-34. It is a disease involving a target gene.
  • the treatment method of the present invention is characterized by using the artificial match-type miRNA of the present invention, and other steps and conditions are not limited at all.
  • 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 use of the present invention is use of the artificial match-type miRNA of the present invention for suppressing expression of the target gene.
  • the single-stranded nucleic acid of the present invention is a single-stranded nucleic acid for use in the treatment of a disease, wherein the single-stranded nucleic acid is the artificial match-type miRNA of the present invention, and the disease is hsa-miR. It is characterized by a disease involving a target gene of -34 mature miRNA.
  • Example 1 Based on the guide strand of mature miR-34a, the artificial match-type miRNA of the present invention was synthesized, and suppression of proliferation of H1299 cells was confirmed.
  • RNA As a positive control miRNA, human mature miR-34a comprising the following guide strand (SEQ ID NO: 1) and passenger strand (SEQ ID NO: 2) was synthesized. As a negative control, a mature miR-34a scramble was synthesized comprising a scrambled guide strand (SEQ ID NO: 3) with the base composition of the guide strand scrambled and a corresponding passenger strand (SEQ ID NO: 4).
  • an X region composed of the guide strand (SEQ ID NO: 1) and an additional sequence, and a Y region composed of a sequence completely complementary to the X region and an overhang are represented by the following formulae: Matched miR-34a linked via the non-nucleotide structure of the proline derivative (represented by [P] in the sequence) was synthesized.
  • the underlined portion corresponds to the guide strand.
  • the non-nucleotide structure in the matched miRNA is represented by the following formula, and was introduced by using L-proline diamide amidite (see WO2012 / 017919) in the synthesis of the matched miRNA.
  • a match-type miR-34a scramble comprising a guide strand in which the base composition of the guide strand was scrambled and a corresponding passenger strand was synthesized.
  • Mature miR-34a Guide strand (SEQ ID NO: 1) 5'- UGGCAGUGUCUUAGCUGGUUGU -3 ' Passenger strand (SEQ ID NO: 2) 5'-CAAUCAGCAAGUAUACUGCCCU-3 ' Mature miR-34a scrambled guide strand (SEQ ID NO: 3) 5'- UGUAUCGUUAUCGGGUCGGUUG -3 ' Passenger strand (SEQ ID NO: 4) 5'-CAACCGACCCGAUAACGAUACA-3 ' Matched miR-34a (SEQ ID NO: 5) 5'- UGGCAGUGUCUUAGCUGGUUGU UCC- [P] -GGAACAACCAGCUAAGACACUGCCAUA-3 ' Matched miR-34a scramble (SEQ ID NO: 6) 5'- UGUAUCGUUAUCGGGUCG
  • the cells were cultured in the above-mentioned medium, and the culture solution was dispensed into a 24-well plate at 500 ⁇ L at 1 ⁇ 10 4 cells / well. Furthermore, after culturing the cells in the well for 24 hours, the miRNA was transfected using a transfection reagent RNAi MAX Transfection Reagent (trade name, Life Technologies) according to the attached protocol.
  • the composition per well was set as follows. In the following composition, (B) is Opti-MEM (trade name, Invitrogen), (C) is the RNA solution, and 49 ⁇ L of both was added. In the well, the final concentration of the miRNA was 100 nmol / L. After transfection, the cells in the wells were cultured for 3 days. And after the culture
  • FIG. 2 is a graph showing the number of cells per well.
  • “Normal” is an untreated cell
  • “Mock” is a cell into which only a transfection reagent is introduced
  • “Scramble” is a miR-34a scramble of a negative control
  • “miR-34a” is a positive control.
  • the mature miR-34a, “Scramble match” indicates the negative control match-type miR-34a scramble, and “miR-34a match” indicates the result of the match-type miR-34a of the example (hereinafter the same).
  • the match type miR-34a of the example was able to reduce the number of cells most.
  • FIG. 3 is a graph showing relative values of cell proliferation. As shown in FIG. 3, the match type miR-34a of the example was able to reduce the number of cells most.
  • FIG. 4 is a graph showing early apoptosis (%) and late apoptosis (%). As shown in FIG. 4, the match-type miR-34a of the example was able to enhance apoptosis most.
  • reverse transcriptase (trade name: M-MLV reverse transcriptase, Invitrogen) was used to synthesize cDNA from the RNA according to the attached protocol. Then, quantitative PCR was performed using the synthesized cDNA as a template, and the amounts of AXL cDNA and MET cDNA were measured. GAPDH cDNA was used as an internal control, and the amount of the cDNA was measured together.
  • FIG. 5 (A) shows the result of AXL mRNA
  • FIG. 5 (B) shows the result of MET mRNA.
  • the match-type miR-34a of the example showed a decrease in the amount of AXL mRNA to the same extent as the mature miR-34a of the positive control. Further, the amount of MET mRNA was most decreased in the match type miR-34a of the example. For this reason, it can be said that the transcription
  • the match type miR-34a of the example suppresses the expression of AXL mRNA, MET mRNA, and the like, and can suppress the proliferation of H1299 cells and enhance apoptosis.
  • the artificial match-type miRNA is a single-stranded nucleic acid molecule, so that it is not necessary to anneal each single strand during use, and TLR3 involved in innate immunity. It is also possible to avoid being recognized.
  • Example 2 For the match type miR-34a of Example 1, the additional sequence in the X region and the overhang in the Y region were shortened.
  • match-type miR-34a has a 3 base-long additional sequence (J) surrounded by a square on the 3 ′ side of the X region, and on the 5 ′ side of the Y region. It has an overhang (O) with a length of 2 bases surrounded by a square. Therefore, a molecule in which the additional sequence is deleted by one base from the 3 ′ side and the corresponding Y region side sequence is deleted by one base from the 5 ′ side, and the overhang is deleted by one base from the 3 ′ side.
  • a lost molecule and a molecule in which the additional sequence and the overhang were deleted one by one were synthesized, and in the same manner as in Example 1, suppression of expression of AXL mRNA and MET mRNA was confirmed.
  • the 5 ′ side region of [P] is the X region, and in the X region, the underlined portion is the guide strand sequence, and the other is the additional sequence, and the 3 ′ side region of [P] Is a Y region, and in the Y region, a region surrounded by a square is an overhang.
  • FIG. 6 shows the results of AXL mRNA
  • FIG. 7 shows the results of MET mRNA.
  • the artificial match-type miRNA of the present invention can be easily synthesized at low cost, and the translation of the protein encoded by the gene can be suppressed. Since the artificial match-type miRNA of the present invention can suppress the expression of a target gene as described above, it is useful, for example, as a research tool for pharmaceuticals, diagnostic agents, agricultural chemicals, agriculture, medicine, life sciences, and the like. .
  • This application is based on Japanese Patent Application No. 2013-273034 filed in Japan (filing date: December 27, 2013), the contents of which are incorporated in full herein.

