WO2020218494A1 - MODIFIED miR302 NUCLEIC ACID - Google Patents

MODIFIED miR302 NUCLEIC ACID Download PDF

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WO2020218494A1
WO2020218494A1 PCT/JP2020/017659 JP2020017659W WO2020218494A1 WO 2020218494 A1 WO2020218494 A1 WO 2020218494A1 JP 2020017659 W JP2020017659 W JP 2020017659W WO 2020218494 A1 WO2020218494 A1 WO 2020218494A1
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nucleic acid
acid variant
seq
mir302
ribose
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PCT/JP2020/017659
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French (fr)
Japanese (ja)
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秀始 石井
雅允 今野
聡 小比賀
森　正樹
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国立大学法人大阪大学
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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
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
    • 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
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • 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

Definitions

  • the present disclosure relates to miR302 nucleic acid variants and their use. More specifically, it relates to a human miR302a nucleic acid variant and its use.
  • the miR302 nucleic acid variants of the present disclosure can be used in the treatment of diseases such as cancer.
  • Non-Patent Document 1 ESC-related transcription factors such as Nanog, Oct3/4, Sox2 and Rex1 are miR302-367 promoters. It is expected to be an upstream regulator of, and miR302 thus regulated is thought to influence a variety of biological events.
  • the miR302 nucleic acid variant can be used for the treatment of diseases such as cancer, and have reached the present disclosure.
  • a miR302 nucleic acid variant and a composition containing the same are provided, and their use in the treatment of diseases such as cancer is also provided.
  • the miR302 nucleic acid variants of the present disclosure can be stable, for example in plasma, and can be effectively used in administration regimens with low subject load.
  • the present disclosure provides: (Item 1) Human miR302a nucleic acid variant.
  • a nucleic acid variant of any of the above items comprising a base sequence containing 0, 1, 2, 3, 4 or 5 substitutions, deletions or insertions from the base sequence of.
  • At least one of the 8th and 18th bases of UAAGUGCUUCCAUGUUUUGGUGA (g1) (SEQ ID NO: 2), or Anucleic acid variant of any of the above items, comprising a base sequence comprising a base substitution, deletion or insertion at a position corresponding to at least one of the 4th and 14th bases of ACUUAAACGUGGAUGUACUUGCU (p1) (SEQ ID NO: 3).
  • UAAGUGCTUCCAUGUTUTGGUTGA (SEQ ID NO: 4) has the same base sequence other than the 8th and 18th bases, or A nucleic acid variant of any of the above items, which comprises a base sequence having the same base other than the 4th and 14th bases of ACCAAAACAUGGAAGCACUUACT (p10) (SEQ ID NO: 5).
  • (Item 7) A nucleic acid variant of any of the above items, wherein R x is hydrogen, methyl, ethyl, n-propyl, isopropyl, or benzyl, respectively.
  • (Item 8) U ⁇ A (M) A ⁇ G (M) U ⁇ G (M) C ⁇ T (M) U ⁇ C (M) C ⁇ A (M) U ⁇ G (M) U ⁇ T (M) U ⁇ T (M) G ⁇ G (M) U ⁇ T (M) G (L) ⁇ A (L) or A (L) ⁇ C (L) ⁇ C ⁇ A ⁇ A ⁇ A ⁇ A ⁇ A ⁇ C ⁇ A ⁇ U ⁇ G ⁇ A ⁇ A ⁇ G ⁇ C ⁇ A ⁇ C ⁇ U ⁇ U ⁇ A ⁇ C ( L) ⁇ T (L) Structure (in the formula, U stands for uracil, T stands for thymine, A stands
  • (L) is the ribose in the ribonucleotide unit to the left of this symbol Represents a modification that has been replaced by ⁇ Represents a modification in which one oxygen atom that is not involved in the bond of the phosphate group between the adjacent ribonucleotide units is replaced with a sulfur atom)
  • (Item 11) A double strand formed by a nucleic acid variant of any of the above items, which is a guide strand, and a nucleic acid variant of any of the above items, which is a passenger strand.
  • (Item 12) A nucleic acid variant molecule comprising a portion of a nucleic acid variant of any of the above items which is a guide portion and a portion of a nucleic acid variant of any of the above items which is a passenger portion.
  • (Item 13) A nucleic acid variant molecule of any of the above items, which has a sequence that can be degraded in vivo.
  • a nucleic acid variant of any of the above items which produces lower luciferase luminescence than natural human miR302a when contacted with HT29 cells introduced by lentivirus with a nucleic acid construct in which the luciferase gene and the HnRNPA2B1 gene are bound.
  • Degradation rate when contacted with fetal bovine serum (FBS) in vitro is slower than that of natural human miR302a, nucleic acid variant of any of the above items, double strand of any of the above items, or any of the above items. Nucleic acid variant molecule.
  • a pharmaceutical composition for treating cancer comprising a nucleic acid variant of any of the above items, a double strand of any of the above items, or a nucleic acid variant of any of the above items.
  • a method for treating cancer comprising administering to a subject a nucleic acid variant of any of the above items, a double strand of any of the above items, or a nucleic acid variant molecule of any of the above items.
  • This disclosure is useful for the treatment of diseases such as cancer.
  • the result of the primary screening is shown.
  • the height of the bar indicates the relative luminescence intensity of luciferase observed in each guide chain-passenger chain pair when the result in the G1P1 pair (denoted as miR302 in the figure) is 1.
  • Each bar shows the results in any combination of any of the 19 guide chains and any of the 20 passenger chains.
  • Each bar is only arranged based on emission intensity, and the horizontal or vertical axis does not refer to a particular guide chain or passenger chain. For example, other bars in the same column or row as the G1P1 pair of bars do not necessarily result in a pair containing G1 or P1.
  • the effect of treatment with native miR302 (G1P1 pair) on Panc-1 cells is shown.
  • the graph on the left shows the results on the 4th day, and the graph on the right shows the results on the 7th day.
  • the vertical axis shows the relative DAPI staining area of the G1P1 pair when the control result is 1.
  • the difference in stained area between the G1P1 pair and the control was a statistically significant difference (P ⁇ 0.01).
  • the effect of treatment with miR302 nucleic acid variant or native miR302 (G1P1 pair) on MIA-PaCa cells is shown.
  • the vertical axis shows the relative DAPI staining area of each guide chain-passenger chain pair when the result of the G1P1 pair on the 4th day is 1.
  • the horizontal axis shows the types of guide-passenger pairs, for each pair the left bar shows the results for day 4 and the bar on the right shows the results for day 7. * Indicates that there is a statistically significant difference compared to the results of the G1P1 pair.
  • the effect of treatment with miR302 nucleic acid variant or native miR302 (G1P1 pair) on Panc-1 cells is shown.
  • the vertical axis shows the relative DAPI staining area of each guide-passenger pair when the result of the G1P1 pair on the 4th day is 1.
  • the horizontal axis shows the types of guide-passenger pairs, for each pair the left bar shows the results for day 4 and the bar on the right shows the results for day 7.
  • the change in body weight over time when a single dose of G1P1 pair, OUM302 or vehicle is administered to mice is shown.
  • the vertical axis shows the body weight (g).
  • the horizontal axis shows the number of days with the day of administration as the 0th day. Circled markers indicate vehicle administration group, triangle markers indicate OUM302 administration group, and square markers indicate G1P1 pair administration group. It shows the change in tumor volume over time when a single dose of G1P1 pair, OUM302 or vehicle is administered to mice.
  • the vertical axis shows the tumor volume (mm 3 ).
  • the horizontal axis shows the number of days with the day of administration as the 0th day.
  • Circled markers indicate vehicle administration group, triangle markers indicate OUM302 administration group, and square markers indicate G1P1 pair administration group. It shows the change in body weight over time when a single dose of G1P1 pair or OUM302 in combination with gemcitabine was administered to mice.
  • the vertical axis shows the body weight (g).
  • the horizontal axis shows the number of days with the day of administration as the 0th day.
  • the circle markers indicate the OUM302 + gemcitabine administration group, and the triangular markers indicate the G1P1 pair + gemcitabine administration group. It shows the change in tumor volume over time when a single dose of G1P1 pair or OUM302 in combination with gemcitabine was administered to mice.
  • the vertical axis shows the tumor volume (mm 3 ).
  • the horizontal axis shows the number of days with the day of administration as the 0th day.
  • the circle markers indicate the OUM302 + gemcitabine administration group, and the triangular markers indicate the G1P1 pair + gemcitabine administration group. It shows the change in body weight over time when a single dose of G1P1 pair or OUM302 in combination with 5FU (5-fluorouracil) was administered to mice.
  • the vertical axis shows the body weight (g).
  • the horizontal axis shows the number of days with the day of administration as the 0th day. Circled markers indicate the OUM302 + 5FU administration group, and triangular markers indicate the G1P1 vs. + 5FU administration group.
  • the vertical axis shows the tumor volume (mm 3 ).
  • the horizontal axis shows the number of days with the day of administration as the 0th day. Circled markers indicate the OUM302 + 5FU administration group, and triangular markers indicate the G1P1 vs. + 5FU administration group.
  • the change in body weight over time when a single dose of G1P1 pair or OUM302 in combination with TAS is shown in mice.
  • the vertical axis shows the body weight (g).
  • the horizontal axis shows the number of days with the day of administration as the 0th day.
  • Circled markers indicate the OUM302 + TAS administration group, and triangular markers indicate the G1P1 pair + TAS administration group. It shows the change in tumor volume over time when a single dose of G1P1 pair or OUM302 in combination with TAS (trifluridine / tipiracil hydrochloride, Ronsurf®) is administered to mice.
  • the vertical axis shows the tumor volume (mm 3 ).
  • the horizontal axis shows the number of days with the day of administration as the 0th day.
  • Circled markers indicate the OUM302 + TAS administration group, and triangular markers indicate the G1P1 pair + TAS administration group.
  • the vertical axis shows the tumor volume (mm 3 ).
  • the horizontal axis shows the number of days with the day of administration as the 0th day.
  • the x marker indicates the control treatment group
  • the square marker indicates the TAS single administration group
  • the circle marker indicates the OUM302 + TAS administration group
  • the triangular marker indicates the G1P1 pair + TAS administration group.
  • the vertical axis shows the body weight (g).
  • the horizontal axis shows the number of days with the day of administration as the 0th day.
  • the x marker indicates the vehicle administration group
  • the square marker indicates the G1P1 vs. single administration group
  • the circle marker indicates the OUM302 alone administration group
  • the triangular marker indicates the OUM302 + TAS administration group
  • the G1P1 vs + TAS administration group the G1P1 vs + TAS administration group.
  • the tumor weight 32 days after administration of vehicle, G1P1 pair alone or OUM302 alone to spontaneously developing pancreatic cancer mice is shown.
  • the vehicle administration group, the G1P1 administration group, and the OUM302 administration group are shown from the left, and in the right graph, the G1P1 administration group is shown on the left and the OUM302 administration group is shown on the right.
  • the graph on the right is an enlargement of the G1P1 administration group and the OUM302 administration group in the graph on the left.
  • the vertical axis shows the tumor weight (mg).
  • An electrophoretic image of four types of nucleic acids treated with FBS with 20% acrylamide / bisgel is shown.
  • Both ends are the result of 20bp DNA ladder (Takara Bio, Shiga). From the left, a set of processing results of g1, g9, p1, and p10 is shown. In each set, the results of no FBS addition, 15-minute treatment, 30-minute treatment, 1-hour treatment, 3-hour treatment, and 24-hour treatment are shown from the left. An electrophoretic image of two FBS-treated nucleic acid pairs on a 2% agarose gel (left) or 20% acrylamide / bisgel (right) is shown. Both ends are the result of 20bp DNA ladder (Takara Bio, Shiga). The left shows a set of processing results of g1p1 pair and the right shows a set of processing results of g9p10 pair. In each set, the left shows the result of FBS addition and the right shows the result of FBS 1 hour treatment.
  • nucleic acid is used in the usual sense as used in the art, and “modification” as used herein means substitution, deletion or insertion of a base, or a chemical structure other than a base (eg, for example. Refers to any modification of cross-linked nucleic acid (BNA), locked nucleic acid (LNA), modified nucleic acid such as AmNA). In the present specification, a substance containing at least one such modification is referred to as a variant.
  • nucleic acid variant refers to a substance in which an arbitrary modification (for example, modification of a base, modification of a chemical structure, etc.) is introduced into a naturally occurring nucleic acid.
  • miR302 nucleic acid variant refers to a substance in which any modification (eg, base modification, chemical structure modification (eg, introduction of modified nucleic acid), etc.) has been introduced into naturally occurring miR302.
  • nucleic acid variant refers to a functional part different from the modified (for example, detection of nucleic acid variant, tissue targeting, stability improvement and / or binding to other molecules. It is intended that substances into which (parts that promote) have been introduced are also included.
  • modified nucleic acid refers to a nucleic acid modified so as to have a structure different from that of a normal nucleic acid used in DNA or RNA.
  • modified nucleic acids include ethylene nucleic acids such as locked nucleic acids (LNA), 2'-O, 4'-C-ethylene bridged nucleic acids (2'-O, 4'-C-ethylene bridged nucleic acid, ENA), and others.
  • Bridged nucleic acid bridged nucleic acid, BNA
  • hexitol nucleic acid hexitol nucleic acid, HNA
  • amide bridged nucleic acid Amido-bridged nucleic acid, AmNA
  • morpholino nucleic acid tricyclo-DNA (tcDNA)
  • polyether nucleic acid for example, US Pat. No. 5,908,845
  • cyclohexene nucleic acid CeNA
  • BNA is said to mean Bicyclic Nucleic Acid as well as Bridged Nucleic Acid, and is also called “bridged nucleic acid", "bicyclic nucleic acid” or “bridged / bicyclic nucleic acid”.
  • the bicyclic nucleic acid refers to an arbitrary modified nucleic acid in which the 2'position and the 4'position of the nucleic acid are linked (crosslinked) to form two ring structures (bicyclic).
  • modified nucleic acids are known in the art, for example, Japanese Patent No. 4731324, Pradeep S. Pallan et al., Chem Communi (Camb). 2012 August 25; 48 (66): 8195-8197. Doi: 10.1039 / c2cc32286b
  • the crosslinked nucleic acid described in the above can be used.
  • substitution of a base means changing one base (A, G, C, T, U or a derivative thereof) to another base.
  • the term “deletion” of a base means changing a base sequence to a base sequence from which the target base has been removed.
  • the term “insertion” of a base means changing a base sequence to a base sequence to which a target base has been added.
  • deletion of a modification means to change a variant into which the target modification has been introduced to a substance in which the modification has not been introduced. For example, when this modification is deleted from a variant (AAAAA) in which a modification that deletes G from the base sequence AAAGAA is introduced, the base sequence of AAAGAA is given. In another example, from a variant that has introduced a modification that replaces ribose with deoxyribose, removing this modification gives a substance in which this deoxyribose of the variant is replaced with ribose.
  • complex or “complex molecule” means any construct that includes two or more moieties.
  • one part is a nucleic acid
  • the other part may be a nucleic acid, with other substances (eg, polypeptides, substrates, sugars, lipids, nucleic acids, other hydrocarbons, etc.).
  • two or more portions constituting the complex may be bonded by a covalent bond or other bonds (for example, hydrogen bond, ionic bond, hydrophobic interaction, van der Waals force, etc.). It may have been done.
  • substituted refers to an atom or functional group in which one chemical group is substituted with another in the chemical structure of a nucleic acid variant.
  • base in the present specification means a monovalent group.
  • alkyl group means a monovalent saturated hydrocarbon group.
  • group may be omitted in the description of the substituent in the present specification.
  • the number of substituents in the groups defined by “substitutable or unsubstituted”, “may be substituted”, or “substituted” is not particularly limited as long as it can be substituted, and may be one or more. Is. Unless otherwise indicated, the description of each group also applies when the group is part of another group or a substituent.
  • substituted or unsubstituted is used interchangeably with “may be substituted”, and a specific group may be further substituted or substituted and has no substituent. It means that it is also good.
  • Substituents that can be used in the miRNA or nucleic acid variants used herein include alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, alkoxy, carbocyclic group, heterocyclic group, halogen, hydroxy, Examples include, but are not limited to, thiols, cyanos, nitros, aminos, carboxys, carbamoyls, acyls, acylaminos, thiocarboxys, amides, substituted carbonyls, substituted thiocarbonyls, substituted sulfonyls or substituted sulfinyls. All of the substituents may have a substituent other than hydrogen.
  • the substitution shall replace one or more hydrogen atoms in an organic compound or substituent with another atom or atomic group, or shall be a double bond or a triple bond.
  • the substitution shall replace one hydrogen atom and replace it with a monovalent substituent, or combine it with a single bond to form a double bond, and remove two hydrogen atoms to make a divalent substituent. It can be substituted or combined with a single bond to form a triple bond.
  • alkyl (group) refers to a monovalent group generated by the loss of one hydrogen atom from an aliphatic hydrocarbon (alkane) such as methane, ethane, or propane, and generally means C n H 2n + 1. It is represented by-(where n is a positive integer).
  • Alkyl can be straight or branched.
  • the alkyl (group) may be substituted or unsubstituted (unsubstituted).
  • specific examples of alkyl are C 1 to C 2 alkyl, C 1 to C 3 alkyl, C 1 to C 4 alkyl, C 1 to C 5 alkyl, C 1 to C 6 alkyl, and C 1 to C 7 alkyl.
  • C 1 to C 10 alkyl means a linear or branched alkyl having 1 to 10 carbon atoms.
  • substituted alkyl refers to an alkyl in which the H of the alkyl is substituted by the substituents specified herein.
  • CH 3 OCH 2- , CH 3 OCH 2 CH 2- , CH 3 OCH 2 CH 2 CH 2- , HOCH 2- , HOCH 2 CH 2- , HOCH 2 CH 2 CH 2- , NCCH 2- , NCCH 2 CH 2- , NCCH 2 CH 2 CH 2- , FCH 2- , FCH 2 CH 2- , FCH 2 CH 2- CH 2- , H 2 NCH 2- , H 2 NCH 2 CH 2- , H 2 NCH 2 CH 2 CH 2- , HOOCCH 2- , HOOCCH 2 CH 2- , HOOCCH 2 CH 2 CH 2- , C 6 H 5- CH 2- (benzyl group) can be mentioned. ..
  • alkenyl refers to a monovalent group produced by the loss of one hydrogen atom from an aliphatic hydrocarbon having one double bond in the molecule, and is generally C n H 2n-1- . Represented (where n is a positive integer greater than or equal to 2). "Substituted alkenyl” refers to an alkenyl in which hydrogen of the alkenyl is substituted by the above-mentioned substituent.
  • the 2 ⁇ C 10 alkyl means a straight or branched alkenyl containing 2 to 10 carbon atoms.
  • a C 2 ⁇ C 10 substituted alkenyl, a C 2 ⁇ C 10 alkenyl, of which 1 or more hydrogen atoms refer to those substituted by a substituent.
  • alkynyl refers to a monovalent group generated by the loss of one hydrogen atom from an aliphatic hydrocarbon having one triple bond in the molecule, and is generally represented by C n H 2n-3-. (Here, n is a positive integer greater than or equal to 2).
  • substituted alkynyl refers to an alkynyl in which the hydrogen of the alkynyl is substituted by the above-mentioned substituent.
  • aryl (group) refers to a group formed by the detachment of one hydrogen atom bonded to a ring of an aromatic hydrocarbon, and is included in the carbon ring group in the present specification. Phenyl from benzene (C 6 H 5 -), tolyl from toluene (CH 3 C 6 H 4 - ), xylyl from xylene ((CH 3) 2 C 6 H 3 -), naphthyl naphthalene The group (C 10 H 8- ) is induced.
  • alkoxy (group) is a group in which hydrogen of an alkyl group is replaced with hydroxy
  • the "alkyl (group)” portion is synonymous with the above-mentioned “alkyl (group)”.
  • it is "C 1 to 6 alkoxy (group)”.
  • Specific examples of "C 1 to 6 alkoxy (group)” include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy and the like.
  • halogen (atom) includes, for example, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like. It is preferably a fluorine atom or a chlorine atom.
  • arabinose as a sugar (pentose).
  • Nucleotides or nucleic acids can be referred to by a generally recognized one-letter code. Amino acids may be referred to herein by either their generally known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • identity search can be performed using, for example, NCBI's BLAST 2.8.1 (issued on January 1.4, 2019).
  • the value of identity in the present specification usually refers to the value when the above BLAST is used and aligned under the default conditions. However, if a higher value is obtained by changing the parameter, the highest value is set as the identity value. When identity is evaluated in multiple regions, the highest value among them is set as the identity value. Similarity is a numerical value that takes into account similar amino acids in addition to identity.
  • U means uracil
  • T means thymine
  • A means adenine
  • C means cytosine.
  • (M) represents a modification in which the 2'hydroxyl group of ribose in the ribonucleotide unit to the left of this symbol is replaced with a methoxy group.
  • (L) represents a modification in which ribose in the ribonucleotide unit to the left of this symbol is replaced by LNA.
  • ⁇ Represents a modification in which one oxygen atom that is not involved in the bond of a phosphate group between adjacent ribonucleotide units is replaced with a sulfur atom.
  • the term "means” means a tool that can be an arbitrary tool for achieving a certain purpose (for example, detection, diagnosis, treatment), and in particular, in the present specification, “means for selective recognition (detection)". "" Means a means by which an object can be recognized (detected) differently from another.
  • label refers to an entity (eg, substance, energy, electromagnetic wave, etc.) for identifying a target molecule or substance from others.
  • labeling methods include RI (radioisotope) method, stable isotope labeling method, fluorescence method, biotin method, optical method using Raman scattering, chemiluminescence method and the like.
  • subject refers to an organism subject to the diagnosis, detection, or treatment of the present disclosure (for example, an organism such as a human being, cells taken from an organism, blood, serum, etc.). ..
  • drug As used herein, “drug”, “drug” or “factor” (both corresponding to agents in English) are used interchangeably in any sense as long as they can achieve their intended purpose. It may also be a substance or other element (eg, energy such as light, radioactivity, heat, electricity). Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including, for example, cDNA, DNA such as genomic DNA, RNA such as mRNA), poly.
  • cDNA DNA such as genomic DNA
  • RNA such as mRNA
  • organic small molecules eg, hormones, ligands, signaling substances, organic small molecules, molecules synthesized with combinatorial chemistries, small molecules that can be used as pharmaceuticals (eg, small molecule ligands, etc.)
  • treatment refers to a disease or disorder (eg, cancer) that, when such a condition occurs, prevents the exacerbation of such disease or disorder, preferably maintains the status quo. It preferably refers to alleviation, more preferably withdrawal, and includes the ability to exert a symptom-improving effect or a preventive effect on a patient's disease or one or more symptoms associated with the disease. Diagnosis in advance and appropriate treatment is called “companion treatment”, and the diagnostic agent for that purpose is sometimes called “companion diagnostic agent”.
  • the "therapeutic agent (drug)” means any drug that can treat a target condition (for example, a disease such as cancer) in a broad sense.
  • the "therapeutic agent” may be a pharmaceutical composition comprising an active ingredient and one or more pharmacologically acceptable carriers.
  • the pharmaceutical composition can be produced, for example, by mixing the active ingredient with the carrier and any method known in the technical field of pharmaceutics.
  • the therapeutic agent is not limited in the form of use as long as it is used for treatment, and may be the active ingredient alone or a mixture of the active ingredient and an arbitrary ingredient.
  • the shape of the carrier is not particularly limited, and may be, for example, a solid or a liquid (for example, a buffer solution).
  • the therapeutic agent for cancer or the like includes a drug (preventive agent) used for prevention of cancer or the like or an inhibitor for cancer or the like.
  • prevention means to prevent a certain disease or disorder (for example, a disease such as cancer) from becoming such a state before it becomes such a state.
  • the agents of the present disclosure can be used to make a diagnosis, and if necessary, the agents of the present disclosure can be used to prevent, for example, cancer, or to take preventive measures.
  • preventive drug means any drug that can prevent a target condition (for example, a disease such as cancer) in a broad sense.
  • kits are a unit in which parts to be provided (for example, a test drug, a diagnostic drug, a therapeutic drug, an antibody, a label, an instruction manual, etc.) are usually divided into two or more sections. To say.
  • the form of this kit is preferred when the purpose is to provide a composition that should not be mixed and provided for stability and the like, but is preferably mixed and used immediately before use.
  • kits are preferably instructions or instructions describing how to use the provided parts (eg, test agents, diagnostic agents, therapeutic agents, or how reagents should be treated).
  • the kit When the kit is used as a reagent kit in the present specification, the kit usually includes an instruction manual or the like that describes how to use a test drug, a diagnostic drug, a therapeutic drug, an antibody, or the like. Is included.
  • the "instruction” describes the method of using this disclosure to a doctor or another user.
  • This instruction sheet contains words instructing the detection method of the present disclosure, how to use a diagnostic agent, or administration of a medicine or the like.
  • the instruction sheet may include a wording instructing the administration site to be administered orally or to the esophagus (for example, by injection).