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Abstract

La présente invention concerne un micro-ARN de type à correspondance artificielle qui est un acide nucléique monocaténaire comprenant une région X et une région Y et qui est caractérisé en ce que : l'extrémité 3' de la région X et l'extrémité 5' de la région Y sont reliées par l'intermédiaire d'une structure non nucléotidique qui comprend un squelette pyrrolidine et/ou un squelette pipéridine ; la région X comprend une séquence de brin guide de micro-ARN mature hsa-miR-34 ; et la région Y comprend une séquence qui est parfaitement complémentaire de la région X. Ce micro-ARN de type à correspondance artificielle permet d'inhiber l'expression d'un gène cible.
PCT/JP2014/084725 2013-12-27 2014-12-27 Micro-arn de type à correspondance artificielle pour le contrôle de l'expression génétique et son utilisation WO2015099188A1 (fr)

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JPWO2015099187A1 (ja) * 2013-12-27 2017-03-23 株式会社ボナック 遺伝子発現制御のための人工マッチ型ｍｉＲＮＡおよびその用途
US10612020B2 (en) 2013-12-26 2020-04-07 Tokyo Medical University Artificial mimic miRNA for controlling gene expression, and use of same
US11027023B2 (en) 2014-12-27 2021-06-08 Bonac Corporation Natural type miRNA for controlling gene expression, and use of same
US11142769B2 (en) 2015-03-27 2021-10-12 Bonac Corporation Single-stranded nucleic acid molecule having delivery function and gene expression regulating ability

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JP2008519606A (ja) * 2004-11-12 2008-06-12 アンビオン インコーポレーティッド ｍｉＲＮＡおよびｍｉＲＮＡ阻害分子に関する方法および組成物
WO2012017919A1 (fr) * 2010-08-03 2012-02-09 株式会社ボナック Molécule d'acide nucléique simple brin ayant un squelette alicyclique contenant de l'azote
WO2012106591A1 (fr) * 2011-02-03 2012-08-09 Mirna Therapeutics, Inc. Mimétiques synthétiques de mir-34
WO2013103146A1 (fr) * 2012-01-07 2013-07-11 株式会社ボナック Molécule d'acide nucléique simple brin ayant un squelette d'acides aminés
JP2013153736A (ja) * 2012-01-07 2013-08-15 Bonac Corp ペプチド骨格を有する一本鎖核酸分子

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JP2008519606A (ja) * 2004-11-12 2008-06-12 アンビオン インコーポレーティッド ｍｉＲＮＡおよびｍｉＲＮＡ阻害分子に関する方法および組成物
WO2012017919A1 (fr) * 2010-08-03 2012-02-09 株式会社ボナック Molécule d'acide nucléique simple brin ayant un squelette alicyclique contenant de l'azote
WO2012106591A1 (fr) * 2011-02-03 2012-08-09 Mirna Therapeutics, Inc. Mimétiques synthétiques de mir-34
WO2013103146A1 (fr) * 2012-01-07 2013-07-11 株式会社ボナック Molécule d'acide nucléique simple brin ayant un squelette d'acides aminés
JP2013153736A (ja) * 2012-01-07 2013-08-15 Bonac Corp ペプチド骨格を有する一本鎖核酸分子

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10612020B2 (en) 2013-12-26 2020-04-07 Tokyo Medical University Artificial mimic miRNA for controlling gene expression, and use of same
JPWO2015099187A1 (ja) * 2013-12-27 2017-03-23 株式会社ボナック 遺伝子発現制御のための人工マッチ型ｍｉＲＮＡおよびその用途
JP2018145199A (ja) * 2013-12-27 2018-09-20 株式会社ボナック 遺伝子発現制御のための人工マッチ型ｍｉＲＮＡおよびその用途
US10934542B2 (en) 2013-12-27 2021-03-02 Bonac Corporation Artificial match-type miRNA for controlling gene expression and use therefor
US11027023B2 (en) 2014-12-27 2021-06-08 Bonac Corporation Natural type miRNA for controlling gene expression, and use of same
US11142769B2 (en) 2015-03-27 2021-10-12 Bonac Corporation Single-stranded nucleic acid molecule having delivery function and gene expression regulating ability

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