  • This instruction is prepared and approved by the regulatory agency of the country in which this disclosure is implemented (eg, Ministry of Health, Labor and Welfare in Japan, Food and Drug Administration (FDA) in the United States, etc.). It is clearly stated that it has been received.
  • the instruction sheet is a so-called package insert, which is usually provided in a paper medium, but is not limited thereto, and is in a form such as an electronic medium (for example, a homepage provided on the Internet, an e-mail). But can be provided.
  • the present disclosure provides a miR302 nucleic acid variant.
  • a human miR302 nucleic acid variant is provided, and more specifically, a human miR302a nucleic acid variant is provided.
  • miR302 is a kind of microRNA and is said to form a miR-302 / 367 cluster, so any microRNA belonging to this cluster falls into this category. It contains at least five members, miR-367, miR-302d, miR-302a, miR-302c and miR-302b (eg Gao Z, Zhu X, Dou Y.
  • the miR-302 / 367 cluster a comprehensive update on its evolution and functions. Open Biol.2015; 5 (12): 150138. See doi: 10.1098 / rsob.150138.
  • Natural human miR302a is a microRNA having the following base sequence. 5'-CCACCACUUAAACGUGGAUGUACUUGCUUUGAAACUAAAGAAGUAAGUGCUUCCAUGUUUUGGUGAUGG-3'(SEQ ID NO: 1)
  • the natural human miR302a molecule is thought to form the stem-loop structure shown below.
  • the nucleic acid considered to correspond to the upper half of this stem-loop structure is referred to as a passenger strand (p)
  • the nucleic acid considered to correspond to the lower half of this stem-loop structure is referred to as a guide strand (g). is there.
  • the miR302 nucleic acid variant of the present disclosure comprises 5 to 70 consecutive arbitrary bases of SEQ ID NO: 1, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45,50,55,60,65 or 70 pieces
  • Modifications eg, substitutions, deletions or insertions
  • 0 to 10 eg, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 from the base sequence consisting of It may contain or consist of a base sequence containing.
  • the modified base sequence includes alkyl bases other than adenine (A), guanine (G), cytosine (C), uracil (U) and thymine (T) (eg, alkyl of any of these bases such as methylated adenine). Chemicals (eg, methylated) may be included.
  • the miR302 nucleic acid variant molecules of the present disclosure have an arbitrary base length of 5 to 500 (eg, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16).
  • the miR302 nucleic acid variants of the present disclosure are: UAAGUGCUUCCAUGUUUUGGUGA (g1) (SEQ ID NO: 2) or ACUUAAACGUGGAUGUACUUGCU (p1) (SEQ ID NO: 3)
  • Both the sequences of SEQ ID NO: 2 and SEQ ID NO: 3 are 23 consecutive base sequences of SEQ ID NO: 1.
  • the miR302 nucleic acid variants of the present disclosure are: At least one of the 8th and 18th bases of UAAGUGCUUCCAUGUUUUGGUGA (g1) (SEQ ID NO: 2), or ACUUAAACGUGGAUGUACUUGCU (p1) (SEQ ID NO: 3) may contain a base sequence containing a base substitution, deletion or insertion at a position corresponding to at least one of the 4th and 14th bases. Even if the 8th and 18th bases of SEQ ID NO: 2 are modified, the effect on their functions is expected to be small. Even if the 4th and 14th bases of SEQ ID NO: 3 are modified, the effect on their functions is expected to be small.
  • the miR302 nucleic acid variants of the present disclosure are: A base sequence containing 0, 1, 2, 3, 4 or 5 substitutions, deletions or insertions in bases other than the 8th and 18th bases of UAAGUGCUUCCAUGUUUUGGUGA (g1) (SEQ ID NO: 2), or ACUUAAACGUGGAUGUACUUGCU (p1) (SEQ ID NO: 3) may contain base sequences other than the 4th and 14th bases containing 0, 1, 2, 3, 4 or 5 substitutions, deletions or insertions.
  • the miR302 nucleic acid variants of the present disclosure are: A base sequence containing 0, 1, 2, 3, 4 or 5 substitutions, deletions or insertions in bases other than the 8th and 18th bases of UAAGUGCTUCCAUGUTUTGGUTGA (g9) (SEQ ID NO: 4), or It may contain a base sequence containing 0, 1, 2, 3, 4 or 5 substitutions, deletions or insertions in bases other than the 4th and 14th bases of ACCAAAACAUGGAAGCACUUACT (p10) (SEQ ID NO: 5).
  • SEQ ID NO: 4 is a sequence having substitutions of 3 bases and insertion of 1 base from 23 consecutive base sequences (bases 44 to 66) of SEQ ID NO: 1.
  • SEQ ID NO: 5 is a sequence having 7 base substitutions from 23 consecutive base sequences (6th to 28th bases) of SEQ ID NO: 1. Even if the 8th and 18th bases of SEQ ID NO: 4 are modified, the effect on their functions is expected to be small. Even if the 4th and 14th bases of SEQ ID NO: 5 are modified, the effect on their functions is expected to be small.
  • the miR302 nucleic acid variants of the present disclosure may exist as a complex of two molecules (eg, a complex of a guide chain molecule and a passenger molecule) or as a plurality of separated molecules. It may be present as one of the guide chain molecule or the passenger molecule alone, or may be present as a molecule in which both the guide chain portion and the passenger moiety are contained in one molecule. If necessary, this molecule may have a sequence that can be decomposed in vivo by the action of, for example, Dicer.
  • sequences can be appropriately designed by those skilled in the art, including, but not limited to, sequences and structures such as UUGAAACUAAAGAAG (SEQ ID NO: 7), such sequences, guide chain moieties and passengers.
  • the nucleic acid can be designed as a part-containing molecule (eg, the degradation sequence is located between the guide strand portion and the passenger moiety).
  • the miR302 nucleic acid variants of the present disclosure are modified by any one or more of the following (1) to (5): (1) Substitution of ribose to crosslinked sugar (2) Substitution of oxygen atom of phosphate group to sulfur atom (3) Substitution of sugar at 2'position (4) Substitution of ribose to stereoisomer sugar (5) One or more phosphorylations at the 5'position of the terminal nucleic acid unit (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65 or 70).
  • the miR302 nucleic acid variant may comprise any of the nucleotide sequences described above.
  • the miR302 nucleic acid variant of the present disclosure is modified from any of the above (1), (3), (4) and (5) from 0 to 10 (eg, 0, 1, 2).
  • the crosslinked sugar in the modification "replacement of ribose with a crosslinked sugar” in (1) above may have any suitable structure.
  • Substituent linear or branched C 1 to C 12 alkyl, optionally substituted linear or branched C 2 to C 12 alkenyl, optionally substituted direct chain or branched C 2 ⁇ C 12 alkynyl, substituted optionally unsaturated also be 5-12 membered, in unsaturated or aromatic cyclic group (ring, two or three N, O Or S may be included), halogen, OJ 1 , N (J 1 ) 2 , SJ 1 , N 3 , or CN; each J 1 is independently H, a linear array which may be substituted.
  • each R 1 in the formula can be H or C 1 to C 12 alkyl.
  • the modification of (1) above is the substitution of ribose with a crosslinked sugar having any of the structures shown below.
  • R x are independently hydrogen, substituted or unsubstituted linear or branched C 1 to C 12 alkyl, substituted or unsubstituted linear or branched C 2 to C 12 alkenyl, substituted or unsubstituted linear or branched C 2 -C 12 alkynyl, substituted or unsubstituted 5- to 12-membered cyclic group, halogen, OJ 1 , N (J 1 ) 2 , SJ 1 , N 3 , or CN, and in a specific embodiment, R x is H, methyl, ethyl, n-propyl, isopropyl, or benzyl).
  • crosslinked sugar having the above structure mutual substitution between crosslinked sugars having Rx of hydrogen, methyl, ethyl, n-propyl, isopropyl, or benzyl is predicted to have a small effect on the function of nucleic acid variants containing the crosslinked sugars. Will be done.
  • substitution of an oxygen atom of a phosphate group with a sulfur atom is typically a modification of replacing one oxygen atom that is not involved in the bond of a phosphate group with a sulfur atom, that is, Structure, It is a modification that replaces the structure of.
  • substitution of the 2'position of sugar for example, the hydroxyl group at the 2'position of sugar (for example, ribose) is replaced with halogen (for example, fluorine), -R 3 , -OR 3 and Substituents that change to a substituent selected from the group consisting of ⁇ N (R 3 ) 2 are mentioned, in which R 3 is hydrogen, substituted or unsubstituted linear or branched C 1 to C.
  • R 3 is hydrogen, methyl, ethyl, n- propyl, are selected from the group consisting of isopropyl or benzyl.
  • the modification of (3) above can be a modification that replaces the 2'hydroxyl group of a sugar (eg, ribose) with a hydrogen, fluorine or alkoxyl group (eg, a methoxyl group).
  • substitution of ribose to a stereoisomer sugar includes at least one of the 1', 2', 3'and 4'positions of ribose (for example, one, two, three or more).
  • substitution with a stereoisomer sugar in which the bonding relationship (back or front of the ring) between two groups (hydrogen atom and substituent) bonded to the same carbon is reversed can be mentioned.
  • ribose-type to arabino-type substitution substitution with a stereoisomer sugar in which the bond relationship (back or front of the ring) between two groups bonded to the carbon at the 2'position of Bose is reversed.
  • the modification may be on one nucleic acid unit or on a plurality of nucleic acid units.
  • the modification of (2) above “substitution of oxygen atom of phosphoric acid group with sulfur atom”
  • the modification of (3) above “2'position of sugar”
  • the miR302 nucleic acid variants of the present disclosure may have any combination of any of the modifications described above.
  • the miR302 nucleic acid variant disclosed in the present application is: U ⁇ A (M) A ⁇ G (M) U ⁇ G (M) C ⁇ T (M) U ⁇ C (M) C ⁇ A (M) U ⁇ G (M) U ⁇ T (M) U ⁇ T (M) G ⁇ G (M) U ⁇ T (M) G (L) ⁇ A (L) or A (L) ⁇ C (L) ⁇ C ⁇ A ⁇ A ⁇ A ⁇ A ⁇ A ⁇ C ⁇ A ⁇ U ⁇ G ⁇ G ⁇ A ⁇ A ⁇ G ⁇ C ⁇ A ⁇ C ⁇ U ⁇ U ⁇ A ⁇ C ( L) ⁇ T (L) (During the ceremony, U stands for uracil, T stands for thymine, A stands for adenine, C stands for cytosine, (M) represents a modification in which the 2'hydroxyl group of ribose in the ribonucleot
  • (L) is the ribose in the ribonucleotide unit to the left of this symbol Represents a modification that has been replaced by ⁇ Represents a modification in which one oxygen atom that is not involved in the bond of the phosphate group between the adjacent ribonucleotide units is replaced with a sulfur atom) ,
  • 0 to 10 modifications of (M), (L) and ⁇ eg, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
  • 0 to 10 eg, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or any modifications of (1) to (5) above have been deleted.
  • 10) have been added and may contain structures containing 0, 1, 2, 3, 4 or 5 base substitutions, deletions or insertions.
  • the miR302 nucleic acid variants of the present disclosure are: U ⁇ A (M) A ⁇ G (M) U ⁇ G (M) C ⁇ T (M) U ⁇ C (M) C ⁇ A (M) U ⁇ G (M) U ⁇ T (M) U ⁇ T (M) G ⁇ G (M) U ⁇ T (M) G (L) ⁇ A (L) or A (L) ⁇ C (L) ⁇ C ⁇ A ⁇ A ⁇ A ⁇ A ⁇ A ⁇ C ⁇ A ⁇ U ⁇ G ⁇ A ⁇ A ⁇ G ⁇ C ⁇ A ⁇ C ⁇ U ⁇ U ⁇ A ⁇ C ( L) ⁇ T (L) (In the formula, U, T,
  • 0, 1, 2, 3 or 4 has been changed to the substitution with another cross-linked sugar in (1) above, and 0 to 10 modifications of ⁇ (eg, 0, 1, 2, 3). 4, 5, 6, 7, 8, 9 or 10) have been deleted, and 0 to 10 modifications (2) above (eg 0, 1, 2, 3, 4, 5, 6, 7) have been deleted. , 8, 9 or 10), and 0 to 5 (eg, 0, 1, 2, 3, 4) of any modification of (1), (3), (4) or (5) above. Or 5) may be a structure-containing molecule that has been added and contains 0, 1, 2, 3, 4 or 5 base substitutions, deletions or insertions.
  • the miR302 nucleic acid variants of the present disclosure facilitate functional moieties different from those of the modifications (eg, detection of nucleic acid variants, tissue targeting, stability enhancement and / or binding to other molecules.
  • the part to be used may be included.
  • Functional moieties include, but are not limited to, for example, fluorescent dyes, luminescent dyes, PEGs, cholesterol, lipids, biotin, linker moieties (such as moieties containing NHS, azides or alkynes).
  • modified nucleic acids described herein using any known technique.
  • synthesis of modified nucleic acids refer to, for example, Current protocols in nucleic acid chemistry (https://currentprotocols.onlinelibrary.wiley.com/journal/19349289).
  • the miR302 nucleic acid variant of the present disclosure is a case where HT29 cells into which a nucleic acid construct in which a luciferase gene and an HnRNPA2B1 gene are bound are introduced by a lentivirus are brought into contact with the miR302 nucleic acid variant.
  • Lower than the double strand (g1p1 pair) formed by the native human miR302a or nucleic acid of SEQ ID NO: 2 (g1) and nucleic acid of SEQ ID NO: 3 (p1) (eg, 1% -99.99% lower, For example, about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, (About 85%, about 90%, about 95%, about 98%, about 99%, about 99.5%, about 99.9% or about 99.99% lower) can result in luciferase luminescence.
  • the miR302 nucleic acid variants of the present disclosure are in vitro with fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • the rate of degradation upon contact is slower than the double strand (g1p1 pair) formed by the native human miR302a or the nucleic acid of SEQ ID NO: 2 (g1) and the nucleic acid of SEQ ID NO: 3 (p1) (eg, 1).
  • % -99.99% slower eg, about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60% , About 70%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, about 99.5%, about 99.9% or about 99.99% slower) Can have stability.
  • the miR302 nucleic acid variants of the present disclosure can be used in the treatment of diseases such as cancer.
  • the miR302 nucleic acid variants of the present disclosure may be provided in the form contained in the composition.
  • Cancers that can be treated with the miR302 nucleic acid variants of the present disclosure include pancreatic cancer (eg, early pancreatic cancer), liver cancer, gallbladder cancer, biliary tract cancer, gastric cancer, colon cancer, bladder cancer, and kidney.
  • Cancer, breast cancer, lung cancer, brain cancer and skin cancer include, but are not limited to.
  • the miR302 nucleic acid variants of the present disclosure are provided as various forms of compositions or pharmaceuticals (therapeutic or prophylactic agents).
  • the administration route of the therapeutic agent is preferably one that is effective for treatment, and may be, for example, intravenous, subcutaneous, intramuscular, intraperitoneal, or oral administration.
  • the administration form may be, for example, an injection, a capsule, a tablet, a granule, or the like.
  • the aqueous solution for injection may be stored, for example, in a vial or a stainless steel container. Further, the aqueous solution for injection may contain, for example, physiological saline, sugar (for example, trehalose), NaCl, NaOH or the like. Further, the therapeutic agent may contain, for example, a buffer (for example, a phosphate buffer), a stabilizer, or the like.
  • compositions, pharmaceuticals, therapeutic agents, prophylactic agents, etc. of the present disclosure include therapeutically effective amounts of therapeutic or active ingredients, and pharmaceutically acceptable carriers or excipients.
  • pharmaceutically acceptable is used in animals, and more specifically in humans, for use in government regulatory agencies or in the pharmacopoeia or other generally accepted pharmacopoeia. It means that it is listed.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle to which a therapeutic agent is administered together.
  • Such carriers can also be sterile liquids such as water and oils, including, but not limited to, petroleum, animal, plant or synthetic origins, sesame oil, soybean oil, minerals.
  • saline and aqueous dextrose are the preferred carriers.
  • saline solution, as well as aqueous dextrose and glycerol solutions are used as liquid carriers for the injectable solution.
  • Suitable excipients include light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, choke, silica gel, sodium stearate, glycerol monostearate, talc, chloride.
  • composition Sodium, defatted milk powder, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl acetal diethylaminoacetate, polyvinyl pyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene curing
  • castor oil 60 sucrose, carboxymethyl cellulose, corn starch, inorganic salts and the like.
  • the composition can also contain small amounts of wetting or emulsifying agents, or pH buffers, if desired.
  • compositions can also take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. It is also possible to formulate the composition as a suppository using traditional binders and carriers such as triglycerides. Oral formulations can also include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate and the like. Examples of suitable carriers are E.I. W. Described in Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, U.S.A.).
  • compositions contain a therapeutically effective amount of a therapeutic agent, preferably a purified form, along with an appropriate amount of carrier to provide a form for appropriate administration to the patient.
  • a therapeutic agent preferably a purified form
  • the formulation should be suitable for the mode of administration.
  • surfactants for example, surfactants, excipients, colorants, fragrances, preservatives, stabilizers, buffers, suspending agents, tonicity agents, binders, disintegrants, lubricants, fluidity It may contain an accelerator, a flavoring agent and the like.
  • the "salt” includes, for example, an anion salt formed of any acidic (eg, carboxyl) group or a cationic salt formed of any basic (eg, amino) group.
  • the salts include inorganic salts or organic salts, and include, for example, the salts described in Berg et al., J. Pharm. Sci., 1977, 66, 1-19. Further, for example, a metal salt, an ammonium salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid and the like can be mentioned.
  • a "solvate" is a compound formed by a solute and a solvent. For the solvate, for example, J.
  • the solvent is water, the solvate formed is a hydrate.
  • the solvent is preferably one that does not interfere with the biological activity of the solute. Examples of such preferred solvents include, but are not limited to, water or various buffers.
  • the miR302 nucleic acid variants of the present disclosure are administered as pharmaceuticals
  • various delivery systems are known, and such systems can also be used to administer the therapeutic agents of the present disclosure to appropriate sites.
  • encapsulation in liposomes, microparticles, and microcapsules the use of receptor-mediated endocytosis; construction of therapeutic nucleic acids as part of retroviral vectors or other vectors. and so on.
  • Induction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the drug can be administered by any of the preferred routes, eg by injection, by bolus injection, by absorption through the epithelial or mucocutaneous lining (eg, oral, rectal and intestinal mucosa).
  • Inhalers or nebulizers can be used, depending on the aerosolizing agent, and can also be administered with other bioactive agents. Administration can also be systemic or topical.
  • the miR302 nucleic acid variant of the present disclosure can be further administered by any appropriate route, such as direct injection into the cancer (lesion).
  • the composition can be blended as a pharmaceutical composition adapted for administration to humans according to a known method.
  • Such compositions can be administered by injection.
  • the composition for injection administration is a solution in a sterile isotonic aqueous buffer.
  • the composition can also include a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site.
  • the ingredients are supplied separately or mixed together in a unit dosage form and lyophilized powder or water-free concentrate in a sealed container such as an ampoule or sachet indicating the amount of activator. It can be supplied as a thing.
  • composition is to be administered by infusion, it can also be dispensed using an infusion bottle containing sterile drug grade water or saline. If the composition is to be administered by injection, it is also possible to provide an ampoule of sterile water or saline for injection so that the ingredients can be mixed prior to administration.
  • compositions, pharmaceuticals, therapeutic agents and prophylactic agents of the present disclosure in neutral or salt forms or other prodrugs (eg, esters, etc.).
  • Pharmaceutically acceptable salts include those formed with free carboxyl groups derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, prokine. It includes those formed with free amine groups such as those derived from such as, and those derived from sodium, potassium, ammonium, calcium, ferric hydroxide and the like.
  • the amount of therapeutic agent of the present disclosure effective for the treatment of a particular disorder or condition may vary depending on the nature of the disorder or condition, but one of ordinary skill in the art can determine by standard clinical techniques as described herein.
  • in vitro assays can optionally be used to assist in identifying optimal dosage ranges.
  • the exact dose to be used in the formulation may also vary depending on the route of administration and the severity of the disease or disorder and should be determined according to the discretion of the attending physician and the circumstances of each patient. However, the dose is not particularly limited, and may be, for example, 0.001, 1, 5, 10, 15, 100, or 1000 mg / kg body weight per dose, within the range of any two of these values. There may be.
  • the dosing interval is not particularly limited, but may be administered once or twice per 1, 7, 14, 21, or 28 days, and once or twice within the range of any two of these values. May be good.
  • the miR302 nucleic acid variants of the present disclosure may have high plasma stability and / or high activity, so administration frequency may be reduced.
  • the dose, administration interval, and administration method may be appropriately selected depending on the age and weight of the patient, symptoms, target organ, and the like.
  • the therapeutic agent preferably contains a therapeutically effective amount or an effective amount of the active ingredient that exerts a desired action. If the malignant tumor marker is significantly reduced after administration, it may be judged that there is a therapeutic effect. Effective doses can be estimated from dose-response curves obtained in vitro or from animal model test systems.
  • the "patient” or “subject” is a human or mammal (eg, mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, cat, dog, Includes marmosets, monkeys, or chimpanzees, etc.).
  • a human or mammal eg, mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, cat, dog, Includes marmosets, monkeys, or chimpanzees, etc.
  • the pharmaceutical composition or therapeutic or prophylactic agent of the present disclosure can be provided as a kit.
  • the present disclosure provides drug packs or kits comprising one or more containers filled with one or more components of the compositions or pharmaceuticals of the present disclosure.
  • compositions containing the ingredients of the present disclosure can be administered via liposomes, microparticles, or microcapsules. In various embodiments of the present disclosure, it may be useful to use such compositions to achieve sustained release of the components of the present disclosure.
  • oligonucleotides of the present disclosure in the manufacture of immunostimulatory oligonucleotide nucleic acid reagents is provided. It is understood that the oligonucleotides used in this aspect can use any of the forms described herein.
  • Example 1 Design of miR302 nucleic acid variant Nucleic acid having a partial sequence of miR302 nucleic acid variant and natural human miR302a (19 types of guide strands and 20 types of passenger strands in total) was requested to Gene Design (Osaka). And synthesized.
  • -(M) represents a modification in which the 2'hydroxyl group of ribose in the ribonucleotide unit to the left of this symbol is replaced with a methoxy group.
  • ⁇ In (L) the ribose in the ribonucleotide unit to the left of this symbol is Represents a modification that has been replaced by ⁇ ⁇ Represents a modification in which one oxygen atom that is not involved in the bond of the phosphate group between the adjacent ribonucleotide units is replaced with a sulfur atom).
  • Alexa647 was attached to the 5'end of the guide chains (g1 and g9) via an ssH amino linker.
  • Example 2 Preparation of cells for primary screening
  • a gene in which a luciferase gene was fused to 3'UTR (hereinafter) of the HnRNPA2B1 gene was used in HT29 cells using a lentiwill. It was introduced into (colon cancer cell line) to prepare cells that stably express this fusion gene.
  • the binding of miR302a to the 3'UTR region of the HnRNPA2B1 gene destabilizes the mRNA of the fusion gene and reduces the amount of luciferase luminescence.
  • Example 3 Primary screening The above HT29 cells were seeded on a 96-well plate (Nunc TM) at a concentration of 1 ⁇ 10 3 cells / well (day 0). On day 1, cells in each well were introduced with the above miR302 nucleic acid variant or native miR302 (combination of guide and passenger strands) using Lipofectamine RNAi MAX (Invitrogen, USA). The medium was changed on the second day, and the amount of luciferase emitted was measured on the third day. The Dual-Glo® Luciferase Assay System (promega, USA) and GLO MAX MULTI (promega, USA) were used to measure luciferase luminescence. The stained area was calculated with Image J and compared. The results are shown in FIG.
  • Example 4 Secondary screening As a result of the primary screening, 10 kinds of miR302 nucleic acid variant combinations having a high inhibitory effect on luciferase luminescence were subjected to the secondary screening.
  • pancreatic cancer cell lines Panc-1 and MIA-PaCa were seeded on 96-well plates (Cell able®, Toyo Gosei, Tokyo) (day 0) and the next day.
  • the above miR302 nucleic acid variant or native miR302 was introduced using Lipofectamine RNAi MAX (Invitrogen, USA).
  • Example 5 Animal administration experiment Nucleic acid (62.5 nmol / kg) (OUM302 or natural miR302 (G1P1 pair)) + unit PIC was intravenously injected into NOD-SCID mice having a tumor volume of 50 mm 3 and 14 Body weight and tumor volume were observed until days later. In addition, the vehicle-administered group was included. The results are shown in FIGS. 5 to 6. In mice, OUM302 showed superior antitumor effects compared to native miR302.
  • Nucleic acid (62.5 nmol / kg) (OUM302 or native miR302) + unit PIC) + anti-cancer agent (TAS-102 (trifluridine / tipiracil hydrochloride, Ronsurf) in NOD-SCID mice with a tumor volume of 50 mm 3 Registered trademark)) (150 mg / kg), 5FU (5-fluorouracil) (8.3 mg / kg) or gemcitabine (100 mg / kg)) was administered as a single dose, and body weight and tumor volume were observed until 14 days later. The results are shown in FIGS. 7-12. In mice, OUM302 showed superior antitumor effects compared to native miR302 when combined with anticancer agents.
  • TAS-102 trifluridine / tipiracil hydrochloride, Ronsurf
  • 5FU 5-fluorouracil
  • gemcitabine 100 mg / kg
  • EL1-SV40 Tg mouse FVB / N-Tg (Cela1-Luc, Cela1-Tag) 116Xen mouse purchased from Taconic
  • nucleic acid (62.5 nmol / kg)
  • TAS-102 150 mg / kg
  • TAS-102 150 mg / kg
  • Example 6 Stability of miR302 nucleic acid variant MiR302 nucleic acid variant (g1, g9, p1 or p10) was added to a solution to which FBS was added so as to have a final concentration of 1% so that the final concentration was 2 ⁇ M. Incubated at 37 ° C. Samples were collected 15 minutes, 30 minutes, 1 hour, 3 hours and 24 hours after the start of incubation and subjected to electrophoresis. Electrophoresis was performed with 20% acrylamide / bisgel. The condition without adding FBS was used as a control. A 20 bp DNA ladder (Takara Bio, Shiga) was used as the migration marker. The exposure time was Cy3_30sec. (Fig. 16)
  • a miR302 nucleic acid variant (natural g1p1 pair or g9p10 vs. OUM302) was added to a solution to which FBS was added to a final concentration of 1% to a final concentration of 2 ⁇ M, and the mixture was incubated at 37 ° C. Samples were collected 1 hour after the start of incubation and subjected to electrophoresis. Electrophoresis was performed on a 2% agarose gel or 20% acrylamide / bisgel. The condition without adding FBS was used as a control. A 20 bp DNA ladder (Takara Bio, Shiga) was used as the migration marker (Fig. 17).
  • This disclosure is available for pharmaceutical products, such as pharmaceutical products for treating cancer.
  • -SEQ ID NO: 1 Natural human miR302a sequence CCACCACUUAAACGUGGAUGUACUUGCUUUGAAACUAAAGAAGUAAGUGCUUCCAUGUUUUGGUGAUGG
  • SEQ ID NO: 2 Guide sequence of natural human miR302a (g1) UAAGUGCUUCCAUGUUUUGGUGA
  • SEQ ID NO: 3 Passenger sequence of natural human miR302a (p1) ACUUAAACGUGGAUGUACUUGCU SEQ ID NO: 4: Nucleotide sequence of miR302 nucleic acid variant (g9) UAAGUGCTUCCAUGUTUTGGUTGA
  • SEQ ID NO: 5 Nucleotide sequence of miR302 nucleic acid variant (p10) ACCAAAACAUGGAAGCACUUACT
  • SEQ ID NO: 6 3'UTR sequence of the HnRNPA2B1 gene used
  • SEQ ID NO: 7 exemplary degraded sequence UUGAAACUAAAGAAG

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Abstract

The present disclosure provides a modified miR302 nucleic acid. According to one embodiment, the present disclosure provides a treatment for diseases such as cancer that uses the modified miR302 nucleic acid. According to one embodiment, the present disclosure provides a composition that includes the modified miR302 nucleic acid and a use for the composition in the treatment of diseases such as cancer. According to one embodiment, the modified miR302 nucleic acid of the present disclosure can include a base sequence that includes 0-10 modifications (e.g., substitutions, deletions, or insertions) from a base sequence that comprises 5-70 arbitrary consecutive bases from SEQ ID NO: 1.

Description

ｍｉＲ３０２核酸改変体miR302 nucleic acid variant
　本開示は、miR302核酸改変体およびその使用に関する。より特定すると、ヒトmiR302a核酸改変体およびその使用に関する。本開示のmiR302核酸改変体は、がんなどの疾患の治療に使用され得る。 The present disclosure relates to miR302 nucleic acid variants and their use. More specifically, it relates to a human miR302a nucleic acid variant and its use. The miR302 nucleic acid variants of the present disclosure can be used in the treatment of diseases such as cancer.
　Barroso-delJesus　Aら、Mol　Cell　Biol.　2008　Nov;28(21):6609-19.［非特許文献１］では、Ｎａｎｏｇ、Ｏｃｔ３／４、Ｓｏｘ２およびＲｅｘ１などのＥＳＣ関連転写因子がmiR302-367プロモーターの上流の制御因子であると予想されており、このように制御されたmiR302は種々の生物学的事象に影響を及ぼしていると考えられる。 In Barroso-del Jesus A et al., Mol Cell Biol. 2008 Nov; 28 (21): 6609-19. [Non-Patent Document 1], ESC-related transcription factors such as Nanog, Oct3/4, Sox2 and Rex1 are miR302-367 promoters. It is expected to be an upstream regulator of, and miR302 thus regulated is thought to influence a variety of biological events.
　しかし、miR302核酸改変体を使用した疾患の治療は今まで試みられてこなかった。 However, treatment of diseases using miR302 nucleic acid variants has not been attempted so far.
　発明者らは、miR302核酸改変体が、がんなどの疾患の治療に使用できることを見出し、本開示に到達した。本開示によれば、miR302核酸改変体およびそれを含む組成物が提供され、また、がんなどの疾患の治療におけるその使用も提供される。本開示のmiR302核酸改変体は、例えば、血漿中などにおいて安定であり得、被験体の負荷が低い投与レジメンにおいて有効に使用され得る。 The inventors have found that the miR302 nucleic acid variant can be used for the treatment of diseases such as cancer, and have reached the present disclosure. According to the present disclosure, a miR302 nucleic acid variant and a composition containing the same are provided, and their use in the treatment of diseases such as cancer is also provided. The miR302 nucleic acid variants of the present disclosure can be stable, for example in plasma, and can be effectively used in administration regimens with low subject load.
　したがって、本開示は以下を提供する。
（項目１）
　ヒトmiR302a核酸改変体。
（項目２）
UAAGUGCUUCCAUGUUUUGGUGA（ｇ１）(配列番号２)または
ACUUAAACGUGGAUGUACUUGCU（ｐ１）(配列番号３)
の塩基配列から０、１、２、３、４または５個の置換、欠失または挿入を含む塩基配列を含む、上記項目のいずれかの核酸改変体。
（項目３）
UAAGUGCUUCCAUGUUUUGGUGA（ｇ１）(配列番号２)の８番目および１８番目の塩基の少なくとも一方、または
ACUUAAACGUGGAUGUACUUGCU（ｐ１）(配列番号３)の４番目および１４番目の塩基の少なくとも一方
に対応する位置において塩基の置換、欠失または挿入を含む塩基配列を含む、上記項目のいずれかの核酸改変体。
（項目４）
UAAGUGCUUCCAUGUUUUGGUGA（ｇ１）(配列番号２)の８番目および１８番目以外の塩基において０、１または２個の置換、欠失または挿入を含む塩基配列、または
ACUUAAACGUGGAUGUACUUGCU（ｐ１）(配列番号３)の４番目および１４番目以外の塩基において０、１または２個の置換、欠失または挿入を含む塩基配列
を含む、上記項目のいずれかの核酸改変体。
（項目５）
UAAGUGCTUCCAUGUTUTGGUTGA（ｇ９）(配列番号４)の８番目および１８番目以外の塩基が同一である塩基配列、または
ACCAAAACAUGGAAGCACUUACT（ｐ１０）(配列番号５)の４番目および１４番目以外の塩基が同一である塩基配列
を含む、上記項目のいずれかの核酸改変体。
（項目６）
　以下の（１）～（７）のいずれかの改変：
（１）リボースから以下のいずれかの架橋リボースへの置換
Figure JPOXMLDOC01-appb-C000003
（式中、Ｒｘは、それぞれ独立して水素、または置換または非置換のＣ１～Ｃ６アルキルである）
（２）リン酸基の結合に関与しない一つの酸素原子の硫黄原子への置換
（３）リボースの２’のヒドロキシル基のアルコキシ基への置換
（４）リボースの２’のヒドロキシル基のハロゲン原子への置換
（５）リボースからデオキシリボースへの置換
（６）末端５’のリン酸化
（７）リボースの２’位の炭素に結合した２つの基の間の結合関係（環の裏または表）が逆転した立体異性体糖への置換
を１つまたは複数含む、上記項目のいずれかの核酸改変体。
（項目７）
　Ｒｘが、それぞれ独立して水素、メチル、エチル、ｎ－プロピル、イソプロピル、またはベンジルである、上記項目のいずれかの核酸改変体。
（項目８）
U^A(M)A^G(M)U^G(M)C^T(M)U^C(M)C^A(M)U^G(M)U^T(M)U^T(M)G^G(M)U^T(M)G(L)^A(L)または
A(L)^C(L)^C^A^A^A^A^C^A^U^G^G^A^A^G^C^A^C^U^U^A^C(L)^T(L)
の構造
（式中、
　Uはウラシル、Tはチミン、Aはアデニン、Cはシトシンの各塩基を表し、
　(M)は、この記号の左のリボヌクレオチド単位中のリボースの２’のヒドロキシル基がメトキシ基に置き換えられている改変を表し、
　(L)は、この記号の左のリボヌクレオチド単位中のリボースが
Figure JPOXMLDOC01-appb-C000004
に置き換えられている改変を表し、
　^は、両隣のリボヌクレオチド単位の間のリン酸基の結合に関与しない一つの酸素原子が硫黄原子に置き換えられている改変を表す）
から、(M)、(L)および^の改変が、０、１、２、３、４または５個追加および／または削除されている構造を含む、上記項目のいずれかの核酸改変体。
（項目９）
U^A(M)A^G(M)U^G(M)C^T(M)U^C(M)C^A(M)U^G(M)U^T(M)U^T(M)G^G(M)U^T(M)G(L)^A(L)または
A(L)^C(L)^C^A^A^A^A^C^A^U^G^G^A^A^G^C^A^C^U^U^A^C(L)^T(L)
の構造
（式中、U、T、A、C、(M)、(L)、および^の定義は前記と同じ）
を含む、項目８に記載の核酸改変体。
（項目１０）
U^A(M)A^G(M)U^G(M)C^T(M)U^C(M)C^A(M)U^G(M)U^T(M)U^T(M)G^G(M)U^T(M)G(L)^A(L)または
A(L)^C(L)^C^A^A^A^A^C^A^U^G^G^A^A^G^C^A^C^U^U^A^C(L)^T(L)
の構造
（式中、U、T、A、C、(M)、(L)、および^の定義は前記と同じ）
から、(M)および(L)の改変が、０、１、２または３個追加および／または削除されており、^の改変が、０、１、２、３、４、５、６、７、８、９または１０個追加および／または削除されている、上記項目のいずれかの核酸改変体。
（項目１１）
　ガイド鎖である上記項目のいずれかの核酸改変体と、パッセンジャー鎖である上記項目のいずれかの核酸改変体とによって形成された二本鎖。
（項目１２）
　ガイド部分である上記項目のいずれかの核酸改変体の部分と、パッセンジャー部分である上記項目のいずれかの核酸改変体の部分とを含む、核酸改変体分子。
（項目１３）
　生体内で分解され得る配列を有する、上記項目のいずれかの核酸改変体分子。
（項目１４）
　ルシフェラーゼ遺伝子と、HnRNPA2B1遺伝子とを結合させた核酸構築物をレンチウイルスにより導入したＨＴ２９細胞と接触させた場合に、天然のヒトmiR302aよりも低いルシフェラーゼ発光をもたらす、上記項目のいずれかの核酸改変体、上記項目のいずれかの二本鎖または上記項目のいずれかの核酸改変体分子。
（項目１５）
　インビトロでウシ胎児血清（FBS）と接触させた場合の分解速度が、天然のヒトmiR302aよりも遅い、上記項目のいずれかの核酸改変体、上記項目のいずれかの二本鎖または上記項目のいずれかの核酸改変体分子。
（項目１６）
　上記項目のいずれかの核酸改変体、上記項目のいずれかの二本鎖または上記項目のいずれかの核酸改変体分子を含む、がんを治療するための医薬組成物。
（項目１７）
　がんの治療における使用のための、上記項目のいずれかの核酸改変体、上記項目のいずれかの二本鎖または上記項目のいずれかの核酸改変体分子。
（項目１８）
　上記項目のいずれかの核酸改変体、上記項目のいずれかの二本鎖または上記項目のいずれかの核酸改変体分子を被験体に投与する工程を含む、がんを治療するための方法。
（項目１９）
　がんを治療するための医薬の製造における上記項目のいずれかの核酸改変体、上記項目のいずれかの二本鎖または上記項目のいずれかの核酸改変体分子の使用。 Accordingly, the present disclosure provides:
(Item 1)
Human miR302a nucleic acid variant.
(Item 2)
UAAGUGCUUCCAUGUUUUGGUGA (g1) (SEQ ID NO: 2) or
ACUUAAACGUGGAUGUACUUGCU (p1) (SEQ ID NO: 3)
A nucleic acid variant of any of the above items, comprising a base sequence containing 0, 1, 2, 3, 4 or 5 substitutions, deletions or insertions from the base sequence of.
(Item 3)
At least one of the 8th and 18th bases of UAAGUGCUUCCAUGUUUUGGUGA (g1) (SEQ ID NO: 2), or
Anucleic acid variant of any of the above items, comprising a base sequence comprising a base substitution, deletion or insertion at a position corresponding to at least one of the 4th and 14th bases of ACUUAAACGUGGAUGUACUUGCU (p1) (SEQ ID NO: 3).
(Item 4)
A base sequence containing 0, 1 or 2 substitutions, deletions or insertions in bases other than the 8th and 18th bases of UAAGUGCUUCCAUGUUUUGGUGA (g1) (SEQ ID NO: 2), or
Anucleic acid variant of any of the above items, comprising a base sequence containing 0, 1 or 2 substitutions, deletions or insertions at bases other than the 4th and 14th bases of ACUUAAACGUGGAUGUACUUGCU (p1) (SEQ ID NO: 3).
(Item 5)
UAAGUGCTUCCAUGUTUTGGUTGA (g9) (SEQ ID NO: 4) has the same base sequence other than the 8th and 18th bases, or
A nucleic acid variant of any of the above items, which comprises a base sequence having the same base other than the 4th and 14th bases of ACCAAAACAUGGAAGCACUUACT (p10) (SEQ ID NO: 5).
(Item 6)
Modification of any of the following (1) to (7):
(1) Substitution of ribose with any of the following cross-linked ribose
Figure JPOXMLDOC01-appb-C000003
(In the formula, R x is hydrogen independently, or substituted or unsubstituted C 1 to C 6 alkyl, respectively)
(2) Substitution of one oxygen atom not involved in the bond of phosphate group to sulfur atom (3) Substitution of ribose 2'hydroxyl group to alkoxy group (4) Halogen atom of ribose 2'hydroxyl group Substitution to (5) Substitution of ribose to deoxyribose (6) Phosphorization of terminal 5'(7) Bonding relationship between two groups bonded to the 2'carbon of ribose (back or front of ring) A nucleic acid variant of any of the above items, comprising one or more substitutions with reversed steric isomer sugar.
(Item 7)
A nucleic acid variant of any of the above items, wherein R x is hydrogen, methyl, ethyl, n-propyl, isopropyl, or benzyl, respectively.
(Item 8)
U ^ A (M) A ^ G (M) U ^ G (M) C ^ T (M) U ^ C (M) C ^ A (M) U ^ G (M) U ^ T (M) U ^ T (M) G ^ G (M) U ^ T (M) G (L) ^ A (L) or
A (L) ^ C (L) ^ C ^ A ^ A ^ A ^ A ^ C ^ A ^ U ^ G ^ G ^ A ^ A ^ G ^ C ^ A ^ C ^ U ^ U ^ A ^ C ( L) ^ T (L)
Structure (in the formula,
U stands for uracil, T stands for thymine, A stands for adenine, C stands for cytosine,
(M) represents a modification in which the 2'hydroxyl group of ribose in the ribonucleotide unit to the left of this symbol is replaced with a methoxy group.
(L) is the ribose in the ribonucleotide unit to the left of this symbol
Figure JPOXMLDOC01-appb-C000004
Represents a modification that has been replaced by
^ Represents a modification in which one oxygen atom that is not involved in the bond of the phosphate group between the adjacent ribonucleotide units is replaced with a sulfur atom)
A nucleic acid variant of any of the above items, comprising a structure in which 0, 1, 2, 3, 4 or 5 modifications are added and / or deleted from (M), (L) and ^.
(Item 9)
U ^ A (M) A ^ G (M) U ^ G (M) C ^ T (M) U ^ C (M) C ^ A (M) U ^ G (M) U ^ T (M) U ^ T (M) G ^ G (M) U ^ T (M) G (L) ^ A (L) or
A (L) ^ C (L) ^ C ^ A ^ A ^ A ^ A ^ C ^ A ^ U ^ G ^ G ^ A ^ A ^ G ^ C ^ A ^ C ^ U ^ U ^ A ^ C ( L) ^ T (L)
(In the formula, the definitions of U, T, A, C, (M), (L), and ^ are the same as above)
Item 8. The nucleic acid variant according to item 8.
(Item 10)
U ^ A (M) A ^ G (M) U ^ G (M) C ^ T (M) U ^ C (M) C ^ A (M) U ^ G (M) U ^ T (M) U ^ T (M) G ^ G (M) U ^ T (M) G (L) ^ A (L) or
A (L) ^ C (L) ^ C ^ A ^ A ^ A ^ A ^ C ^ A ^ U ^ G ^ G ^ A ^ A ^ G ^ C ^ A ^ C ^ U ^ U ^ A ^ C ( L) ^ T (L)
(In the formula, the definitions of U, T, A, C, (M), (L), and ^ are the same as above)
From, the modifications of (M) and (L) are added and / or deleted by 0, 1, 2 or 3, and the modifications of ^ are 0, 1, 2, 3, 4, 5, 6, 7 , 8, 9 or 10 added and / or deleted, a nucleic acid variant of any of the above items.
(Item 11)
A double strand formed by a nucleic acid variant of any of the above items, which is a guide strand, and a nucleic acid variant of any of the above items, which is a passenger strand.
(Item 12)
A nucleic acid variant molecule comprising a portion of a nucleic acid variant of any of the above items which is a guide portion and a portion of a nucleic acid variant of any of the above items which is a passenger portion.
(Item 13)
A nucleic acid variant molecule of any of the above items, which has a sequence that can be degraded in vivo.
(Item 14)
A nucleic acid variant of any of the above items, which produces lower luciferase luminescence than natural human miR302a when contacted with HT29 cells introduced by lentivirus with a nucleic acid construct in which the luciferase gene and the HnRNPA2B1 gene are bound. A double-stranded molecule of any of the above items or a nucleic acid variant molecule of any of the above items.
(Item 15)
Degradation rate when contacted with fetal bovine serum (FBS) in vitro is slower than that of natural human miR302a, nucleic acid variant of any of the above items, double strand of any of the above items, or any of the above items. Nucleic acid variant molecule.
(Item 16)
A pharmaceutical composition for treating cancer, comprising a nucleic acid variant of any of the above items, a double strand of any of the above items, or a nucleic acid variant of any of the above items.
(Item 17)
A nucleic acid variant of any of the above items, a double strand of any of the above items or a nucleic acid variant molecule of any of the above items for use in the treatment of cancer.
(Item 18)
A method for treating cancer, comprising administering to a subject a nucleic acid variant of any of the above items, a double strand of any of the above items, or a nucleic acid variant molecule of any of the above items.
(Item 19)
Use of a nucleic acid variant of any of the above items, a double strand of any of the above items or a nucleic acid variant molecule of any of the above items in the manufacture of a medicament for treating cancer.
　本開示において、上記の１つまたは複数の特徴は、明示された組み合わせに加え、さらに組み合わせて提供され得ることが意図される。本開示のなおさらなる実施形態および利点は、必要に応じて以下の詳細な説明を読んで理解すれば、当業者に認識される。 In the present disclosure, it is intended that one or more of the above features may be provided in addition to the specified combinations. Further embodiments and advantages of the present disclosure will be appreciated by those skilled in the art upon reading and understanding the following detailed description as necessary.
　本開示は、がんなどの疾患の治療に有用である。 This disclosure is useful for the treatment of diseases such as cancer.
一次スクリーニングの結果を示す。バーの高さは、Ｇ１Ｐ１対（図中、miR302と表記される）における結果を１とした場合のそれぞれのガイド鎖－パッセンジャー鎖対で観察されたルシフェラーゼの相対的な発光強度を示す。それぞれのバーは、１９種類のガイド鎖のいずれかと、２０種類のパッセンジャー鎖のいずれかとの任意の組合せにおける結果を示す。それぞれのバーは、発光強度に基づいて配置しているだけであり、横軸または縦軸は、特定のガイド鎖またはパッセンジャー鎖を指すものではない。例えば、Ｇ１Ｐ１対のバーと同じ列または行の他のバーは、必ずしもＧ１またはＰ１を含む対の結果とは限らない。The result of the primary screening is shown. The height of the bar indicates the relative luminescence intensity of luciferase observed in each guide chain-passenger chain pair when the result in the G1P1 pair (denoted as miR302 in the figure) is 1. Each bar shows the results in any combination of any of the 19 guide chains and any of the 20 passenger chains. Each bar is only arranged based on emission intensity, and the horizontal or vertical axis does not refer to a particular guide chain or passenger chain. For example, other bars in the same column or row as the G1P1 pair of bars do not necessarily result in a pair containing G1 or P1. Ｐａｎｃ－１細胞に対する天然型miR302（Ｇ１Ｐ１対）による処置の効果を示す。左のグラフは４日目の結果を示し、右のグラフは７日目の結果を示す。各グラフにおいて、縦軸は、対照の結果を１とした場合のＧ１Ｐ１対の相対的なＤＡＰＩ染色面積を示す。４日目および７日目の両方において、Ｇ１Ｐ１対と対照との間の染色面積の差は、統計的に有意な差であった（Ｐ＜０．０１）。The effect of treatment with native miR302 (G1P1 pair) on Panc-1 cells is shown. The graph on the left shows the results on the 4th day, and the graph on the right shows the results on the 7th day. In each graph, the vertical axis shows the relative DAPI staining area of the G1P1 pair when the control result is 1. On both days 4 and 7, the difference in stained area between the G1P1 pair and the control was a statistically significant difference (P <0.01). ＭＩＡ－ＰａＣａ細胞に対するmiR302核酸改変体または天然型miR302（Ｇ１Ｐ１対）による処置の効果を示す。縦軸は、Ｇ１Ｐ１対の４日目の結果を１とした場合のそれぞれのガイド鎖－パッセンジャー鎖対の相対的なＤＡＰＩ染色面積を示す。横軸は、ガイド－パッセンジャー対の種類を示し、各対について左のバーは４日目の結果を示し、右のバーは７日目の結果を示す。＊は、Ｇ１Ｐ１対の結果と比較して統計的に有意な差があることを示す。The effect of treatment with miR302 nucleic acid variant or native miR302 (G1P1 pair) on MIA-PaCa cells is shown. The vertical axis shows the relative DAPI staining area of each guide chain-passenger chain pair when the result of the G1P1 pair on the 4th day is 1. The horizontal axis shows the types of guide-passenger pairs, for each pair the left bar shows the results for day 4 and the bar on the right shows the results for day 7. * Indicates that there is a statistically significant difference compared to the results of the G1P1 pair. Ｐａｎｃ－１細胞に対するmiR302核酸改変体または天然型miR302（Ｇ１Ｐ１対）による処置の効果を示す。縦軸は、Ｇ１Ｐ１対の４日目の結果を１とした場合のそれぞれのガイド－パッセンジャー対の相対的なＤＡＰＩ染色面積を示す。横軸は、ガイド－パッセンジャー対の種類を示し、各対について左のバーは４日目の結果を示し、右のバーは７日目の結果を示す。＊は、Ｇ１Ｐ１対の結果と比較して統計的に有意な差があることを示す。The effect of treatment with miR302 nucleic acid variant or native miR302 (G1P1 pair) on Panc-1 cells is shown. The vertical axis shows the relative DAPI staining area of each guide-passenger pair when the result of the G1P1 pair on the 4th day is 1. The horizontal axis shows the types of guide-passenger pairs, for each pair the left bar shows the results for day 4 and the bar on the right shows the results for day 7. * Indicates that there is a statistically significant difference compared to the results of the G1P1 pair. Ｇ１Ｐ１対、ＯＵＭ３０２またはベヒクルをマウスに単回投与した場合の時間経過に伴う体重変化を示す。縦軸は体重（ｇ）を示す。横軸は、投与の日を０日目とした日数を示す。丸のマーカーはベヒクル投与群を、三角のマーカーはＯＵＭ３０２投与群を、四角のマーカーはＧ１Ｐ１対投与群をそれぞれ示す。The change in body weight over time when a single dose of G1P1 pair, OUM302 or vehicle is administered to mice is shown. The vertical axis shows the body weight (g). The horizontal axis shows the number of days with the day of administration as the 0th day. Circled markers indicate vehicle administration group, triangle markers indicate OUM302 administration group, and square markers indicate G1P1 pair administration group. Ｇ１Ｐ１対、ＯＵＭ３０２またはベヒクルをマウスに単回投与した場合の時間経過に伴う腫瘍体積変化を示す。縦軸は腫瘍体積（ｍｍ３）を示す。横軸は、投与の日を０日目とした日数を示す。丸のマーカーはベヒクル投与群を、三角のマーカーはＯＵＭ３０２投与群を、四角のマーカーはＧ１Ｐ１対投与群をそれぞれ示す。It shows the change in tumor volume over time when a single dose of G1P1 pair, OUM302 or vehicle is administered to mice. The vertical axis shows the tumor volume (mm 3 ). The horizontal axis shows the number of days with the day of administration as the 0th day. Circled markers indicate vehicle administration group, triangle markers indicate OUM302 administration group, and square markers indicate G1P1 pair administration group. Ｇ１Ｐ１対またはＯＵＭ３０２をゲムシタビンと組み合わせてマウスに単回投与した場合の時間経過に伴う体重変化を示す。縦軸は体重（ｇ）を示す。横軸は、投与の日を０日目とした日数を示す。丸のマーカーはＯＵＭ３０２＋ゲムシタビン投与群を、三角のマーカーはＧ１Ｐ１対＋ゲムシタビン投与群をそれぞれ示す。It shows the change in body weight over time when a single dose of G1P1 pair or OUM302 in combination with gemcitabine was administered to mice. The vertical axis shows the body weight (g). The horizontal axis shows the number of days with the day of administration as the 0th day. The circle markers indicate the OUM302 + gemcitabine administration group, and the triangular markers indicate the G1P1 pair + gemcitabine administration group. Ｇ１Ｐ１対またはＯＵＭ３０２をゲムシタビンと組み合わせてマウスに単回投与した場合の時間経過に伴う腫瘍体積変化を示す。縦軸は腫瘍体積（ｍｍ３）を示す。横軸は、投与の日を０日目とした日数を示す。丸のマーカーはＯＵＭ３０２＋ゲムシタビン投与群を、三角のマーカーはＧ１Ｐ１対＋ゲムシタビン投与群をそれぞれ示す。It shows the change in tumor volume over time when a single dose of G1P1 pair or OUM302 in combination with gemcitabine was administered to mice. The vertical axis shows the tumor volume (mm 3 ). The horizontal axis shows the number of days with the day of administration as the 0th day. The circle markers indicate the OUM302 + gemcitabine administration group, and the triangular markers indicate the G1P1 pair + gemcitabine administration group. Ｇ１Ｐ１対またはＯＵＭ３０２を５ＦＵ（５－フルオロウラシル）と組み合わせてマウスに単回投与した場合の時間経過に伴う体重変化を示す。縦軸は体重（ｇ）を示す。横軸は、投与の日を０日目とした日数を示す。丸のマーカーはＯＵＭ３０２＋５ＦＵ投与群を、三角のマーカーはＧ１Ｐ１対＋５ＦＵ投与群をそれぞれ示す。It shows the change in body weight over time when a single dose of G1P1 pair or OUM302 in combination with 5FU (5-fluorouracil) was administered to mice. The vertical axis shows the body weight (g). The horizontal axis shows the number of days with the day of administration as the 0th day. Circled markers indicate the OUM302 + 5FU administration group, and triangular markers indicate the G1P1 vs. + 5FU administration group. Ｇ１Ｐ１対またはＯＵＭ３０２を５ＦＵと組み合わせてマウスに単回投与した場合の時間経過に伴う腫瘍体積変化を示す。縦軸は腫瘍体積（ｍｍ３）を示す。横軸は、投与の日を０日目とした日数を示す。丸のマーカーはＯＵＭ３０２＋５ＦＵ投与群を、三角のマーカーはＧ１Ｐ１対＋５ＦＵ投与群をそれぞれ示す。It shows the change in tumor volume over time when a single dose of G1P1 pair or OUM302 in combination with 5FU was administered to mice. The vertical axis shows the tumor volume (mm 3 ). The horizontal axis shows the number of days with the day of administration as the 0th day. Circled markers indicate the OUM302 + 5FU administration group, and triangular markers indicate the G1P1 vs. + 5FU administration group. Ｇ１Ｐ１対またはＯＵＭ３０２をＴＡＳと組み合わせてマウスに単回投与した場合の時間経過に伴う体重変化を示す。縦軸は体重（ｇ）を示す。横軸は、投与の日を０日目とした日数を示す。丸のマーカーはＯＵＭ３０２＋ＴＡＳ投与群を、三角のマーカーはＧ１Ｐ１対＋ＴＡＳ投与群をそれぞれ示す。The change in body weight over time when a single dose of G1P1 pair or OUM302 in combination with TAS is shown in mice. The vertical axis shows the body weight (g). The horizontal axis shows the number of days with the day of administration as the 0th day. Circled markers indicate the OUM302 + TAS administration group, and triangular markers indicate the G1P1 pair + TAS administration group. Ｇ１Ｐ１対またはＯＵＭ３０２をＴＡＳ（トリフルリジン・チピラシル塩酸塩、ロンサーフ（登録商標））と組み合わせてマウスに単回投与した場合の時間経過に伴う腫瘍体積変化を示す。縦軸は腫瘍体積（ｍｍ３）を示す。横軸は、投与の日を０日目とした日数を示す。丸のマーカーはＯＵＭ３０２＋ＴＡＳ投与群を、三角のマーカーはＧ１Ｐ１対＋ＴＡＳ投与群をそれぞれ示す。It shows the change in tumor volume over time when a single dose of G1P1 pair or OUM302 in combination with TAS (trifluridine / tipiracil hydrochloride, Ronsurf®) is administered to mice. The vertical axis shows the tumor volume (mm 3 ). The horizontal axis shows the number of days with the day of administration as the 0th day. Circled markers indicate the OUM302 + TAS administration group, and triangular markers indicate the G1P1 pair + TAS administration group. ＴＡＳ単独、ＯＵＭ３０２＋ＴＡＳまたはＧ１Ｐ１対＋ＴＡＳを、１００ｍｍ３の腫瘍体積を有するマウスに単回投与した場合の時間経過に伴う腫瘍体積変化を示す。縦軸は腫瘍体積（ｍｍ３）を示す。横軸は、投与の日を０日目とした日数を示す。×のマーカーは対照処置群を、四角のマーカーはＴＡＳ単独投与群を、丸のマーカーはＯＵＭ３０２＋ＴＡＳ投与群を、三角のマーカーはＧ１Ｐ１対＋ＴＡＳ投与群をそれぞれ示す。It shows the change in tumor volume over time when TAS alone, OUM302 + TAS or G1P1 pair + TAS is administered once to a mouse having a tumor volume of 100 mm 3 . The vertical axis shows the tumor volume (mm 3 ). The horizontal axis shows the number of days with the day of administration as the 0th day. The x marker indicates the control treatment group, the square marker indicates the TAS single administration group, the circle marker indicates the OUM302 + TAS administration group, and the triangular marker indicates the G1P1 pair + TAS administration group. ベヒクル、Ｇ１Ｐ１対単独、ＯＵＭ３０２単独、ＯＵＭ３０２＋ＴＡＳまたはＧ１Ｐ１対＋ＴＡＳを、膵がん自然発症マウスに投与した場合の時間経過に伴う体重変化を示す。縦軸は体重（ｇ）を示す。横軸は、投与の日を０日目とした日数を示す。×のマーカーはベヒクル投与群を、四角のマーカーはＧ１Ｐ１対単独投与群を、丸のマーカーはＯＵＭ３０２単独投与群を、三角のマーカーはＯＵＭ３０２＋ＴＡＳ投与群を、Ｇ１Ｐ１対＋ＴＡＳ投与群をそれぞれ示す。It shows the change in body weight over time when vehicle, G1P1 pair alone, OUM302 alone, OUM302 + TAS or G1P1 pair + TAS is administered to spontaneously developing pancreatic cancer mice. The vertical axis shows the body weight (g). The horizontal axis shows the number of days with the day of administration as the 0th day. The x marker indicates the vehicle administration group, the square marker indicates the G1P1 vs. single administration group, the circle marker indicates the OUM302 alone administration group, the triangular marker indicates the OUM302 + TAS administration group, and the G1P1 vs + TAS administration group. ベヒクル、Ｇ１Ｐ１対単独またはＯＵＭ３０２単独を、膵がん自然発症マウスに投与した３２日後の腫瘍重量を示す。左のグラフでは左から、ベヒクル投与群、Ｇ１Ｐ１投与群、ＯＵＭ３０２投与群をそれぞれ示し、右のグラフでは左がＧ１Ｐ１投与群、右がＯＵＭ３０２投与群を示す。右のグラフは、左のグラフのＧ１Ｐ１投与群およびＯＵＭ３０２投与群を拡大したものである。縦軸は、腫瘍重量（ｍｇ）を示す。The tumor weight 32 days after administration of vehicle, G1P1 pair alone or OUM302 alone to spontaneously developing pancreatic cancer mice is shown. In the graph on the left, the vehicle administration group, the G1P1 administration group, and the OUM302 administration group are shown from the left, and in the right graph, the G1P1 administration group is shown on the left and the OUM302 administration group is shown on the right. The graph on the right is an enlargement of the G1P1 administration group and the OUM302 administration group in the graph on the left. The vertical axis shows the tumor weight (mg). ＦＢＳで処理した４種類の核酸の２０％アクリルアミド／ビスゲルによる電気泳動像を示す。両端は、20bp　DNA　ladder(タカラバイオ、滋賀)の結果である。左から、ｇ１、ｇ９、ｐ１、ｐ１０の処理結果のセットを示す。それぞれのセットにおいて、左から、ＦＢＳ添加なし、１５分間処理、３０分間処理、１時間処理、３時間処理、２４時間処理の結果を示す。An electrophoretic image of four types of nucleic acids treated with FBS with 20% acrylamide / bisgel is shown. Both ends are the result of 20bp DNA ladder (Takara Bio, Shiga). From the left, a set of processing results of g1, g9, p1, and p10 is shown. In each set, the results of no FBS addition, 15-minute treatment, 30-minute treatment, 1-hour treatment, 3-hour treatment, and 24-hour treatment are shown from the left. ＦＢＳで処理した２種類の核酸対の２％アガロースゲル（左）または２０％アクリルアミド／ビスゲル（右）による電気泳動像を示す。両端は、20bp　DNA　ladder(タカラバイオ、滋賀)の結果である。左はｇ１ｐ１対、右はｇ９ｐ１０対の処理結果のセットを示す。それぞれのセットにおいて、左はＦＢＳ添加なし、右はＦＢＳ１時間処理の結果を示す。An electrophoretic image of two FBS-treated nucleic acid pairs on a 2% agarose gel (left) or 20% acrylamide / bisgel (right) is shown. Both ends are the result of 20bp DNA ladder (Takara Bio, Shiga). The left shows a set of processing results of g1p1 pair and the right shows a set of processing results of g9p10 pair. In each set, the left shows the result of FBS addition and the right shows the result of FBS 1 hour treatment.
　以下、本開示を最良の形態を示しながら説明する。本明細書の全体にわたり、単数形の表現は、特に言及しない限り、その複数形の概念をも含むことが理解されるべきである。従って、単数形の冠詞（例えば、英語の場合は「ａ」、「ａｎ」、「ｔｈｅ」など）は、特に言及しない限り、その複数形の概念をも含むことが理解されるべきである。また、本明細書において使用される用語は、特に言及しない限り、当該分野で通常用いられる意味で用いられることが理解されるべきである。したがって、他に定義されない限り、本明細書中で使用される全ての専門用語および科学技術用語は、本開示の属する分野の当業者によって一般的に理解されるのと同じ意味を有する。矛盾する場合、本明細書（定義を含めて）が優先する。 Hereinafter, the present disclosure will be described while showing the best form. Throughout the specification, it should be understood that the singular representation also includes its plural concept, unless otherwise stated. Therefore, it should be understood that singular articles (eg, "a", "an", "the", etc. in English) also include the concept of their plural, unless otherwise noted. It should also be understood that the terms used herein are used in the meaning commonly used in the art unless otherwise noted. Thus, unless otherwise defined, all terminology and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In case of conflict, this specification (including definitions) takes precedence.
　以下に本明細書において特に使用される用語の定義および／または基本的技術内容を適宜説明する。 The definitions and / or basic technical contents of terms particularly used in the present specification will be described below as appropriate.
　本明細書において、「核酸」は当該分野において使用される通常の意味で用いられ、本明細書において「改変」とは、塩基の置換、欠失または挿入、あるいは塩基以外の化学構造（例えば、架橋核酸（ＢＮＡ）、ロックト核酸（ＬＮＡ）、ＡｍＮＡなどの修飾核酸）の任意の変更を指す。本明細書では、このような改変を少なくとも１つ含む物質を改変体と呼ぶ。 As used herein, "nucleic acid" is used in the usual sense as used in the art, and "modification" as used herein means substitution, deletion or insertion of a base, or a chemical structure other than a base (eg, for example. Refers to any modification of cross-linked nucleic acid (BNA), locked nucleic acid (LNA), modified nucleic acid such as AmNA). In the present specification, a substance containing at least one such modification is referred to as a variant.
　本明細書において「核酸改変体」とは、天然に存在する核酸に任意の改変（例えば、塩基の改変、化学構造の改変など）が導入された物質を指す。例えば、「miR302核酸改変体」は、天然に存在するmiR302に任意の改変（例えば、塩基の改変、化学構造の改変（例えば、修飾核酸の導入）など）が導入された物質を指す。本明細書において、特に断らない限り、「核酸改変体」には、改変とは異なる機能性部分（例えば、核酸改変体の検出、組織標的化、安定性向上および/または他の分子との結合を促進する部分）が導入された物質も包含されることが企図される。 As used herein, the term "nucleic acid variant" refers to a substance in which an arbitrary modification (for example, modification of a base, modification of a chemical structure, etc.) is introduced into a naturally occurring nucleic acid. For example, "miR302 nucleic acid variant" refers to a substance in which any modification (eg, base modification, chemical structure modification (eg, introduction of modified nucleic acid), etc.) has been introduced into naturally occurring miR302. In the present specification, unless otherwise specified, "nucleic acid variant" refers to a functional part different from the modified (for example, detection of nucleic acid variant, tissue targeting, stability improvement and / or binding to other molecules. It is intended that substances into which (parts that promote) have been introduced are also included.
　本明細書において「修飾核酸」は、ＤＮＡまたはＲＮＡにおいて使用される通常の核酸とは異なる構造をもつように改変された核酸をいう。修飾核酸の例としては、ロックト核酸（ＬＮＡ）、２’－Ｏ，４’－Ｃ－エチレン架橋核酸(2'-O,4'-C-ethylene　bridged　nucleic　acid、ENA)などのエチレン核酸、その他の架橋核酸（bridged　nucleic　acid、BNA)、ヘキシトール核酸（hexitol　nucleic　acid、HNA）、アミド架橋核酸（Amido-bridged　nucleic　acid、AmNA）、モルホリノ核酸、トリシクロ-DNA（tcDNA）、ポリエーテル核酸（例えば、米国特許第5,908,845号参照）、シクロヘキセン核酸（CeNA）、およびそれらの組み合わせを挙げることができる。ＢＮＡは、Bridged　Nucleic　Acidの他Bicyclic　Nucleic　Acidも意味するといわれており、「架橋型核酸」、「二環式核酸」あるいは「架橋／二環式核酸」ともいう。二環式核酸とは、核酸の２’位と４’位との間が連結され（架橋）され、環構造が２つ（二環式）となっている任意の修飾された核酸をいう。これらの修飾核酸は当該分野において公知であり、例えば、特許４７３１３２４号、Pradeep　S.　Pallan　et　al.,　Chem　Commun　(Camb).　2012　August　25;　48(66):　8195-8197.　doi:10.1039/c2cc32286bに記載される架橋核酸を用いることができる。 As used herein, the term "modified nucleic acid" refers to a nucleic acid modified so as to have a structure different from that of a normal nucleic acid used in DNA or RNA. Examples of modified nucleic acids include ethylene nucleic acids such as locked nucleic acids (LNA), 2'-O, 4'-C-ethylene bridged nucleic acids (2'-O, 4'-C-ethylene bridged nucleic acid, ENA), and others. Bridged nucleic acid (bridged nucleic acid, BNA), hexitol nucleic acid (hexitol nucleic acid, HNA), amide bridged nucleic acid (Amido-bridged nucleic acid, AmNA), morpholino nucleic acid, tricyclo-DNA (tcDNA), polyether nucleic acid (for example, US Pat. No. 5,908,845), cyclohexene nucleic acid (CeNA), and combinations thereof. BNA is said to mean Bicyclic Nucleic Acid as well as Bridged Nucleic Acid, and is also called "bridged nucleic acid", "bicyclic nucleic acid" or "bridged / bicyclic nucleic acid". The bicyclic nucleic acid refers to an arbitrary modified nucleic acid in which the 2'position and the 4'position of the nucleic acid are linked (crosslinked) to form two ring structures (bicyclic). These modified nucleic acids are known in the art, for example, Japanese Patent No. 4731324, Pradeep S. Pallan et al., Chem Communi (Camb). 2012 August 25; 48 (66): 8195-8197. Doi: 10.1039 / c2cc32286b The crosslinked nucleic acid described in the above can be used.
　本明細書において、塩基の「置換」とは、ある塩基（A、G、C、T、Uまたはその誘導体）を別の塩基に変更することを指す。本明細書において、塩基の「欠失」とは、ある塩基配列を対象の塩基が除かれた塩基配列に変更することを指す。本明細書において、塩基の「挿入」とは、ある塩基配列を対象の塩基が追加かれた塩基配列に変更することを指す。 In the present specification, "substitution" of a base means changing one base (A, G, C, T, U or a derivative thereof) to another base. As used herein, the term "deletion" of a base means changing a base sequence to a base sequence from which the target base has been removed. As used herein, the term "insertion" of a base means changing a base sequence to a base sequence to which a target base has been added.
　本明細書において、改変の「削除」とは、対象の改変が導入された改変体を、その改変が導入されていない状態の物質に変更することを指す。例えば、AAAGAAという塩基配列に対してGを欠失させる改変を導入した改変体（AAAAA）から、この改変を削除するとAAAGAAの塩基配列が与えられる。別の例では、リボースをデオキシリボースに置換する改変を導入した改変体から、この改変を削除すると改変体のこのデオキシリボースがリボースに置換された物質が与えられる。 In the present specification, "deletion" of a modification means to change a variant into which the target modification has been introduced to a substance in which the modification has not been introduced. For example, when this modification is deleted from a variant (AAAAA) in which a modification that deletes G from the base sequence AAAGAA is introduced, the base sequence of AAAGAA is given. In another example, from a variant that has introduced a modification that replaces ribose with deoxyribose, removing this modification gives a substance in which this deoxyribose of the variant is replaced with ribose.
　本明細書において使用される場合、「複合体」または「複合分子」とは、２以上の部分を含む任意の構成体を意味する。例えば、一方の部分が核酸である場合は、他方の部分は、核酸であってもよく、それ以外の物質（例えば、ポリペプチド、基材、糖、脂質、核酸、他の炭化水素等）であってもよい。本明細書において複合体を構成する２以上の部分は、共有結合で結合されていてもよくそれ以外の結合（例えば、水素結合、イオン結合、疎水性相互作用、ファンデルワールス力等）で結合されていてもよい。 As used herein, "complex" or "complex molecule" means any construct that includes two or more moieties. For example, if one part is a nucleic acid, the other part may be a nucleic acid, with other substances (eg, polypeptides, substrates, sugars, lipids, nucleic acids, other hydrocarbons, etc.). There may be. In the present specification, two or more portions constituting the complex may be bonded by a covalent bond or other bonds (for example, hydrogen bond, ionic bond, hydrophobic interaction, van der Waals force, etc.). It may have been done.
　本明細書において「置換基」とは核酸改変体の化学構造中で，ある化学基を他のものを置換した原子または官能基をいう。 As used herein, the term "substituent" refers to an atom or functional group in which one chemical group is substituted with another in the chemical structure of a nucleic acid variant.
　本明細書において「基」なる用語は、１価基を意味する。例えば、「アルキル基」は、１価の飽和炭化水素基を意味する。また、本明細書における置換基の説明において、「基」なる用語を省略する場合もある。尚、「置換または非置換の」もしくは「置換されていてもよい」または「置換されている」で定義される基における置換基の数は、置換可能であれば特に制限はなく、１または複数である。また、特に指示した場合を除き、各々の基の説明はその基が他の基の一部分または置換基である場合にも該当する。本明細書において「置換または非置換の」は「置換されていてもよい」と交換可能に使用され、ある特定の基がさらなる置換基でも置き換えられていてもよく、置換基を有しなくてもよいことを意味する。 The term "base" in the present specification means a monovalent group. For example, "alkyl group" means a monovalent saturated hydrocarbon group. In addition, the term "group" may be omitted in the description of the substituent in the present specification. The number of substituents in the groups defined by "substitutable or unsubstituted", "may be substituted", or "substituted" is not particularly limited as long as it can be substituted, and may be one or more. Is. Unless otherwise indicated, the description of each group also applies when the group is part of another group or a substituent. In the present specification, "substituted or unsubstituted" is used interchangeably with "may be substituted", and a specific group may be further substituted or substituted and has no substituent. It means that it is also good.
　本明細書で使用されるmiRNAまたは核酸改変体において使用され得る置換基としては、アルキル、シクロアルキル、アルケニル、シクロアルケニル、アルキニル、シクロアルキニル、アルコキシ、炭素環基、ヘテロ環基、ハロゲン、ヒドロキシ、チオール、シアノ、ニトロ、アミノ、カルボキシ、カルバモイル、アシル、アシルアミノ、チオカルボキシ、アミド、置換されたカルボニル、置換されたチオカルボニル、置換されたスルホニルまたは置換されたスルフィニルが挙げられるがそれらに限定されない。置換基は、すべてが水素以外の置換基を有していても良い。 Substituents that can be used in the miRNA or nucleic acid variants used herein include alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, alkoxy, carbocyclic group, heterocyclic group, halogen, hydroxy, Examples include, but are not limited to, thiols, cyanos, nitros, aminos, carboxys, carbamoyls, acyls, acylaminos, thiocarboxys, amides, substituted carbonyls, substituted thiocarbonyls, substituted sulfonyls or substituted sulfinyls. All of the substituents may have a substituent other than hydrogen.
　本明細書においては、特に言及がない限り、置換は、ある有機化合物または置換基中の１または２以上の水素原子を他の原子または原子団で置き換えるか、または二重結合もしくは三重結合とすることをいう。水素原子を１つ除去して１価の置換基に置換するかまたは単結合と一緒にして二重結合とすることも可能であり、そして水素原子を２つ除去して２価の置換基に置換するか、または単結合と一緒にして三重結合とすることも可能である。 In the present specification, unless otherwise specified, the substitution shall replace one or more hydrogen atoms in an organic compound or substituent with another atom or atomic group, or shall be a double bond or a triple bond. Say that. It is also possible to remove one hydrogen atom and replace it with a monovalent substituent, or combine it with a single bond to form a double bond, and remove two hydrogen atoms to make a divalent substituent. It can be substituted or combined with a single bond to form a triple bond.
　本明細書において「アルキル（基）」とは、メタン、エタン、プロパンのような脂肪族炭化水素（アルカン）から水素原子が一つ失われて生ずる１価の基をいい、一般にＣｎＨ２ｎ＋１－で表される（ここで、ｎは正の整数である）。アルキルは、直鎖または分枝鎖であり得る。アルキル（基）は置換されていてもよく置換されていなく（非置換であっ）てもよい。ここで、アルキルの具体例は、Ｃ１～Ｃ２アルキル、Ｃ１～Ｃ３アルキル、Ｃ１～Ｃ４アルキル、Ｃ１～Ｃ５アルキル、Ｃ１～Ｃ６アルキル、Ｃ１～Ｃ７アルキル、Ｃ１～Ｃ８アルキル、Ｃ１～Ｃ９アルキル、Ｃ１～Ｃ１０アルキル、Ｃ１～Ｃ１１アルキルまたはＣ１～Ｃ２０アルキル、Ｃ１～Ｃ２置換アルキル、Ｃ１～Ｃ３置換アルキル、Ｃ１～Ｃ４置換アルキル、Ｃ１～Ｃ５置換アルキル、Ｃ１～Ｃ６置換アルキル、Ｃ１～Ｃ７置換アルキル、Ｃ１～Ｃ８置換アルキル、Ｃ１～Ｃ９置換アルキル、Ｃ１～Ｃ１０置換アルキル、Ｃ１～Ｃ１１置換アルキルまたはＣ１～Ｃ２０置換アルキルであり得る。ここで、たとえばＣ１～Ｃ１０アルキルとは、炭素原子を１～１０個有する直鎖または分枝状のアルキルを意味する。本明細書において「置換アルキル」とは、本明細書に規定する置換基によってアルキルのＨが置換されたアルキルをいう。具体的には、これらに限定されるものではないが、ＣＨ３ＯＣＨ２－、ＣＨ３ＯＣＨ２ＣＨ２－、ＣＨ３ＯＣＨ２ＣＨ２ＣＨ２－、ＨＯＣＨ２－、ＨＯＣＨ２ＣＨ２－、ＨＯＣＨ２ＣＨ２ＣＨ２－、ＮＣＣＨ２－、ＮＣＣＨ２ＣＨ２－、ＮＣＣＨ２ＣＨ２ＣＨ２－、ＦＣＨ２－、ＦＣＨ２ＣＨ２－、ＦＣＨ２ＣＨ２ＣＨ２－、Ｈ２ＮＣＨ２－、Ｈ２ＮＣＨ２ＣＨ２－、Ｈ２ＮＣＨ２ＣＨ２ＣＨ２－、ＨＯＯＣＣＨ２－、ＨＯＯＣＣＨ２ＣＨ２－、ＨＯＯＣＣＨ２ＣＨ２ＣＨ２－、Ｃ６Ｈ５－ＣＨ２－（ベンジル基）が挙げられる。 As used herein, the term "alkyl (group)" refers to a monovalent group generated by the loss of one hydrogen atom from an aliphatic hydrocarbon (alkane) such as methane, ethane, or propane, and generally means C n H 2n + 1. It is represented by-(where n is a positive integer). Alkyl can be straight or branched. The alkyl (group) may be substituted or unsubstituted (unsubstituted). Here, specific examples of alkyl are C 1 to C 2 alkyl, C 1 to C 3 alkyl, C 1 to C 4 alkyl, C 1 to C 5 alkyl, C 1 to C 6 alkyl, and C 1 to C 7 alkyl. , C 1 to C 8 alkyl, C 1 to C 9 alkyl, C 1 to C 10 alkyl, C 1 to C 11 alkyl or C 1 to C 20 alkyl, C 1 to C 2 substituted alkyl, C 1 to C 3 substituted Alkyl, C 1 to C 4 substituted alkyl, C 1 to C 5 substituted alkyl, C 1 to C 6 substituted alkyl, C 1 to C 7 substituted alkyl, C 1 to C 8 substituted alkyl, C 1 to C 9 substituted alkyl, It can be a C 1 to C 10 substituted alkyl, a C 1 to C 11 substituted alkyl or a C 1 to C 20 substituted alkyl. Here, for example, C 1 to C 10 alkyl means a linear or branched alkyl having 1 to 10 carbon atoms. As used herein, the term "substituted alkyl" refers to an alkyl in which the H of the alkyl is substituted by the substituents specified herein. Specifically, but not limited to these, CH 3 OCH 2- , CH 3 OCH 2 CH 2- , CH 3 OCH 2 CH 2 CH 2- , HOCH 2- , HOCH 2 CH 2- , HOCH 2 CH 2 CH 2- , NCCH 2- , NCCH 2 CH 2- , NCCH 2 CH 2 CH 2- , FCH 2- , FCH 2 CH 2- , FCH 2 CH 2- CH 2- , H 2 NCH 2- , H 2 NCH 2 CH 2- , H 2 NCH 2 CH 2 CH 2- , HOOCCH 2- , HOOCCH 2 CH 2- , HOOCCH 2 CH 2 CH 2- , C 6 H 5- CH 2- (benzyl group) can be mentioned. ..
　本明細書において「アルケニル」とは、分子内に二重結合を一つ有する脂肪族炭化水素から水素原子が一つ失われて生ずる１価の基をいい、一般にＣｎＨ２ｎ－１－で表される（ここで、ｎは２以上の正の整数である）。「置換アルケニル」とは、上述の置換基によってアルケニルの水素が置換されたアルケニルをいう。具体例としては、Ｃ２～Ｃ３アルケニル、Ｃ２～Ｃ４アルケニル、Ｃ２～Ｃ５アルケニル、Ｃ２～Ｃ６アルケニル、Ｃ２～Ｃ７アルケニル、Ｃ２～Ｃ８アルケニル、Ｃ２～Ｃ９アルケニル、Ｃ２～Ｃ１０アルケニル、Ｃ２～Ｃ１１アルケニルまたはＣ２～Ｃ２０アルケニル、Ｃ２～Ｃ３置換アルケニル、Ｃ２～Ｃ４置換アルケニル、Ｃ２～Ｃ５置換アルケニル、Ｃ２～Ｃ６置換アルケニル、Ｃ２～Ｃ７置換アルケニル、Ｃ２～Ｃ８置換アルケニル、Ｃ２～Ｃ９置換アルケニル、Ｃ２～Ｃ１０置換アルケニル、Ｃ２～Ｃ１１置換アルケニルまたはＣ２～Ｃ２０置換アルケニルであり得る。ここで、たとえばＣ２～Ｃ１０アルキルとは、炭素原子を２～１０個含む直鎖または分枝状のアルケニルを意味する。また、たとえば、Ｃ２～Ｃ１０置換アルケニルとは、Ｃ２～Ｃ１０アルケニルであって、そのうち１または複数の水素原子が置換基により置換されているものをいう。 As used herein, the term "alkenyl" refers to a monovalent group produced by the loss of one hydrogen atom from an aliphatic hydrocarbon having one double bond in the molecule, and is generally C n H 2n-1- . Represented (where n is a positive integer greater than or equal to 2). "Substituted alkenyl" refers to an alkenyl in which hydrogen of the alkenyl is substituted by the above-mentioned substituent. Specific examples, C 2 ~ C 3 alkenyl, C 2 ~ C 4 alkenyl, C 2 ~ C 5 alkenyl, C 2 ~ C 6 alkenyl, C 2 ~ C 7 alkenyl, C 2 ~ C 8 alkenyl, C 2 ~ C 9 alkenyl, C 2 to C 10 alkenyl, C 2 to C 11 alkenyl or C 2 to C 20 alkenyl, C 2 to C 3 substituted alkenyl, C 2 to C 4 substituted alkenyl, C 2 to C 5 substituted alkenyl, C 2 to C 6 substituted alkenyl, C 2 to C 7 substituted alkenyl, C 2 to C 8 substituted alkenyl, C 2 to C 9 substituted alkenyl, C 2 to C 10 substituted alkenyl, C 2 to C 11 substituted alkenyl or C 2 to It is a C 20 substituted alkenyl. Here, for example C The 2 ~ C 10 alkyl means a straight or branched alkenyl containing 2 to 10 carbon atoms. Further, for example, a C 2 ~ C 10 substituted alkenyl, a C 2 ~ C 10 alkenyl, of which 1 or more hydrogen atoms refer to those substituted by a substituent.
　本明細書において「アルキニル」とは、分子内に三重結合を一つ有する脂肪族炭化水素から水素原子が一つ失われて生ずる１価の基をいい、一般にＣｎＨ２ｎ－３－で表される（ここで、ｎは２以上の正の整数である）。「置換アルキニル」とは、上述の置換基によってアルキニルの水素が置換されたアルキニルをいう。 As used herein, the term "alkynyl" refers to a monovalent group generated by the loss of one hydrogen atom from an aliphatic hydrocarbon having one triple bond in the molecule, and is generally represented by C n H 2n-3-. (Here, n is a positive integer greater than or equal to 2). The "substituted alkynyl" refers to an alkynyl in which the hydrogen of the alkynyl is substituted by the above-mentioned substituent.
　本明細書において「アリール（基）」とは、芳香族炭化水素の環に結合する水素原子が１個離脱して生ずる基をいい、本明細書において、炭素環基に包含される。ベンゼンからはフェニル基（Ｃ６Ｈ５－）、トルエンからはトリル基（ＣＨ３Ｃ６Ｈ４－）、キシレンからはキシリル基（（ＣＨ３）２Ｃ６Ｈ３－）、ナフタレンからはナフチル基（Ｃ１０Ｈ８－）が誘導される。 As used herein, the term "aryl (group)" refers to a group formed by the detachment of one hydrogen atom bonded to a ring of an aromatic hydrocarbon, and is included in the carbon ring group in the present specification. Phenyl from benzene (C 6 H 5 -), tolyl from toluene (CH 3 C 6 H 4 - ), xylyl from xylene ((CH 3) 2 C 6 H 3 -), naphthyl naphthalene The group (C 10 H 8- ) is induced.
　本明細書において「アルコキシ（基）」は、アルキル基の水素がヒドロキシに置き換わった基であり、「アルキル（基）」部分は、前記「アルキル（基）」と同義である。好ましくは、「Ｃ１～６アルコキシ（基）」である。「Ｃ１～６アルコキシ（基）」の具体例としては、メトキシ、エトキシ、プロポキシ、イソプロポキシ、ブトキシ、イソブトキシ、ｓｅｃ－ブトキシ、ｔｅｒｔ－ブトキシ等が挙げられる。 In the present specification, "alkoxy (group)" is a group in which hydrogen of an alkyl group is replaced with hydroxy, and the "alkyl (group)" portion is synonymous with the above-mentioned "alkyl (group)". Preferably, it is "C 1 to 6 alkoxy (group)". Specific examples of "C 1 to 6 alkoxy (group)" include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy and the like.
　本明細書において「ハロゲン（原子）」は、例えばフッ素原子、塩素原子、臭素原子またはヨウ素原子等が挙げられる。好ましくは、フッ素原子、または塩素原子である。 In the present specification, the "halogen (atom)" includes, for example, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom and the like. It is preferably a fluorine atom or a chlorine atom.
　本明細書において「アラビノ型」ヌクレオチドまたは核酸とは糖（ペントース）として、アラビノースを有するヌクレオチドまたは核酸をいう。 In the present specification, the "arabino-type" nucleotide or nucleic acid means a nucleotide or nucleic acid having arabinose as a sugar (pentose).
　ヌクレオチドまたは核酸は一般に認知された１文字コードにより言及され得る。アミノ酸は、その一般に公知の３文字記号か、またはIUPAC－IUB　Biochemical　Nomenclature　Commissionにより推奨される１文字記号のいずれかにより、本明細書中で言及され得る。本明細書では、アミノ酸配列および塩基配列の類似性、同一性および相同性の比較は、配列分析用ツールであるＢＬＡＳＴを用いてデフォルトパラメータを用いて算出される。同一性の検索は例えば、ＮＣＢＩのBLAST　2.8.1（2019．1.4発行）を用いて行うことができる。本明細書における同一性の値は通常は上記BLASTを用い、デフォルトの条件でアラインした際の値をいう。ただし、パラメータの変更により、より高い値が出る場合は、最も高い値を同一性の値とする。複数の領域で同一性が評価される場合はそのうちの最も高い値を同一性の値とする。類似性は、同一性に加え、類似のアミノ酸についても計算に入れた数値である。 Nucleotides or nucleic acids can be referred to by a generally recognized one-letter code. Amino acids may be referred to herein by either their generally known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. In the present specification, comparison of amino acid sequence and base sequence similarity, identity and homology is calculated using default parameters using BLAST, a tool for sequence analysis. The identity search can be performed using, for example, NCBI's BLAST 2.8.1 (issued on January 1.4, 2019). The value of identity in the present specification usually refers to the value when the above BLAST is used and aligned under the default conditions. However, if a higher value is obtained by changing the parameter, the highest value is set as the identity value. When identity is evaluated in multiple regions, the highest value among them is set as the identity value. Similarity is a numerical value that takes into account similar amino acids in addition to identity.
　本明細書において、「U」はウラシル、「T」はチミン、「A」はアデニン、「C」はシトシンの各塩基をそれぞれ意味する。「(M)」は、この記号の左のリボヌクレオチド単位中のリボースの２’のヒドロキシル基がメトキシ基に置き換えられている改変を表す。「(L)」は、この記号の左のリボヌクレオチド単位中のリボースがLNAで置き換えられている改変を表す。「^」は、両隣のリボヌクレオチド単位の間のリン酸基の結合に関与しない一つの酸素原子が硫黄原子に置き換えられている改変を表す。 In this specification, "U" means uracil, "T" means thymine, "A" means adenine, and "C" means cytosine. "(M)" represents a modification in which the 2'hydroxyl group of ribose in the ribonucleotide unit to the left of this symbol is replaced with a methoxy group. "(L)" represents a modification in which ribose in the ribonucleotide unit to the left of this symbol is replaced by LNA. “^” Represents a modification in which one oxygen atom that is not involved in the bond of a phosphate group between adjacent ribonucleotide units is replaced with a sulfur atom.
　本明細書において「手段」とは、ある目的（例えば、検出、診断、治療）を達成する任意の道具となり得るものをいい、特に、本明細書では、「選択的に認識（検出）する手段」とは、ある対象を他のものとは異なって認識（検出）することができる手段をいう。 As used herein, the term "means" means a tool that can be an arbitrary tool for achieving a certain purpose (for example, detection, diagnosis, treatment), and in particular, in the present specification, "means for selective recognition (detection)". "" Means a means by which an object can be recognized (detected) differently from another.
　本明細書において「標識」とは、目的となる分子または物質を他から識別するための存在（例えば、物質、エネルギー、電磁波など）をいう。そのような標識方法としては、ＲＩ（ラジオアイソトープ）法、安定同位体標識法、蛍光法、ビオチン法、ラマン散乱を利用した光学手法、化学発光法等を挙げることができる。 As used herein, the term "label" refers to an entity (eg, substance, energy, electromagnetic wave, etc.) for identifying a target molecule or substance from others. Examples of such labeling methods include RI (radioisotope) method, stable isotope labeling method, fluorescence method, biotin method, optical method using Raman scattering, chemiluminescence method and the like.
　本明細書において「被験体（者）」とは、本開示の診断または検出、あるいは治療等の対象となる対象（例えば、ヒト等の生物または生物から取り出した細胞、血液、血清等）をいう。 As used herein, the term "subject (person)" refers to an organism subject to the diagnosis, detection, or treatment of the present disclosure (for example, an organism such as a human being, cells taken from an organism, blood, serum, etc.). ..
　本明細書において「薬剤」、「剤」または「因子」（いずれも英語ではagentに相当する）は、広義には、交換可能に使用され、意図する目的を達成することができる限りどのような物質または他の要素（例えば、光、放射能、熱、電気などのエネルギー）でもあってもよい。そのような物質としては、例えば、タンパク質、ポリペプチド、オリゴペプチド、ペプチド、ポリヌクレオチド、オリゴヌクレオチド、ヌクレオチド、核酸（例えば、ｃＤＮＡ、ゲノムＤＮＡのようなＤＮＡ、ｍＲＮＡのようなＲＮＡを含む）、ポリサッカリド、オリゴサッカリド、脂質、有機低分子（例えば、ホルモン、リガンド、情報伝達物質、有機低分子、コンビナトリアルケミストリで合成された分子、医薬品として利用され得る低分子（例えば、低分子リガンドなど）など）、これらの複合分子が挙げられるがそれらに限定されない。 As used herein, "drug", "drug" or "factor" (both corresponding to agents in English) are used interchangeably in any sense as long as they can achieve their intended purpose. It may also be a substance or other element (eg, energy such as light, radioactivity, heat, electricity). Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including, for example, cDNA, DNA such as genomic DNA, RNA such as mRNA), poly. Saccharides, oligosaccharides, lipids, organic small molecules (eg, hormones, ligands, signaling substances, organic small molecules, molecules synthesized with combinatorial chemistries, small molecules that can be used as pharmaceuticals (eg, small molecule ligands, etc.)) , But are not limited to these complex molecules.
　本明細書において「治療」とは、ある疾患または障害（例えば、がん）について、そのような状態になった場合に、そのような疾患または障害の悪化を防止、好ましくは、現状維持、より好ましくは、軽減、さらに好ましくは消退させることをいい、患者の疾患、もしくは疾患に伴う1つ以上の症状の、症状改善効果あるいは予防効果を発揮しうることを含む。事前に診断を行って適切な治療を行うことは「コンパニオン治療」といい、そのための診断薬を「コンパニオン診断薬」ということがある。 As used herein, the term "treatment" refers to a disease or disorder (eg, cancer) that, when such a condition occurs, prevents the exacerbation of such disease or disorder, preferably maintains the status quo. It preferably refers to alleviation, more preferably withdrawal, and includes the ability to exert a symptom-improving effect or a preventive effect on a patient's disease or one or more symptoms associated with the disease. Diagnosis in advance and appropriate treatment is called "companion treatment", and the diagnostic agent for that purpose is sometimes called "companion diagnostic agent".
　本明細書において「治療薬（剤）」とは、広義には、目的の状態（例えば、がん等の疾患など）を治療できるあらゆる薬剤をいう。本開示の一実施形態において「治療薬」は、有効成分と、薬理学的に許容される1つもしくはそれ以上の担体とを含む医薬組成物であってもよい。医薬組成物は、例えば有効成分と上記担体とを混合し、製剤学の技術分野において知られる任意の方法により製造できる。また治療薬は、治療のために用いられる物であれば使用形態は限定されず、有効成分単独であってもよいし、有効成分と任意の成分との混合物であってもよい。また上記担体の形状は特に限定されず、例えば、固体または液体（例えば、緩衝液）であってもよい。なおがん等の治療薬は、がん等の予防のために用いられる薬物（予防薬）、またはがん等の抑制剤を含む。 In the present specification, the "therapeutic agent (drug)" means any drug that can treat a target condition (for example, a disease such as cancer) in a broad sense. In one embodiment of the present disclosure, the "therapeutic agent" may be a pharmaceutical composition comprising an active ingredient and one or more pharmacologically acceptable carriers. The pharmaceutical composition can be produced, for example, by mixing the active ingredient with the carrier and any method known in the technical field of pharmaceutics. Further, the therapeutic agent is not limited in the form of use as long as it is used for treatment, and may be the active ingredient alone or a mixture of the active ingredient and an arbitrary ingredient. The shape of the carrier is not particularly limited, and may be, for example, a solid or a liquid (for example, a buffer solution). The therapeutic agent for cancer or the like includes a drug (preventive agent) used for prevention of cancer or the like or an inhibitor for cancer or the like.
　本明細書において「予防」とは、ある疾患または障害（例えば、がん等の疾患など）について、そのような状態になる前に、そのような状態にならないようにすることをいう。本開示の薬剤を用いて、診断を行い、必要に応じて本開示の薬剤を用いて例えば、がん等の予防をするか、あるいは予防のための対策を講じることができる。 In the present specification, "prevention" means to prevent a certain disease or disorder (for example, a disease such as cancer) from becoming such a state before it becomes such a state. The agents of the present disclosure can be used to make a diagnosis, and if necessary, the agents of the present disclosure can be used to prevent, for example, cancer, or to take preventive measures.
　本明細書において「予防薬（剤）」とは、広義には、目的の状態（例えば、がん等の疾患など）を予防できるあらゆる薬剤をいう。 In the present specification, the term "preventive drug (drug)" means any drug that can prevent a target condition (for example, a disease such as cancer) in a broad sense.
　本明細書において「キット」とは、通常２つ以上の区画に分けて、提供されるべき部分（例えば、検査薬、診断薬、治療薬、抗体、標識、説明書など）が提供されるユニットをいう。安定性等のため、混合されて提供されるべきでなく、使用直前に混合して使用することが好ましいような組成物の提供を目的とするときに、このキットの形態は好ましい。そのようなキットは、好ましくは、提供される部分（例えば、検査薬、診断薬、治療薬をどのように使用するか、あるいは、試薬をどのように処理すべきかを記載する指示書または説明書を備えていることが有利である。本明細書においてキットが試薬キットとして使用される場合、キットには、通常、検査薬、診断薬、治療薬、抗体等の使い方などを記載した指示書などが含まれる。 As used herein, a "kit" is a unit in which parts to be provided (for example, a test drug, a diagnostic drug, a therapeutic drug, an antibody, a label, an instruction manual, etc.) are usually divided into two or more sections. To say. The form of this kit is preferred when the purpose is to provide a composition that should not be mixed and provided for stability and the like, but is preferably mixed and used immediately before use. Such kits are preferably instructions or instructions describing how to use the provided parts (eg, test agents, diagnostic agents, therapeutic agents, or how reagents should be treated). When the kit is used as a reagent kit in the present specification, the kit usually includes an instruction manual or the like that describes how to use a test drug, a diagnostic drug, a therapeutic drug, an antibody, or the like. Is included.
　本明細書において「指示書」は、本開示を使用する方法を医師または他の使用者に対する説明を記載したものである。この指示書は、本開示の検出方法、診断薬の使い方、または医薬などを投与することを指示する文言が記載されている。また、指示書には、投与部位として、経口、食道への投与（例えば、注射などによる）することを指示する文言が記載されていてもよい。この指示書は、本開示が実施される国の監督官庁（例えば、日本であれば厚生労働省、米国であれば食品医薬品局（ＦＤＡ）など）が規定した様式に従って作成され、その監督官庁により承認を受けた旨が明記される。指示書は、いわゆる添付文書（package　insert）であり、通常は紙媒体で提供されるが、それに限定されず、例えば、電子媒体（例えば、インターネットで提供されるホームページ、電子メール）のような形態でも提供され得る。 In this specification, the "instruction" describes the method of using this disclosure to a doctor or another user. This instruction sheet contains words instructing the detection method of the present disclosure, how to use a diagnostic agent, or administration of a medicine or the like. In addition, the instruction sheet may include a wording instructing the administration site to be administered orally or to the esophagus (for example, by injection). This instruction is prepared and approved by the regulatory agency of the country in which this disclosure is implemented (eg, Ministry of Health, Labor and Welfare in Japan, Food and Drug Administration (FDA) in the United States, etc.). It is clearly stated that it has been received. The instruction sheet is a so-called package insert, which is usually provided in a paper medium, but is not limited thereto, and is in a form such as an electronic medium (for example, a homepage provided on the Internet, an e-mail). But can be provided.
（miR302核酸改変体）
　一つの局面において、本開示は、miR302核酸改変体を提供する。一つの実施形態では、ヒトmiR302核酸改変体が提供され、より具体的には、ヒトmiR302a核酸改変体が提供される。 (MiR302 nucleic acid variant)
In one aspect, the present disclosure provides a miR302 nucleic acid variant. In one embodiment, a human miR302 nucleic acid variant is provided, and more specifically, a human miR302a nucleic acid variant is provided.
　本明細書において「miR302」とは、マイクロRNAの一種であり、miR-302/367クラスターを形成するといわれるため、このクラスターに属する任意のマイクロRNAがこの範疇に入る。少なくともmiR-367、miR-302d、miR-302a、miR-302cおよびmiR-302bの５つのメンバーが含まれており（例えば、Gao　Z,　Zhu　X,　Dou　Y.　The　miR-302/367　cluster:　a　comprehensive　update　on　its　evolution　and　functions.　Open　Biol.2015;5(12):150138.　doi:10.1098/rsob.150138を参照。https://www.addgene.org/98748/もまた参照）、http://www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000071（2019年4月10日アクセス）によれば、miRNA302だけで４９種類の配列が登録されている。ヒトmiR302は、miR302のうち、ヒトに由来する任意のマイクロRNAを指す。 In the present specification, "miR302" is a kind of microRNA and is said to form a miR-302 / 367 cluster, so any microRNA belonging to this cluster falls into this category. It contains at least five members, miR-367, miR-302d, miR-302a, miR-302c and miR-302b (eg Gao Z, Zhu X, Dou Y. The miR-302 / 367 cluster: a comprehensive update on its evolution and functions. Open Biol.2015; 5 (12): 150138. See doi: 10.1098 / rsob.150138. See also https://www.addgene.org/98748/), http: / According to /www.mirbase.org/cgi-bin/mirna_summary.pl?fam=MIPF0000071 (accessed April 10, 2019), 49 types of sequences are registered for miRNA302 alone. Human miR302 refers to any human-derived microRNA of miR302.
　天然のヒトmiR302aは以下の塩基配列を有するマイクロRNAである。
5'-CCACCACUUAAACGUGGAUGUACUUGCUUUGAAACUAAAGAAGUAAGUGCUUCCAUGUUUUGGUGAUGG-3'（配列番号１） Natural human miR302a is a microRNA having the following base sequence.
5'-CCACCACUUAAACGUGGAUGUACUUGCUUUGAAACUAAAGAAGUAAGUGCUUCCAUGUUUUGGUGAUGG-3'(SEQ ID NO: 1)
　天然のヒトmiR302a分子は以下に示すステム－ループ構造を形成すると考えられる。
Figure JPOXMLDOC01-appb-C000005
本明細書において、このステム－ループ構造の上半分に対応すると考えられる核酸をパッセンジャー鎖（ｐ）、このステム－ループ構造の下半分に対応すると考えられる核酸をガイド鎖（ｇ）と呼ぶことがある。 The natural human miR302a molecule is thought to form the stem-loop structure shown below.
Figure JPOXMLDOC01-appb-C000005
In the present specification, the nucleic acid considered to correspond to the upper half of this stem-loop structure is referred to as a passenger strand (p), and the nucleic acid considered to correspond to the lower half of this stem-loop structure is referred to as a guide strand (g). is there.
　一つの実施形態では、本開示のmiR302核酸改変体は、配列番号１の連続した５～７０個の任意の塩基（例えば、５、６、７、８、９、１０、１１、１２、１３、１４、１５、１６、１７、１８、１９、２０、２１、２２、２３、２４、２５、２６、２７、２８、２９、３０、３５、４０、４５、５０、５５、６０、６５または７０個の塩基）からなる塩基配列から０～１０個（例えば、０、１、２、３、４、５、６、７、８、９または１０個）の改変（例えば、置換、欠失または挿入）を含む塩基配列を含んでもよいし、からなってもよい。改変後の塩基配列には、アデニン（A）、グアニン（G)、シトシン（C)、ウラシル（U)およびチミン（T)以外の塩基（例えば、メチル化アデニンなどの任意のこれらの塩基のアルキル化体（例えば、メチル化体））が含まれていてもよい。一つの実施形態では、本開示のmiR302核酸改変体分子は、５～５００の任意の塩基長（例えば、５、６、７、８、９、１０、１１、１２、１３、１４、１５、１６、１７、１８、１９、２０、２１、２２、２３、２４、２５、２６、２７、２８、２９、３０、３５、４０、４５、５０、５５、６０、６５、７０、８０、９０、１００、１１０、１２０、１３０、１４０、１５０、２００、２５０、３００、３５０、４００、４５０または５００の塩基長）を有し得る。 In one embodiment, the miR302 nucleic acid variant of the present disclosure comprises 5 to 70 consecutive arbitrary bases of SEQ ID NO: 1, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45,50,55,60,65 or 70 pieces Modifications (eg, substitutions, deletions or insertions) of 0 to 10 (eg, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) from the base sequence consisting of It may contain or consist of a base sequence containing. The modified base sequence includes alkyl bases other than adenine (A), guanine (G), cytosine (C), uracil (U) and thymine (T) (eg, alkyl of any of these bases such as methylated adenine). Chemicals (eg, methylated) may be included. In one embodiment, the miR302 nucleic acid variant molecules of the present disclosure have an arbitrary base length of 5 to 500 (eg, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16). , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90, 100 , 110, 120, 130, 140, 150, 200, 250, 300, 350, 400, 450 or 500 base lengths).
　一つの実施形態では、本開示のmiR302核酸改変体は、
UAAGUGCUUCCAUGUUUUGGUGA（ｇ１）(配列番号２)または
ACUUAAACGUGGAUGUACUUGCU（ｐ１）（配列番号３)
の塩基配列から０、１、２、３、４、５、６、７、８、９または１０個の置換、欠失または挿入を含む塩基配列を含み得る。配列番号２および配列番号３の配列は両方とも、配列番号１の連続した２３の塩基配列である。 In one embodiment, the miR302 nucleic acid variants of the present disclosure are:
UAAGUGCUUCCAUGUUUUGGUGA (g1) (SEQ ID NO: 2) or
ACUUAAACGUGGAUGUACUUGCU (p1) (SEQ ID NO: 3)
Can contain a base sequence containing 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, deletions or insertions from the base sequence of. Both the sequences of SEQ ID NO: 2 and SEQ ID NO: 3 are 23 consecutive base sequences of SEQ ID NO: 1.
　一つの実施形態では、本開示のmiR302核酸改変体は、
UAAGUGCUUCCAUGUUUUGGUGA（ｇ１）（配列番号２)の８番目および１８番目の塩基の少なくとも一方、または
ACUUAAACGUGGAUGUACUUGCU（ｐ１）(配列番号３)の４番目および１４番目の塩基の少なく
とも一方
に対応する位置において塩基の置換、欠失または挿入を含む塩基配列を含み得る。配列番号２の８番目および１８番目の塩基は改変しても、その機能への影響が小さいと予測される。配列番号３の４番目および１４番目の塩基は改変しても、その機能への影響が小さいと予測される。 In one embodiment, the miR302 nucleic acid variants of the present disclosure are:
At least one of the 8th and 18th bases of UAAGUGCUUCCAUGUUUUGGUGA (g1) (SEQ ID NO: 2), or
ACUUAAACGUGGAUGUACUUGCU (p1) (SEQ ID NO: 3) may contain a base sequence containing a base substitution, deletion or insertion at a position corresponding to at least one of the 4th and 14th bases. Even if the 8th and 18th bases of SEQ ID NO: 2 are modified, the effect on their functions is expected to be small. Even if the 4th and 14th bases of SEQ ID NO: 3 are modified, the effect on their functions is expected to be small.
　一つの実施形態では、本開示のmiR302核酸改変体は、
UAAGUGCUUCCAUGUUUUGGUGA（ｇ1）(配列番号２)の８番目および１８番目以外の塩基において０、１、２、３、４または５個の置換、欠失または挿入を含む塩基配列、または
ACUUAAACGUGGAUGUACUUGCU（ｐ１）（配列番号３)の４番目および１４番目以外の塩基において０、１、２、３、４または５個の置換、欠失または挿入を含む塩基配列
を含み得る。 In one embodiment, the miR302 nucleic acid variants of the present disclosure are:
A base sequence containing 0, 1, 2, 3, 4 or 5 substitutions, deletions or insertions in bases other than the 8th and 18th bases of UAAGUGCUUCCAUGUUUUGGUGA (g1) (SEQ ID NO: 2), or
ACUUAAACGUGGAUGUACUUGCU (p1) (SEQ ID NO: 3) may contain base sequences other than the 4th and 14th bases containing 0, 1, 2, 3, 4 or 5 substitutions, deletions or insertions.
　一つの実施形態では、本開示のmiR302核酸改変体は、
UAAGUGCTUCCAUGUTUTGGUTGA（ｇ９）（配列番号４)の８番目および１８番目以外の塩基において０、１、２、３、４または５個の置換、欠失または挿入を含む塩基配列、または
ACCAAAACAUGGAAGCACUUACT（ｐ１０）（配列番号５)の４番目および１４番目以外の塩基において０、１、２、３、４または５個の置換、欠失または挿入を含む塩基配列
を含み得る。配列番号４は、配列番号１の連続した２３の塩基配列（４４～６６番目の塩基）から３個の塩基の置換および１個の塩基の挿入を有する配列である。配列番号５は、配列番号１の連続した２３の塩基配列（６～２８番目の塩基）から７個の塩基の置換を有する配列である。配列番号４の８番目および１８番目の塩基は改変しても、その機能への影響が小さいと予測される。配列番号５の４番目および１４番目の塩基は改変しても、その機能への影響が小さいと予測される。 In one embodiment, the miR302 nucleic acid variants of the present disclosure are:
A base sequence containing 0, 1, 2, 3, 4 or 5 substitutions, deletions or insertions in bases other than the 8th and 18th bases of UAAGUGCTUCCAUGUTUTGGUTGA (g9) (SEQ ID NO: 4), or
It may contain a base sequence containing 0, 1, 2, 3, 4 or 5 substitutions, deletions or insertions in bases other than the 4th and 14th bases of ACCAAAACAUGGAAGCACUUACT (p10) (SEQ ID NO: 5). SEQ ID NO: 4 is a sequence having substitutions of 3 bases and insertion of 1 base from 23 consecutive base sequences (bases 44 to 66) of SEQ ID NO: 1. SEQ ID NO: 5 is a sequence having 7 base substitutions from 23 consecutive base sequences (6th to 28th bases) of SEQ ID NO: 1. Even if the 8th and 18th bases of SEQ ID NO: 4 are modified, the effect on their functions is expected to be small. Even if the 4th and 14th bases of SEQ ID NO: 5 are modified, the effect on their functions is expected to be small.
　一つの実施形態では、本開示のmiR302核酸改変体は、２つの分子の複合体（例えば、ガイド鎖分子とパッセンジャー分子との複合体）として存在してもよいし、分離した複数種類の分子として存在してもよいし、ガイド鎖分子またはパッセンジャー分子の一方単独として存在してもよいし、１分子中にガイド鎖部分およびパッセンジャー部分の両方が含まれる分子として存在してもよい。この分子において、必要に応じて、例えば、Ｄｉｃｅｒ等の作用により生体内で分解され得る配列を有していてもよい。そのような配列は、当業者は適宜設計することができ、例えば、UUGAAACUAAAGAAG（配列番号７）のような配列や構造が挙げられるが、それに限定されず、このような配列、ガイド鎖部分およびパッセンジャー部分を含む分子（例えば、分解配列は、ガイド鎖部分とパッセンジャー部分との間に位置付けられる）として核酸を設計することができる。 In one embodiment, the miR302 nucleic acid variants of the present disclosure may exist as a complex of two molecules (eg, a complex of a guide chain molecule and a passenger molecule) or as a plurality of separated molecules. It may be present as one of the guide chain molecule or the passenger molecule alone, or may be present as a molecule in which both the guide chain portion and the passenger moiety are contained in one molecule. If necessary, this molecule may have a sequence that can be decomposed in vivo by the action of, for example, Dicer. Such sequences can be appropriately designed by those skilled in the art, including, but not limited to, sequences and structures such as UUGAAACUAAAGAAG (SEQ ID NO: 7), such sequences, guide chain moieties and passengers. The nucleic acid can be designed as a part-containing molecule (eg, the degradation sequence is located between the guide strand portion and the passenger moiety).
　一つの実施形態では、本開示のmiR302核酸改変体は、以下の（１）～（５）のいずれか１種または複数種の改変：
（１）リボースから架橋糖への置換
（２）リン酸基の酸素原子の硫黄原子への置換
（３）糖の２’位の置換
（４）リボースから立体異性体糖への置換
（５）末端の核酸単位の５’位のリン酸化
を１つまたは複数（例えば、１、２、３、４、５、６、７、８、９、１０、１１、１２、１３、１４、１５、１６、１７、１８、１９、２０、２１、２２、２３、２４、２５、２６、２７、２８、２９、３０、３５、４０、４５、５０、５５、６０、６５または７０）含んでもよく、一つの実施形態では、該miR302核酸改変体は、上述の塩基配列のいずれかを含み得る。これらの改変により、エキソヌクレアーゼ耐性および/またはエンドヌクレアーゼ耐性が付与され得る。また、（２）の改変は、その改変を有するmiR302核酸改変体の機能を大きく変化させないと予測される。そのため、一つの実施形態では、本開示のmiR302核酸改変体は、上記（１）、（３）、（４）および（５）のいずれかの改変を０～１０（例えば、０、１、２、３、４、５、６、７、８、９または１０）含み、かつ上記（２）の改変を１～５０の間の任意の数（例えば、１、２、３、４、５、６、７、８、９、１０、１１、１２、１３、１４、１５、１６、１７、１８、１９、２０、２１、２２、２３、２４、２５、２６、２７、２８、２９、３０、３５、４０、４５または５０）含み、上述の塩基配列のいずれかを含む分子であり得る。 In one embodiment, the miR302 nucleic acid variants of the present disclosure are modified by any one or more of the following (1) to (5):
(1) Substitution of ribose to crosslinked sugar (2) Substitution of oxygen atom of phosphate group to sulfur atom (3) Substitution of sugar at 2'position (4) Substitution of ribose to stereoisomer sugar (5) One or more phosphorylations at the 5'position of the terminal nucleic acid unit (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65 or 70). In one embodiment, the miR302 nucleic acid variant may comprise any of the nucleotide sequences described above. These modifications can confer exonuclease resistance and / or endonuclease resistance. Moreover, it is predicted that the modification of (2) does not significantly change the function of the miR302 nucleic acid variant having the modification. Therefore, in one embodiment, the miR302 nucleic acid variant of the present disclosure is modified from any of the above (1), (3), (4) and (5) from 0 to 10 (eg, 0, 1, 2). , 3, 4, 5, 6, 7, 8, 9 or 10), and any number of modifications (2) above between 1 and 50 (eg, 1, 2, 3, 4, 5, 6) , 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35 , 40, 45 or 50) and can be a molecule comprising any of the above base sequences.
　上記（１）の改変「リボースから架橋糖への置換」における架橋糖は、任意の好適な構造を有し得る。一つの実施形態では、この架橋糖は、糖の４’位と２’位との間に少なくとも１つの架橋を有する構造を有し得、その架橋の各々は、独立して、－［Ｃ（Ｒ１）２］ｎ－、－Ｃ（Ｒ１）＝Ｃ（Ｒ２）－、－Ｃ（Ｒ１）＝Ｎ－、－Ｃ（＝ＮＲ１）－、－Ｃ（＝Ｏ）－、－Ｃ（＝Ｓ）－、－Ｏ－、－Ｓｉ（Ｒ１）２－、－Ｓ（＝Ｏ）ｘ－、および－Ｎ（Ｒ１）－から独立して選択される１、２、３または４個の連結基を含み得、式中、ｘは、０、１、または２であり；ｎは、１、２、３、または４であり；各Ｒ１は、独立して、Ｈ、ヒドロキシル、置換されていてもよい直鎖状または分枝状のＣ１～Ｃ１２アルキル、置換されていてもよい直鎖状または分枝状のＣ２～Ｃ１２アルケニル、置換されていてもよい直鎖状または分枝状のＣ２～Ｃ１２アルキニル、置換されていてもよい５～１２員の飽和、不飽和または芳香族環式基（環中に、１、２または３個のＮ、ＯまたはＳを含んでもよい）、ハロゲン、ＯＪ１、Ｎ（Ｊ１）２、ＳＪ１、Ｎ３、またはＣＮであり；各Ｊ１は、独立して、Ｈ、置換されていてもよい直鎖状または分枝状のＣ１～Ｃ１２アルキル、置換されていてもよい直鎖状または分枝状のＣ２～Ｃ１２アルケニル、または置換されていてもよい直鎖状または分枝状のＣ２～Ｃ１２アルキニルであり、「置換されていてもよい」における置換は、独立に、水素原子を、ヒドロキシル、ハロゲン、直鎖状または分枝状のＣ１～Ｃ６アルキル、直鎖状または分枝状のＣ１～Ｃ６アルコキシルおよびベンゼンからなる群から選択される置換基に置き換える置換、あるいは同じ原子に結合した２つの水素原子を＝Ｏに置き換える置換である。 The crosslinked sugar in the modification "replacement of ribose with a crosslinked sugar" in (1) above may have any suitable structure. In one embodiment, the crosslinked sugar may have a structure having at least one crosslink between the 4'and 2'positions of the sugar, each of which is independently − [C ( R 1 ) 2 ] n- , -C (R 1 ) = C (R 2 )-, -C (R 1 ) = N-, -C (= NR 1 )-, -C (= O)-,- 1, 2 , 3 or independently selected from C (= S)-, -O-, -Si (R 1 ) 2- , -S (= O) x- , and -N (R 1 )- It may contain 4 linking groups, where x is 0, 1, or 2; n is 1, 2, 3, or 4; each R 1 is independently H, hydroxyl. , Substituent linear or branched C 1 to C 12 alkyl, optionally substituted linear or branched C 2 to C 12 alkenyl, optionally substituted direct chain or branched C 2 ~ C 12 alkynyl, substituted optionally unsaturated also be 5-12 membered, in unsaturated or aromatic cyclic group (ring, two or three N, O Or S may be included), halogen, OJ 1 , N (J 1 ) 2 , SJ 1 , N 3 , or CN; each J 1 is independently H, a linear array which may be substituted. Linear or branched C 1 to C 12 alkyl, optionally substituted linear or branched C 2 to C 12 alkenyl, or optionally substituted linear or branched C Substitutions in 2 to C 12 alkynyls, which may be substituted, are independent of hydrogen atoms, hydroxyl, halogen, linear or branched C 1 to C 6 alkyl, linear or Substitution to replace with a substituent selected from the group consisting of branched C 1 to C 6 alkoxyls and benzene, or substitution to replace two hydrogen atoms bonded to the same atom with = O.
　一つの実施形態では、４’－２’架橋構造の例として、－［Ｃ（Ｒ１）２］ｎ－、－［Ｃ（Ｒ１）２］ｎ－Ｏ－、－Ｃ（Ｒ１）２－Ｎ（Ｒ１）－Ｏ－、および－Ｃ（Ｒ１）２－Ｏ－Ｎ（Ｒ１）－が挙げられる。一つの実施形態では、架橋構造の例として、４’－ＣＨ２－２’、４’－（ＣＨ２）２－２’、４’－（ＣＨ２）３－２’、４’－ＣＨ２－Ｏ－２’、４’－（ＣＨ２）２－Ｏ－２’、４’－ＣＨ２－Ｏ－Ｎ（Ｒ１）－２’、および４’－ＣＨ２－Ｎ（Ｒ１）－Ｏ－２’が挙げられ、一つの実施形態では、式中、各Ｒ１は、ＨまたはＣ１～Ｃ１２アルキルであり得る。 In one embodiment, as an example of a 4'-2'crosslinked structure,-[C (R 1 ) 2 ] n -,-[C (R 1 ) 2 ] n- O-, -C (R 1 ) 2 -N (R 1 ) -O- and -C (R 1 ) 2 -ON (R 1 )- are examples. In one embodiment, as an example of a cross-linked structure, 4'-CH 2 -2 ', 4' - (CH 2) 2 -2 ', 4' - (CH 2) 3 -2 ', 4'-CH 2 -O-2', 4'-(CH 2 ) 2- O-2', 4'-CH 2 -ON (R 1 ) -2', and 4'-CH 2- N (R 1 )- O-2'is mentioned, and in one embodiment, each R 1 in the formula can be H or C 1 to C 12 alkyl.
　一つの実施形態では、上記（１）の改変は、リボースの、以下に示すいずれかの構造を有する架橋糖への置換である。
Figure JPOXMLDOC01-appb-C000006
（式中、Ｒｘは、それぞれ独立して水素、置換または非置換の直鎖状または分枝状のＣ１～Ｃ１２アルキル、置換または非置換の直鎖状または分枝状のＣ２～Ｃ１２アルケニル、置換または非置換の直鎖状または分枝状のＣ２～Ｃ１２アルキニル、置換または非置換の５～１２員の環式基、ハロゲン、ＯＪ１、Ｎ（Ｊ１）２、ＳＪ１、Ｎ３、またはＣＮであり、具体的な実施形態では、Ｒｘは、Ｈ、メチル、エチル、ｎ－プロピル、イソプロピル、またはベンジルである） In one embodiment, the modification of (1) above is the substitution of ribose with a crosslinked sugar having any of the structures shown below.
Figure JPOXMLDOC01-appb-C000006
(In the formula, R x are independently hydrogen, substituted or unsubstituted linear or branched C 1 to C 12 alkyl, substituted or unsubstituted linear or branched C 2 to C 12 alkenyl, substituted or unsubstituted linear or branched C 2 -C 12 alkynyl, substituted or unsubstituted 5- to 12-membered cyclic group, halogen, OJ 1 , N (J 1 ) 2 , SJ 1 , N 3 , or CN, and in a specific embodiment, R x is H, methyl, ethyl, n-propyl, isopropyl, or benzyl).
　上記構造の架橋糖において、Ｒｘが、水素、メチル、エチル、ｎ－プロピル、イソプロピル、またはベンジルである架橋糖同士の相互置換は、これを含む核酸改変体の機能への影響が小さいと予測される。 In the crosslinked sugar having the above structure, mutual substitution between crosslinked sugars having Rx of hydrogen, methyl, ethyl, n-propyl, isopropyl, or benzyl is predicted to have a small effect on the function of nucleic acid variants containing the crosslinked sugars. Will be done.
　上記（２）の改変「リン酸基の酸素原子の硫黄原子への置換」は、代表的には、リン酸基の結合に関与しない一つの酸素原子を硫黄原子に置き換える改変、すなわち、
Figure JPOXMLDOC01-appb-C000007
の構造を、
Figure JPOXMLDOC01-appb-C000008
の構造に置き換える改変である。 The modification of (2) above, "replacement of an oxygen atom of a phosphate group with a sulfur atom," is typically a modification of replacing one oxygen atom that is not involved in the bond of a phosphate group with a sulfur atom, that is,
Figure JPOXMLDOC01-appb-C000007
Structure,
Figure JPOXMLDOC01-appb-C000008
It is a modification that replaces the structure of.
　上記（３）の改変「糖の２’位の置換」としては、例えば、糖（例えば、リボース）の２’位のヒドロキシル基を、ハロゲン（例えば、フッ素）、－Ｒ３、－ＯＲ３および－Ｎ（Ｒ３）２からなる群から選択される置換基に変更する置換が挙げられ、式中、Ｒ３は、水素、置換または非置換の直鎖状または分枝状のＣ１～Ｃ６アルキル、置換または非置換の直鎖状または分枝状のＣ２～Ｃ６アルケニル、置換または非置換の直鎖状または分枝状のＣ２～Ｃ６アルキニル、ベンジルまたはＣＮであり、具体的な実施形態では、Ｒ３は、水素、メチル、エチル、ｎ－プロピル、イソプロピル、またはベンジルであるからなる群から選択される。一つの実施形態では、上記（３）の改変は、糖（例えば、リボース）の２’のヒドロキシル基を、水素、フッ素またはアルコキシル基（例えば、メトキシル基）に置き換える改変であり得る。 As the modification "substitution of the 2'position of sugar" in (3) above, for example, the hydroxyl group at the 2'position of sugar (for example, ribose) is replaced with halogen (for example, fluorine), -R 3 , -OR 3 and Substituents that change to a substituent selected from the group consisting of −N (R 3 ) 2 are mentioned, in which R 3 is hydrogen, substituted or unsubstituted linear or branched C 1 to C. 6 alkyl, a substituted or unsubstituted, straight or branched C 2 ~ C 6 alkenyl, substituted or unsubstituted, straight or branched C 2 ~ C 6 alkynyl, benzyl or CN, particularly in embodiments, R 3 is hydrogen, methyl, ethyl, n- propyl, are selected from the group consisting of isopropyl or benzyl. In one embodiment, the modification of (3) above can be a modification that replaces the 2'hydroxyl group of a sugar (eg, ribose) with a hydrogen, fluorine or alkoxyl group (eg, a methoxyl group).
　上記（４）の改変「リボースから立体異性体糖への置換」としては、リボースの１’、２’、３’および４’位の少なくとも１つ（例えば、１つ、２つ、３つまたは４つ）において同じ炭素に結合した２つの基（水素原子および置換基）の間の結合関係（環の裏または表）が逆転した立体異性体糖への置換が挙げられる。代表的には、リボース型からアラビノ型への置換（ボースの２’位の炭素に結合した２つの基の間の結合関係（環の裏または表）が逆転した立体異性体糖への置換）が挙げられる。 The modification "substitution of ribose to a stereoisomer sugar" in (4) above includes at least one of the 1', 2', 3'and 4'positions of ribose (for example, one, two, three or more). In (4), substitution with a stereoisomer sugar in which the bonding relationship (back or front of the ring) between two groups (hydrogen atom and substituent) bonded to the same carbon is reversed can be mentioned. Typically, ribose-type to arabino-type substitution (substitution with a stereoisomer sugar in which the bond relationship (back or front of the ring) between two groups bonded to the carbon at the 2'position of Bose is reversed). Can be mentioned.
　上記（５）の改変「末端の核酸単位の５’位のリン酸化」とは、
Figure JPOXMLDOC01-appb-C000009
の構造を、
Figure JPOXMLDOC01-appb-C000010
の構造に置き換える改変である。ここで、Ｂａｓｅは、塩基を示す。 The modification "phosphorylation of the 5'position of the terminal nucleic acid unit" in (5) above is defined as
Figure JPOXMLDOC01-appb-C000009
Structure,
Figure JPOXMLDOC01-appb-C000010
It is a modification that replaces the structure of. Here, Base indicates a base.
　miR302核酸改変体が複数の上記改変（１）～（５）を有する場合、その改変は、１つの核酸単位上であってもよいし、複数の核酸単位上であってもよい。例えば、１つの核酸単位上に複数の改変を含む例として、上記（２）の改変「リン酸基の酸素原子の硫黄原子への置換」および上記（３）の改変「糖の２’位の置換」が組み合わされた以下の構造が与えられる。
Figure JPOXMLDOC01-appb-C000011
 When the miR302 nucleic acid variant has a plurality of the above modifications (1) to (5), the modification may be on one nucleic acid unit or on a plurality of nucleic acid units. For example, as an example of including a plurality of modifications on one nucleic acid unit, the modification of (2) above "substitution of oxygen atom of phosphoric acid group with sulfur atom" and the modification of (3) above "2'position of sugar" The following structure is given in combination with "substitution".
Figure JPOXMLDOC01-appb-C000011
　本開示のmiR302核酸改変体は、上で記載した任意の改変の任意の組み合わせを有し得る。 The miR302 nucleic acid variants of the present disclosure may have any combination of any of the modifications described above.
　一つの実施形態では、本願開示のmiR302核酸改変体は、
U^A(M)A^G(M)U^G(M)C^T(M)U^C(M)C^A(M)U^G(M)U^T(M)U^T(M)G^G(M)U^T(M)G(L)^A(L)または
A(L)^C(L)^C^A^A^A^A^C^A^U^G^G^A^A^G^C^A^C^U^U^A^C(L)^T(L)
（式中、
　Uはウラシル、Tはチミン、Aはアデニン、Cはシトシンの各塩基を表し、
　(M)は、この記号の左のリボヌクレオチド単位中のリボースの２’のヒドロキシル基が
メトキシ基に置き換えられている改変を表し、
　(L)は、この記号の左のリボヌクレオチド単位中のリボースが
Figure JPOXMLDOC01-appb-C000012
に置き換えられている改変を表し、
　^は、両隣のリボヌクレオチド単位の間のリン酸基の結合に関与しない一つの酸素原子が硫黄原子に置き換えられている改変を表す）
の構造を含んでもよいし、これらの構造から、(M)、(L)および^の改変が０～１０個（例えば、０、１、２、３、４、５、６、７、８、９または１０個）削除されており、上記（１）～（５）の任意の改変が０～１０個（例えば、０、１、２、３、４、５、６、７、８、９または１０個）追加されており、０、１、２、３、４または５個の塩基の置換、欠失または挿入を含む、構造を含んでもよい。上記（１）の改変「リボースから架橋糖への置換」において、ある架橋糖から別の架橋糖への変更は、その改変を有するmiR302核酸改変体の機能を大きく変化させないと予測される。そのため、一つの実施形態では、本開示のmiR302核酸改変体は、
U^A(M)A^G(M)U^G(M)C^T(M)U^C(M)C^A(M)U^G(M)U^T(M)U^T(M)G^G(M)U^T(M)G(L)^A(L)または
A(L)^C(L)^C^A^A^A^A^C^A^U^G^G^A^A^G^C^A^C^U^U^A^C(L)^T(L)
（式中、U、T、A、C、(M)、(L)および^は、上記定義と同じ）
の構造から、(M)および(L)の改変が０～５個（例えば、０、１、２、３、４または５個）削除されており、(L)の改変の０～４個（例えば、０、１、２、３または４個）が上記（１）の別の架橋糖への置換に変更されており、^の改変が０～１０個（例えば、０、１、２、３、４、５、６、７、８、９または１０個）削除されており、上記（２）の改変が０～１０個（例えば、０、１、２、３、４、５、６、７、８、９または１０個）追加されており、上記（１）、（３）、（４）または（５）の任意の改変が０～５個（例えば、０、１、２、３、４または５個）追加されており、０、１、２、３、４または５個の塩基の置換、欠失または挿入を含む、構造を含む分子であり得る。 In one embodiment, the miR302 nucleic acid variant disclosed in the present application is:
U ^ A (M) A ^ G (M) U ^ G (M) C ^ T (M) U ^ C (M) C ^ A (M) U ^ G (M) U ^ T (M) U ^ T (M) G ^ G (M) U ^ T (M) G (L) ^ A (L) or
A (L) ^ C (L) ^ C ^ A ^ A ^ A ^ A ^ C ^ A ^ U ^ G ^ G ^ A ^ A ^ G ^ C ^ A ^ C ^ U ^ U ^ A ^ C ( L) ^ T (L)
(During the ceremony,
U stands for uracil, T stands for thymine, A stands for adenine, C stands for cytosine,
(M) represents a modification in which the 2'hydroxyl group of ribose in the ribonucleotide unit to the left of this symbol is replaced with a methoxy group.
(L) is the ribose in the ribonucleotide unit to the left of this symbol
Figure JPOXMLDOC01-appb-C000012
Represents a modification that has been replaced by
^ Represents a modification in which one oxygen atom that is not involved in the bond of the phosphate group between the adjacent ribonucleotide units is replaced with a sulfur atom)
, And from these structures, 0 to 10 modifications of (M), (L) and ^ (eg, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) have been deleted, and 0 to 10 (eg, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or) any modifications of (1) to (5) above have been deleted. 10) have been added and may contain structures containing 0, 1, 2, 3, 4 or 5 base substitutions, deletions or insertions. In the modification "replacement of ribose to crosslinked sugar" in (1) above, it is predicted that the change from one crosslinked sugar to another does not significantly change the function of the miR302 nucleic acid variant having the modification. Therefore, in one embodiment, the miR302 nucleic acid variants of the present disclosure are:
U ^ A (M) A ^ G (M) U ^ G (M) C ^ T (M) U ^ C (M) C ^ A (M) U ^ G (M) U ^ T (M) U ^ T (M) G ^ G (M) U ^ T (M) G (L) ^ A (L) or
A (L) ^ C (L) ^ C ^ A ^ A ^ A ^ A ^ C ^ A ^ U ^ G ^ G ^ A ^ A ^ G ^ C ^ A ^ C ^ U ^ U ^ A ^ C ( L) ^ T (L)
(In the formula, U, T, A, C, (M), (L) and ^ are the same as the above definitions)
From the structure of (M) and (L), 0 to 5 modifications (for example, 0, 1, 2, 3, 4 or 5) have been deleted, and 0 to 4 modifications of (L) (for example, 0 to 4). For example, 0, 1, 2, 3 or 4) has been changed to the substitution with another cross-linked sugar in (1) above, and 0 to 10 modifications of ^ (eg, 0, 1, 2, 3). 4, 5, 6, 7, 8, 9 or 10) have been deleted, and 0 to 10 modifications (2) above ( eg 0, 1, 2, 3, 4, 5, 6, 7) have been deleted. , 8, 9 or 10), and 0 to 5 (eg, 0, 1, 2, 3, 4) of any modification of (1), (3), (4) or (5) above. Or 5) may be a structure-containing molecule that has been added and contains 0, 1, 2, 3, 4 or 5 base substitutions, deletions or insertions.
　一つの実施形態では、本開示のmiR302核酸改変体は、上記改変とは異なる機能性部分（例えば、核酸改変体の検出、組織標的化、安定性向上および／または他の分子との結合を促進する部分）を含み得る。機能性部分としては、例えば、蛍光色素、発光色素、ＰＥＧ、コレステロール、脂質、ビオチン、リンカー部分（ＮＨＳ、アジドまたはアルキンを含む部分など）が挙げられるが、これらに限定されない。 In one embodiment, the miR302 nucleic acid variants of the present disclosure facilitate functional moieties different from those of the modifications (eg, detection of nucleic acid variants, tissue targeting, stability enhancement and / or binding to other molecules. The part to be used) may be included. Functional moieties include, but are not limited to, for example, fluorescent dyes, luminescent dyes, PEGs, cholesterol, lipids, biotin, linker moieties (such as moieties containing NHS, azides or alkynes).
　当業者は、本明細書に記載される改変核酸を、任意の公知の手法を使用して合成することができる。改変核酸の合成は、例えば、Current　protocols　in　nucleic　acid　chemistry（https://currentprotocols.onlinelibrary.wiley.com/journal/19349289）などを参照のこと。 One of ordinary skill in the art can synthesize the modified nucleic acids described herein using any known technique. For the synthesis of modified nucleic acids, refer to, for example, Current protocols in nucleic acid chemistry (https://currentprotocols.onlinelibrary.wiley.com/journal/19349289).
　一つの実施形態では、本開示のmiR302核酸改変体は、ルシフェラーゼ遺伝子と、ＨｎＲＮＰＡ２Ｂ１遺伝子とを結合させた核酸構築物をレンチウイルスにより導入したＨＴ２９細胞を、該miR302核酸改変体と接触させた場合に、天然のヒトmiR302aまたは配列番号２の核酸（ｇ１）と配列番号３の核酸（ｐ１）とにより形成される二本鎖（ｇ１ｐ１対）よりも低い（例えば、１％～９９．９９％だけ低い、例えば、約１％、約２％、約５％、約１０％、約１５％、約２０％、約３０％、約４０％、約５０％、約６０％、約７０％、約８０％、約８５％、約９０％、約９５％、約９８％、約９９％、約９９．５％、約９９．９％または約９９．９９％だけ低い）ルシフェラーゼ発光をもたらし得る。 In one embodiment, the miR302 nucleic acid variant of the present disclosure is a case where HT29 cells into which a nucleic acid construct in which a luciferase gene and an HnRNPA2B1 gene are bound are introduced by a lentivirus are brought into contact with the miR302 nucleic acid variant. Lower than the double strand (g1p1 pair) formed by the native human miR302a or nucleic acid of SEQ ID NO: 2 (g1) and nucleic acid of SEQ ID NO: 3 (p1) (eg, 1% -99.99% lower, For example, about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, (About 85%, about 90%, about 95%, about 98%, about 99%, about 99.5%, about 99.9% or about 99.99% lower) can result in luciferase luminescence.
　一つの実施形態では、本開示のmiR302核酸改変体（例えば、ガイド鎖とパッセンジャー鎖との二本鎖、またはガイド部分とパッセンジャー部分とを含む単分子）は、インビトロでウシ胎児血清（ＦＢＳ）と接触させた場合の分解速度が、天然のヒトmiR302aまたは配列番号２の核酸（ｇ１）と配列番号３の核酸（ｐ１）とにより形成される二本鎖（ｇ１ｐ１対）よりも遅い（例えば、１％～９９．９９％だけ遅い、例えば、約１％、約２％、約５％、約１０％、約１５％、約２０％、約３０％、約４０％、約５０％、約６０％、約７０％、約８０％、約８５％、約９０％、約９５％、約９８％、約９９％、約９９．５％、約９９．９％または約９９．９９％だけ遅い）という安定性を有し得る。 In one embodiment, the miR302 nucleic acid variants of the present disclosure (eg, a double strand of a guide strand and a passenger strand, or a single molecule containing a guide moiety and a passenger moiety) are in vitro with fetal bovine serum (FBS). The rate of degradation upon contact is slower than the double strand (g1p1 pair) formed by the native human miR302a or the nucleic acid of SEQ ID NO: 2 (g1) and the nucleic acid of SEQ ID NO: 3 (p1) (eg, 1). % -99.99% slower, eg, about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60% , About 70%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, about 99.5%, about 99.9% or about 99.99% slower) Can have stability.
　一つの実施形態では、本開示のmiR302核酸改変体は、がん、などの疾患の治療に使用され得る。一つの実施形態では、本開示のmiR302核酸改変体は、組成物に含まれた形態で提供され得る。本開示のmiR302核酸改変体によって治療され得るがんとして、膵臓がん（例えば、早期膵臓がん）、肝臓がん、胆嚢がん、胆道がん、胃がん、大腸がん、膀胱がん、腎がん、乳がん、肺がん、脳腫瘍および皮膚がんが挙げられるが、これらに限定されない。 In one embodiment, the miR302 nucleic acid variants of the present disclosure can be used in the treatment of diseases such as cancer. In one embodiment, the miR302 nucleic acid variants of the present disclosure may be provided in the form contained in the composition. Cancers that can be treated with the miR302 nucleic acid variants of the present disclosure include pancreatic cancer (eg, early pancreatic cancer), liver cancer, gallbladder cancer, biliary tract cancer, gastric cancer, colon cancer, bladder cancer, and kidney. Cancer, breast cancer, lung cancer, brain cancer and skin cancer include, but are not limited to.
（医薬品、剤型等）
　本開示のmiR302核酸改変体は、種々の形態の組成物または医薬（治療薬または予防薬）として提供される。 (Pharmaceuticals, dosage forms, etc.)
The miR302 nucleic acid variants of the present disclosure are provided as various forms of compositions or pharmaceuticals (therapeutic or prophylactic agents).
　治療薬の投与経路は、治療に際して効果的なものを使用するのが好ましく、例えば、静脈内、皮下、筋肉内、腹腔内、または経口投与等であってもよい。投与形態としては、例えば、注射剤、カプセル剤、錠剤、顆粒剤等であってもよい。注射用の水溶液は、例えば、バイアル、またはステンレス容器で保存してもよい。また注射用の水溶液は、例えば生理食塩水、糖（例えばトレハロース）、ＮａＣｌ、またはＮａＯＨ等を配合してもよい。また治療薬は、例えば、緩衝剤（例えばリン酸塩緩衝液)、安定剤等を配合してもよい。 The administration route of the therapeutic agent is preferably one that is effective for treatment, and may be, for example, intravenous, subcutaneous, intramuscular, intraperitoneal, or oral administration. The administration form may be, for example, an injection, a capsule, a tablet, a granule, or the like. The aqueous solution for injection may be stored, for example, in a vial or a stainless steel container. Further, the aqueous solution for injection may contain, for example, physiological saline, sugar (for example, trehalose), NaCl, NaOH or the like. Further, the therapeutic agent may contain, for example, a buffer (for example, a phosphate buffer), a stabilizer, or the like.
　一般的に、本開示の組成物、医薬、治療剤、予防剤等は、治療有効量の治療剤または有効成分、および薬学的に許容しうるキャリアもしくは賦形剤を含む。本明細書において「薬学的に許容しうる」は、動物、そしてより詳細にはヒトにおける使用のため、政府の監督官庁に認可されたか、あるいは薬局方または他の一般的に認められる薬局方に列挙されていることを意味する。本明細書において使用される「キャリア」は、治療剤を一緒に投与する、希釈剤、アジュバント、賦形剤、またはビヒクルを指す。このようなキャリアは、無菌液体、例えば水および油であることも可能であり、石油、動物、植物または合成起源のものが含まれ、限定されるわけではないが、ピーナツ油、ダイズ油、ミネラルオイル、ゴマ油等が含まれる。医薬を経口投与する場合は、水が好ましいキャリアである。医薬組成物を静脈内投与する場合は、生理食塩水および水性デキストロースが好ましいキャリアである。好ましくは、生理食塩水溶液、並びに水性デキストロースおよびグリセロール溶液が、注射可能溶液の液体キャリアとして使用される。適切な賦形剤には、軽質無水ケイ酸、結晶セルロース、マンニトール、デンプン、グルコース、ラクトース、スクロース、ゼラチン、モルト、米、小麦粉、チョーク、シリカゲル、ステアリン酸ナトリウム、モノステアリン酸グリセロール、タルク、塩化ナトリウム、脱脂粉乳、グリセロール、プロピレン、グリコール、水、エタノール、カルメロースカルシウム、カルメロースナトリウム、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルアセタールジエチルアミノアセテート、ポリビニルピロリドン、ゼラチン、中鎖脂肪酸トリグリセライド、ポリオキシエチレン硬化ヒマシ油６０、白糖、カルボキシメチルセルロース、コーンスターチ、無機塩等が含まれる。組成物は、望ましい場合、少量の湿潤剤または乳化剤、あるいはｐＨ緩衝剤もまた含有することも可能である。これらの組成物は、溶液、懸濁物、エマルジョン、錠剤、ピル、カプセル、粉末、持続放出配合物等の形を取ることも可能である。伝統的な結合剤およびキャリア、例えばトリグリセリドを用いて、組成物を座薬として配合することも可能である。経口配合物は、医薬等級のマンニトール、ラクトース、デンプン、ステアリン酸マグネシウム、サッカリン・ナトリウム、セルロース、炭酸マグネシウムなどの標準的キャリアを含むことも可能である。適切なキャリアの例は、E．W．Martin,　Remington’s　Pharmaceutical　Sciences(Mark　Publishing　Company,　Easton,　U.S.A）に記載される。このような組成物は、患者に適切に投与する形を提供するように、適切な量のキャリアと一緒に、治療有効量の療法剤、好ましくは精製型のものを含有する。配合物は、投与様式に適していなければならない。これらのほか、例えば、界面活性剤、賦形剤、着色料、着香料、保存料、安定剤、緩衝剤、懸濁剤、等張化剤、結合剤、崩壊剤、滑沢剤、流動性促進剤、矯味剤等を含んでいてもよい。 In general, the compositions, pharmaceuticals, therapeutic agents, prophylactic agents, etc. of the present disclosure include therapeutically effective amounts of therapeutic or active ingredients, and pharmaceutically acceptable carriers or excipients. As used herein, "pharmaceutically acceptable" is used in animals, and more specifically in humans, for use in government regulatory agencies or in the pharmacopoeia or other generally accepted pharmacopoeia. It means that it is listed. As used herein, "carrier" refers to a diluent, adjuvant, excipient, or vehicle to which a therapeutic agent is administered together. Such carriers can also be sterile liquids such as water and oils, including, but not limited to, petroleum, animal, plant or synthetic origins, sesame oil, soybean oil, minerals. Includes oil, sesame oil, etc. Water is the preferred carrier for oral administration of the drug. For intravenous administration of the pharmaceutical composition, saline and aqueous dextrose are the preferred carriers. Preferably, saline solution, as well as aqueous dextrose and glycerol solutions, are used as liquid carriers for the injectable solution. Suitable excipients include light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, choke, silica gel, sodium stearate, glycerol monostearate, talc, chloride. Sodium, defatted milk powder, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinyl acetal diethylaminoacetate, polyvinyl pyrrolidone, gelatin, medium chain fatty acid triglyceride, polyoxyethylene curing Includes castor oil 60, sucrose, carboxymethyl cellulose, corn starch, inorganic salts and the like. The composition can also contain small amounts of wetting or emulsifying agents, or pH buffers, if desired. These compositions can also take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. It is also possible to formulate the composition as a suppository using traditional binders and carriers such as triglycerides. Oral formulations can also include standard carriers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin sodium, cellulose, magnesium carbonate and the like. Examples of suitable carriers are E.I. W. Described in Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, U.S.A.). Such compositions contain a therapeutically effective amount of a therapeutic agent, preferably a purified form, along with an appropriate amount of carrier to provide a form for appropriate administration to the patient. The formulation should be suitable for the mode of administration. In addition to these, for example, surfactants, excipients, colorants, fragrances, preservatives, stabilizers, buffers, suspending agents, tonicity agents, binders, disintegrants, lubricants, fluidity It may contain an accelerator, a flavoring agent and the like.
　本開示の一実施形態において「塩」は、例えば、任意の酸性（例えばカルボキシル）基で形成されるアニオン塩、または任意の塩基性（例えばアミノ）基で形成されるカチオン塩を含む。塩類には無機塩または有機塩を含み、例えば、Berge　et　al.,　J.　Pharm.　Sci.,　1977,　66,　1-19に記載されている塩が含まれる。また例えば、金属塩、アンモニウム塩、有機塩基との塩、無機酸との塩、有機酸との塩等が挙げられる。本開示の一実施形態において「溶媒和物」は、溶質および溶媒によって形成される化合物である。溶媒和物については例えば、J.　Honig　et　al.,　The　Van　Nostrand　Chemist’s　Dictionary　P650(1953)を参照できる。溶媒が水であれば形成される溶媒和物は水和物である。この溶媒は、溶質の生物活性を妨げないものが好ましい。そのような好ましい溶媒の例として、特に限定するものではないが、水、または各種バッファーが挙げられる。 In one embodiment of the present disclosure, the "salt" includes, for example, an anion salt formed of any acidic (eg, carboxyl) group or a cationic salt formed of any basic (eg, amino) group. The salts include inorganic salts or organic salts, and include, for example, the salts described in Berg et al., J. Pharm. Sci., 1977, 66, 1-19. Further, for example, a metal salt, an ammonium salt, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid and the like can be mentioned. In one embodiment of the present disclosure, a "solvate" is a compound formed by a solute and a solvent. For the solvate, for example, J. Honig et al., The Van Nostrand Chemist's Dictionary P650 (1953) can be referred to. If the solvent is water, the solvate formed is a hydrate. The solvent is preferably one that does not interfere with the biological activity of the solute. Examples of such preferred solvents include, but are not limited to, water or various buffers.
　本開示のmiR302核酸改変体を医薬として投与する場合、種々の送達（デリバリー）系が知られ、そしてこのような系を用いて、本開示の治療剤を適切な部位に投与することも可能であり、このような系には、例えばリポソーム、微小粒子、および微小カプセル中の被包：受容体が仲介するエンドサイトーシスの使用；レトロウイルスベクターまたは他のベクターの一部としての療法核酸の構築などがある。導入法には、限定されるわけではないが、皮内、筋内、腹腔内、静脈内、皮下、鼻内、硬膜外、および経口経路が含まれる。好適な経路いずれによって、例えば注入によって、ボーラス（bolus）注射によって、上皮または皮膚粘膜裏打ち（例えば口腔、直腸および腸粘膜など）を通じた吸収によって、医薬を投与することも可能であるし、必要に応じてエアロゾル化剤を用いて吸入器または噴霧器を使用しうるし、そして他の生物学的活性剤と一緒に投与することも可能である。投与は全身性または局所であることも可能である。本開示のmiR302核酸改変体ががんに使用される場合、さらに、がん（病変部）に直接注入する等、適切な経路いずれかによって投与されうる。 When the miR302 nucleic acid variants of the present disclosure are administered as pharmaceuticals, various delivery systems are known, and such systems can also be used to administer the therapeutic agents of the present disclosure to appropriate sites. There are, for example, encapsulation in liposomes, microparticles, and microcapsules: the use of receptor-mediated endocytosis; construction of therapeutic nucleic acids as part of retroviral vectors or other vectors. and so on. Induction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. It is also possible and necessary to administer the drug by any of the preferred routes, eg by injection, by bolus injection, by absorption through the epithelial or mucocutaneous lining (eg, oral, rectal and intestinal mucosa). Inhalers or nebulizers can be used, depending on the aerosolizing agent, and can also be administered with other bioactive agents. Administration can also be systemic or topical. When the miR302 nucleic acid variant of the present disclosure is used for cancer, it can be further administered by any appropriate route, such as direct injection into the cancer (lesion).
　好ましい実施形態において、公知の方法に従って、ヒトへの投与に適応させた医薬組成物として、組成物を配合することができる。このような組成物は注射により投与することができる。代表的には、注射投与のための組成物は、無菌等張水性緩衝剤中の溶液である。必要な場合、組成物はまた、可溶化剤および注射部位での疼痛を和らげるリドカインなどの局所麻酔剤も含むことも可能である。一般的に、成分を別個に供給するか、または単位投薬型中で一緒に混合して供給し、例えば活性剤の量を示すアンプルまたはサシェなどの密封容器中、凍結乾燥粉末または水不含濃縮物として供給することができる。組成物を注入によって投与しようとする場合、無菌薬剤等級の水または生理食塩水を含有する注入ビンを用いて、分配することも可能である。組成物を注射によって投与しようとする場合、投与前に、成分を混合可能であるように、注射用の無菌水または生理食塩水のアンプルを提供することも可能である。 In a preferred embodiment, the composition can be blended as a pharmaceutical composition adapted for administration to humans according to a known method. Such compositions can be administered by injection. Typically, the composition for injection administration is a solution in a sterile isotonic aqueous buffer. If desired, the composition can also include a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the ingredients are supplied separately or mixed together in a unit dosage form and lyophilized powder or water-free concentrate in a sealed container such as an ampoule or sachet indicating the amount of activator. It can be supplied as a thing. If the composition is to be administered by infusion, it can also be dispensed using an infusion bottle containing sterile drug grade water or saline. If the composition is to be administered by injection, it is also possible to provide an ampoule of sterile water or saline for injection so that the ingredients can be mixed prior to administration.
　本開示の組成物、医薬、治療剤、予防剤を中性型または塩型あるいは他のプロドラッグ（例えば、エステル等）で配合することも可能である。薬学的に許容しうる塩には、塩酸、リン酸、酢酸、シュウ酸、酒石酸などに由来する遊離型のカルボキシル基とともに形成されるもの、イソプロピルアミン、トリエチルアミン、２－エチルアミノエタノール、ヒスチジン、プロカインなどに由来するものなどの遊離型のアミン基とともに形成されるもの、並びにナトリウム、カリウム、アンモニウム、カルシウム、および水酸化第二鉄などに由来するものが含まれる。 It is also possible to formulate the compositions, pharmaceuticals, therapeutic agents and prophylactic agents of the present disclosure in neutral or salt forms or other prodrugs (eg, esters, etc.). Pharmaceutically acceptable salts include those formed with free carboxyl groups derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, prokine. It includes those formed with free amine groups such as those derived from such as, and those derived from sodium, potassium, ammonium, calcium, ferric hydroxide and the like.
　特定の障害または状態の治療に有効な本開示の治療剤の量は、障害または状態の性質によって変動しうるが、当業者は本明細書の記載に基づき標準的臨床技術によって決定可能である。さらに、場合によって、in　vitroアッセイを使用して、最適投薬量範囲を同定するのを補助することも可能である。配合物に使用しようとする正確な用量はまた、投与経路、および疾患または障害の重大性によっても変動しうるため、担当医の判断および各患者の状況に従って、決定すべきである。しかし、投与量は特に限定されないが、例えば、１回あたり０．００１、１、５、１０、１５、１００、または１０００ｍｇ／ｋｇ体重であってもよく、それらいずれか２つの値の範囲内であってもよい。投与間隔は特に限定されないが、例えば、１、７、１４、２１、または２８日あたりに１または２回投与してもよく、それらいずれか２つの値の範囲あたりに１または２回投与してもよい。本開示のmiR302核酸改変体は、高い血漿中安定性および/または高い活性を有し得るため、投与頻度は低減され得る。投与量、投与間隔、投与方法は、患者の年齢や体重、症状、対象臓器等により、適宜選択してもよい。また治療薬は、治療有効量、または所望の作用を発揮する有効量の有効成分を含むことが好ましい。悪性腫瘍マーカーが、投与後に有意に減少した場合に、治療効果があったと判断してもよい。有効用量は、in　vitroまたは動物モデ
ル試験系から得られる用量－反応曲線から推定可能である。 The amount of therapeutic agent of the present disclosure effective for the treatment of a particular disorder or condition may vary depending on the nature of the disorder or condition, but one of ordinary skill in the art can determine by standard clinical techniques as described herein. In addition, in vitro assays can optionally be used to assist in identifying optimal dosage ranges. The exact dose to be used in the formulation may also vary depending on the route of administration and the severity of the disease or disorder and should be determined according to the discretion of the attending physician and the circumstances of each patient. However, the dose is not particularly limited, and may be, for example, 0.001, 1, 5, 10, 15, 100, or 1000 mg / kg body weight per dose, within the range of any two of these values. There may be. The dosing interval is not particularly limited, but may be administered once or twice per 1, 7, 14, 21, or 28 days, and once or twice within the range of any two of these values. May be good. The miR302 nucleic acid variants of the present disclosure may have high plasma stability and / or high activity, so administration frequency may be reduced. The dose, administration interval, and administration method may be appropriately selected depending on the age and weight of the patient, symptoms, target organ, and the like. In addition, the therapeutic agent preferably contains a therapeutically effective amount or an effective amount of the active ingredient that exerts a desired action. If the malignant tumor marker is significantly reduced after administration, it may be judged that there is a therapeutic effect. Effective doses can be estimated from dose-response curves obtained in vitro or from animal model test systems.
　一つの実施形態において、「患者」または「被験体」は、ヒト、または哺乳動物（例えば、マウス、モルモット、ハムスター、ラット、ネズミ、ウサギ、ブタ、ヒツジ、ヤギ、ウシ、ウマ、ネコ、イヌ、マーモセット、サル、またはチンパンジー等）を含む。 In one embodiment, the "patient" or "subject" is a human or mammal (eg, mouse, guinea pig, hamster, rat, mouse, rabbit, pig, sheep, goat, cow, horse, cat, dog, Includes marmosets, monkeys, or chimpanzees, etc.).
　本開示の医薬組成物または治療剤もしくは予防剤はキットとして提供することができる。 The pharmaceutical composition or therapeutic or prophylactic agent of the present disclosure can be provided as a kit.
　特定の実施形態では、本開示は、本開示の組成物または医薬の１以上の成分が充填された、１以上の容器を含む、薬剤パックまたはキットを提供する。場合によって、このような容器に付随して、医薬または生物学的製品の製造、使用または販売を規制する政府機関によって規定された形で、政府機関による、ヒト投与のための製造、使用または販売の認可を示す情報を示すことも可能である。 In certain embodiments, the present disclosure provides drug packs or kits comprising one or more containers filled with one or more components of the compositions or pharmaceuticals of the present disclosure. Manufacture, use or sale for human administration by Government, as prescribed by Government, which regulates the manufacture, use or sale of pharmaceutical or biological products, optionally incidental to such containers. It is also possible to provide information indicating the authorization of.
　特定の実施形態において、本開示の成分を含む医薬組成物を、リポソーム、微小粒子、または微小カプセルを介して投与することができる。本開示の種々の実施形態において、このような組成物を用いて、本開示の成分の持続放出を達成することが有用である可能性もある。 In certain embodiments, pharmaceutical compositions containing the ingredients of the present disclosure can be administered via liposomes, microparticles, or microcapsules. In various embodiments of the present disclosure, it may be useful to use such compositions to achieve sustained release of the components of the present disclosure.
　本開示の治療薬、予防薬等の医薬等としての製剤化手順は、当該分野において公知であり、例えば、日本薬局方、米国薬局方、他の国の薬局方などに記載されている。従って、当業者は、本明細書の記載があれば、過度な実験を行うことなく、使用すべき量等の実施形態を決定することができる。 The procedures for formulating therapeutic agents, preventive agents, etc. of the present disclosure as pharmaceuticals, etc. are known in the art, and are described in, for example, the Japanese Pharmacopoeia, the United States Pharmacopeia, and the Pharmacopoeia of other countries. Therefore, those skilled in the art can determine an embodiment such as an amount to be used without undue experimentation, if described in the present specification.
　（使用）
　別の局面では、免疫賦活化オリゴヌクレオチド核酸医薬の製造における、本開示のオリゴヌクレオチドの使用が提供される。この局面において使用されるオリゴヌクレオチドは、本明細書に記載される任意の形態を用いることができることが理解される。 (use)
In another aspect, the use of the oligonucleotides of the present disclosure in the manufacture of immunostimulatory oligonucleotide nucleic acid reagents is provided. It is understood that the oligonucleotides used in this aspect can use any of the forms described herein.
　本明細書において「または」は、文章中に列挙されている事項の「少なくとも1つ以上」を採用できるときに使用される。「もしくは」も同様である。本明細書において「2つの値」の「範囲内」と明記した場合、その範囲には2つの値自体も含む。 In this specification, "or" is used when "at least one" of the items listed in the text can be adopted. The same applies to "or". When "within a range" of "two values" is specified in the present specification, the range also includes the two values themselves.
　本明細書において引用された、科学文献、特許、特許出願などの参考文献は、その全体が、各々具体的に記載されたのと同じ程度に本明細書において参考として援用される。 References such as scientific literature, patents, and patent applications cited in this specification are incorporated herein by reference in their entirety to the same extent as they are specifically described.
　以上、本開示を、理解の容易のために好ましい実施形態を示して説明してきた。以下に、実施例に基づいて本開示を説明するが、上述の説明および以下の実施例は、例示の目的のみに提供され、本開示を限定する目的で提供したのではない。従って、本発明の範囲は、本明細書に具体的に記載された実施形態にも実施例にも限定されず、特許請求の範囲によってのみ限定される。 The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples, but the above description and the following examples are provided for purposes of illustration only and not for the purpose of limiting the present disclosure. Therefore, the scope of the present invention is not limited to the embodiments and examples specifically described in the present specification, but is limited only by the scope of claims.
　必要な場合、以下の実施例で用いる動物の取り扱いは、大阪大学の動物実験ガイドラインに従って実施した。試薬類は具体的には実施例中に記載した製品を使用したが、他メーカー（Sigma－Aldrich、和光純薬、ナカライ、R&D　Systems、USCN　Life　Science　INC等）の同等品でも代用可能である。 If necessary, the handling of animals used in the following examples was carried out in accordance with the animal experiment guidelines of Osaka University. Specifically, the reagents described in the examples were used, but equivalent products of other manufacturers (Sigma-Aldrich, Wako Junyaku, Nacalai, R & D Systems, USCN Life Science INC, etc.) can be substituted.
　（実施例１）miR302核酸改変体の設計
　miR302核酸改変体および天然のヒトmiR302aの部分配列を有する核酸（合計で、ガイド鎖１９種類、パッセンジャー鎖２０種類）を、ジーンデザイン（大阪府）に依頼して合成した。 (Example 1) Design of miR302 nucleic acid variant Nucleic acid having a partial sequence of miR302 nucleic acid variant and natural human miR302a (19 types of guide strands and 20 types of passenger strands in total) was requested to Gene Design (Osaka). And synthesized.
　合成した代表的な核酸の構造を以下の表に示す。
・天然型ガイド鎖（ｇ１）：
UAAGUGCUUCCAUGUUUUGGUGA　(配列番号２)
・天然型パッセンジャー鎖（ｐ１）：
ACUUAAACGUGGAUGUACUUGCU　(配列番号３)
・ガイド鎖９（ｇ９）：
U^A(M)A^G(M)U^G(M)C^T(M)U^C(M)C^A(M)U^G(M)U^T(M)U^T(M)G^G(M)U^T(M)G(L)^A(L)
・パッセンジャー鎖１０（ｐ１０）：
A(L)^C(L)^C^A^A^A^A^C^A^U^G^G^A^A^G^C^A^C^U^U^A^C(L)^T(L)
（ここで、
・Uはウラシル、Tはチミン、Aはアデニン、Cはシトシンの各塩基を表し、
・(M)は、この記号の左のリボヌクレオチド単位中のリボースの２’のヒドロキシル基がメトキシ基に置き換えられている改変を表し、
・(L)は、この記号の左のリボヌクレオチド単位中のリボースが
Figure JPOXMLDOC01-appb-C000013
に置き換えられている改変を表し、
・^は、両隣のリボヌクレオチド単位の間のリン酸基の結合に関与しない一つの酸素原子
が硫黄原子に置き換えられている改変を表す）
　ガイド鎖（ｇ１およびｇ９）の５’末端には、ｓｓＨアミノリンカーを介してＡｌｅｘａ６４７を結合させた。 The structure of the synthesized representative nucleic acid is shown in the table below.
-Natural guide chain (g1):
UAAGUGCUUCCAUGUUUUGGUGA (SEQ ID NO: 2)
-Natural passenger chain (p1):
ACUUAAACGUGGAUGUACUUGCU (SEQ ID NO: 3)
-Guide chain 9 (g9):
U ^ A (M) A ^ G (M) U ^ G (M) C ^ T (M) U ^ C (M) C ^ A (M) U ^ G (M) U ^ T (M) U ^ T (M) G ^ G (M) U ^ T (M) G (L) ^ A (L)
-Passenger chain 10 (p10):
A (L) ^ C (L) ^ C ^ A ^ A ^ A ^ A ^ C ^ A ^ U ^ G ^ G ^ A ^ A ^ G ^ C ^ A ^ C ^ U ^ U ^ A ^ C ( L) ^ T (L)
(here,
・ U stands for uracil, T stands for thymine, A stands for adenine, and C stands for cytosine.
-(M) represents a modification in which the 2'hydroxyl group of ribose in the ribonucleotide unit to the left of this symbol is replaced with a methoxy group.
・ In (L), the ribose in the ribonucleotide unit to the left of this symbol is
Figure JPOXMLDOC01-appb-C000013
Represents a modification that has been replaced by
・ ^ Represents a modification in which one oxygen atom that is not involved in the bond of the phosphate group between the adjacent ribonucleotide units is replaced with a sulfur atom).
Alexa647 was attached to the 5'end of the guide chains (g1 and g9) via an ssH amino linker.
　（実施例２）１次スクリーニング用細胞の作製
　合成したmiR302核酸改変体の機能を検討するために、ＨｎＲＮＰＡ２Ｂ１遺伝子の３’ＵＴＲ（以下）にルシフェラーゼ遺伝子を融合した遺伝子を、レンチウイルを用いてＨＴ２９細胞（大腸がん細胞株）に導入して、この融合遺伝子を安定的に発現する細胞を作製した。ＨｎＲＮＰＡ２Ｂ１遺伝子の３’ＵＴＲ領域にｍｉＲ３０２ａが結合することで融合遺伝子のｍＲＮＡが不安定化し、ルシフェラーゼによる発光量が減少する。そのため、この細胞に薬剤を接触させてルシフェラーゼ発光量を測定すれば、その薬剤がどの程度のｍｉＲ３０２ａ活性を有するか試験することができる。
・使用したＨｎＲＮＰＡ２Ｂ１遺伝子の３’ＵＴＲ配列
gcttcttcctatttgccatgggcttcactgtataaataggagaggatgagagcccagaggtaacagaacagcttcaggttatcgaaataacaatgttaaggaaactcttatctcagtcatgcataaatatgcagtgatatggcagaagacaccagagcagatgcagagagccattttgtgaatggattggattatttaataacattaccttactgtggaggaaggattgtaaaaaaaaatgcctttgagacagtttcttagctttttaattgttgtttctttctagtggtctttgtaagagtgtagaagcattccttctttgataatgttaaatttgtaagtttcaggtgacatgtgaaaccttttttaagatttttctcaaagttttgaaaagctattagccaggatcatggtgtaataagacataacgtttttcctttaaaaaaatttaagtgcgtgtgtagagttaagaagctgttgtacatttatgatttaataaaataattctaaaggaaattgtgtaattatagactttttattttaaataagttaaggagtgggtagtataattaaggtccgttgcaaagctgttgttatatttgtataagataaatgctggtcagatgtaagtgtgttgtctgcaattcatcaggattaaattatgtagataacttaagggatatctctgcaaggagaaacacctttttagatcttttagatgctgcttcttcaatgcaaggaaaggaaataaccccagcgaggtactcttcagggacacaggtctagtacaagagaactcttgacggctactaagttcagccagtcttaaaaaactgtgctgtttctacaaaactttaactacagtagtttataaggatgccaacgaaagctgagggtgtagagcaaaatagttctaagcttcagttaaacttctttaggtaagatcttatttacttttcctttcttaattttcctccctaaaagataaactaatactcttaaatggtctttcagtatagtggttcttacgtagtttaacatagctataaattgagtttaacaatttataaactcaagagaataatttttataaaccctgttttccaatctgtcatttacttaaattattttggttgtttttccctttttttccttcttttcccaccccctccccctccatgtgaagatttgggtgcttaacatatcatttttttccctgccggaattttagcattgatatgaaccatggacaagtatattctgctgccacaaagactgtaaagtgcttcatttcaacagctgaggcaagccaagtgatcattaataaagcttttcttggttccttcagtggtgttggtagtaaaatggtaggtaaaagttaggctgcaagttcaataaatcatgagatttcccatcgttacacccttgtgtattcacatttcttggatcaaacattttgagtgaactaggggtttttattaaagacatttgttgtatttatggttgtaactgtacatgcttatcaggatgagactgaaagaaggtagggcaaaaatggttgaatctattttcagatagtagttcatacttgagtgaagtgtcttgtctgcattatgaagcctggtatgtatccagtactaaataggtgggttaaatgtggtaattctagttcagtgtcttaccctgaagagaaagttgtaggttggctgttgaaattcattccttagatatgatcagtttgattgcccggctttattgcctttacaggaatgtgatactcagggcttactctatacaccaatgagtcttctttgatcctaagaccaccactgaagttgtttaggttcttttggacaaacatgataaacttcttcagatactttttttttcctttggcaggaaggtgtcttgctgcaggtaactaatgaagaagtggtcaaccacagagtcttcaagaaataagaaattctgtaccatctgaaagtagttcttgttggtgccttcatttaaaaagcactctttaaaataaaagggaaatgttttctgataaaacaaacatttagttgaggttcttgatataaaacaattacaaaatgagtgttgtttgtaaaacagtaacatcaaattggctagagagataaatgtatcatgttttaaattaggttttgtgagtagacagattacaattctattttaaatataaagtttataaaataaatactttttgtatccaaatacttggtgtaatgtttacacataaaatgtgtgaatcttgttctataaatatttggttgtctaaaagatcaccatcccctaaatttttaaaagcagtttcacaaagctatgcatattttaatattaacaggtaaatgagaagagcattgtggacattattggctgtccccaataaaatgctgttcatta　(配列番号６) (Example 2) Preparation of cells for primary screening In order to examine the function of the synthesized miR302 nucleic acid variant, a gene in which a luciferase gene was fused to 3'UTR (hereinafter) of the HnRNPA2B1 gene was used in HT29 cells using a lentiwill. It was introduced into (colon cancer cell line) to prepare cells that stably express this fusion gene. The binding of miR302a to the 3'UTR region of the HnRNPA2B1 gene destabilizes the mRNA of the fusion gene and reduces the amount of luciferase luminescence. Therefore, if a drug is brought into contact with the cells and the amount of luciferase luminescence is measured, it is possible to test how much the drug has miR302a activity.
-The 3'UTR sequence of the HnRNPA2B1 gene used
(SEQ ID NO: 6)
　（実施例３）一次スクリーニング
　上記のＨＴ２９細胞を９６ウェルプレート（Nunc（商標））に１×１０３細胞／ウェルの濃度で播種した（０日目）。１日目に各ウェルの細胞に、Lipofectamine　RNAi　MAX（Invitrogen、米国）を使用して上記のmiR302核酸改変体または天然型miR302（ガイド鎖とパッセンジャー鎖との組み合わせ）を導入した。２日目に培地を交換し、３日目にルシフェラーゼの発光量を測定した。ルシフェラーゼ発光の測定には、Dual-Glo（登録商標）Luciferase　Assay　System(promega、米国)およびGLO　MAX　MULTI(promega、米国)を使用した。Image　Jで染色面積を算出して比較した。結果を図１に示す。 (Example 3) Primary screening The above HT29 cells were seeded on a 96-well plate (Nunc ™) at a concentration of 1 × 10 3 cells / well (day 0). On day 1, cells in each well were introduced with the above miR302 nucleic acid variant or native miR302 (combination of guide and passenger strands) using Lipofectamine RNAi MAX (Invitrogen, USA). The medium was changed on the second day, and the amount of luciferase emitted was measured on the third day. The Dual-Glo® Luciferase Assay System (promega, USA) and GLO MAX MULTI (promega, USA) were used to measure luciferase luminescence. The stained area was calculated with Image J and compared. The results are shown in FIG.
　（実施例４）二次スクリーニング
　一次スクリーニングの結果、ルシフェラーゼ発光の抑制効果が高かった１０種類のmiR302核酸改変体組み合わせを二次スクリーニングに供した。二次スクリーニングのために、膵がん細胞株であるＰａｎｃ－１およびＭＩＡ－ＰａＣａを、９６ウェルプレート（Cell　able（登録商標）、東洋合成工業、東京）に播種し（０日目）、翌日にLipofectamine　RNAi　MAX（Invitrogen、米国）を使用して上記のmiR302核酸改変体または天然型miR302（ガイド鎖とパッセンジャー鎖との組み合わせ）を導入した。４日目および７日目にＤＡＰＩ染色を行い、蛍光強度を測定して、細胞死を指標としてスクレーニングを行った。結果を図２～４に示す。
　Ｇ９Ｐ１０対は、強い抗腫瘍効果を示すことが観察された。以降では、このＧ９Ｐ１０対をＯＵＭ３０２と称する。 (Example 4) Secondary screening As a result of the primary screening, 10 kinds of miR302 nucleic acid variant combinations having a high inhibitory effect on luciferase luminescence were subjected to the secondary screening. For secondary screening, pancreatic cancer cell lines Panc-1 and MIA-PaCa were seeded on 96-well plates (Cell able®, Toyo Gosei, Tokyo) (day 0) and the next day. The above miR302 nucleic acid variant or native miR302 (combination of guide and passenger strands) was introduced using Lipofectamine RNAi MAX (Invitrogen, USA). DAPI staining was performed on the 4th and 7th days, the fluorescence intensity was measured, and screening was performed using cell death as an index. The results are shown in FIGS. 2-4.
It was observed that the G9P10 pair showed a strong antitumor effect. Hereinafter, this G9P10 pair will be referred to as OUM302.
　（実施例５）動物投与実験
　５０ｍｍ３の腫瘍体積を有するＮＯＤ－ＳＣＩＤマウスに核酸（６２．５ｎｍｏｌ／ｋｇ）（ＯＵＭ３０２または天然型miR302（Ｇ１Ｐ１対））＋ユニットＰＩＣを単回静脈注射し、１４日後まで体重および腫瘍体積を観察した。また、ベヒクル投与群を対象とした。結果を図５～６に示す。
　マウスにおいて、ＯＵＭ３０２は、天然型miR302と比較して優れた抗腫瘍効果を示した。 (Example 5) Animal administration experiment Nucleic acid (62.5 nmol / kg) (OUM302 or natural miR302 (G1P1 pair)) + unit PIC was intravenously injected into NOD-SCID mice having a tumor volume of 50 mm 3 and 14 Body weight and tumor volume were observed until days later. In addition, the vehicle-administered group was included. The results are shown in FIGS. 5 to 6.
In mice, OUM302 showed superior antitumor effects compared to native miR302.
　５０ｍｍ３の腫瘍体積を有するＮＯＤ－ＳＣＩＤマウスに核酸（６２．５ｎｍｏｌ／ｋｇ）（ＯＵＭ３０２または天然型miR302）＋ユニットＰＩＣ）＋抗がん剤（ＴＡＳ－１０２（トリフルリジン・チピラシル塩酸塩、ロンサーフ（登録商標））（１５０ｍｇ／ｋｇ）、５ＦＵ（５－フルオロウラシル）（８．３ｍｇ／ｋｇ）またはゲムシタビン（１００ｍｇ／ｋｇ））を単回投与し、１４日後まで体重および腫瘍体積を観察した。結果を図７～１２に示す。
　マウスにおいて、抗がん剤と組み合わせた場合、ＯＵＭ３０２は、天然型miR302と比較して優れた抗腫瘍効果を示した。 Nucleic acid (62.5 nmol / kg) (OUM302 or native miR302) + unit PIC) + anti-cancer agent (TAS-102 (trifluridine / tipiracil hydrochloride, Ronsurf) in NOD-SCID mice with a tumor volume of 50 mm 3 Registered trademark)) (150 mg / kg), 5FU (5-fluorouracil) (8.3 mg / kg) or gemcitabine (100 mg / kg)) was administered as a single dose, and body weight and tumor volume were observed until 14 days later. The results are shown in FIGS. 7-12.
In mice, OUM302 showed superior antitumor effects compared to native miR302 when combined with anticancer agents.
　膵がん自然発症マウスであるＥＬ１－ＳＶ４０　Ｔｇマウス（Ｔａｃｏｎｉｃ社より購入したＦＶＢ／Ｎ－Ｔｇ（Ｃｅｌａ１－Ｌｕｃ，　Ｃｅｌａ１－Ｔａｇ）１１６Ｘｅｎマウス）に核酸（６２．５ｎｍｏｌ／ｋｇ）（ＯＵＭ３０２または天然型miR302）＋ユニットＰＩＣ＋抗がん剤（ＴＡＳ－１０２、１５０ｍｇ／ｋｇ）を週１回投与し、３２日間体重を観察し、３２日後に腫瘍を摘出して重量を測定した。また、ベヒクル投与群を対象とした。結果を図１４～１５に示す。
　ＯＵＭ３０２は、天然型miR302と比較して優れた抗腫瘍効果を示した。 EL1-SV40 Tg mouse (FVB / N-Tg (Cela1-Luc, Cela1-Tag) 116Xen mouse purchased from Taconic), which is a spontaneous pancreatic cancer mouse, and nucleic acid (62.5 nmol / kg) (OUM302 or natural type miR302) + unit PIC + anticancer drug (TAS-102, 150 mg / kg) was administered once a week, the body weight was observed for 32 days, and after 32 days, the tumor was removed and weighed. In addition, the vehicle-administered group was included. The results are shown in FIGS. 14 to 15.
OUM302 showed an excellent antitumor effect as compared with the natural miR302.
　（実施例６）miR302核酸改変体の安定性
　ＦＢＳを終濃度１％となるように添加した溶液にmiR302核酸改変体（ｇ１、ｇ９、ｐ１またはｐ１０）を終濃度が２μＭとなるように添加して３７℃でインキュベートした。インキュベート開始の１５分、３０分、１時間、３時間、２４時間後に試料を収集し、試料を電気泳動に供した。
　電気泳動は、２０％アクリルアミド／ビスゲルで行った。ＦＢＳを添加しなかった条件を対照として使用した。泳動マーカーは、20bp　DNA　ladder(タカラバイオ、滋賀)を使用した。露光時間はCy3_30secであった。（図１６） (Example 6) Stability of miR302 nucleic acid variant MiR302 nucleic acid variant (g1, g9, p1 or p10) was added to a solution to which FBS was added so as to have a final concentration of 1% so that the final concentration was 2 μM. Incubated at 37 ° C. Samples were collected 15 minutes, 30 minutes, 1 hour, 3 hours and 24 hours after the start of incubation and subjected to electrophoresis.
Electrophoresis was performed with 20% acrylamide / bisgel. The condition without adding FBS was used as a control. A 20 bp DNA ladder (Takara Bio, Shiga) was used as the migration marker. The exposure time was Cy3_30sec. (Fig. 16)
　天然型のｇ１およびｐ１はＦＢＳによる処理後バンドが確認できなかった。miR302核酸改変体であるｇ９およびｐ１０はＦＢＳによる処理後バンドが経時的に消失しているが、天然型のＲＮＡよりは安定であることが観察された。 No band was confirmed after treatment with FBS for natural g1 and p1. It was observed that the miR302 nucleic acid variants g9 and p10 were more stable than the native RNA, although the bands after treatment with FBS disappeared over time.
　ＦＢＳを終濃度１％となるように添加した溶液にmiR302核酸改変体（天然型のｇ１ｐ１対またはｇ９ｐ１０対ＯＵＭ３０２）を終濃度が２μＭとなるように添加して３７℃でインキュベートした。インキュベート開始の１時間後に試料を収集し、試料を電気泳動に供した。
　電気泳動は、２％アガロースゲルまたは２０％アクリルアミド／ビスゲルで行った。ＦＢＳを添加しなかった条件を対照として使用した。泳動マーカーは、20bp　DNA　ladder(タカラバイオ、滋賀)を使用した（図１７）。 A miR302 nucleic acid variant (natural g1p1 pair or g9p10 vs. OUM302) was added to a solution to which FBS was added to a final concentration of 1% to a final concentration of 2 μM, and the mixture was incubated at 37 ° C. Samples were collected 1 hour after the start of incubation and subjected to electrophoresis.
Electrophoresis was performed on a 2% agarose gel or 20% acrylamide / bisgel. The condition without adding FBS was used as a control. A 20 bp DNA ladder (Takara Bio, Shiga) was used as the migration marker (Fig. 17).
　天然型のｇ１ｐ１対はＦＢＳによる処理後バンドが確認できなかった。miR302核酸改変体であるｇ９ｐ１０対はＦＢＳによる処理後においてもバンドが確認できた。 No band could be confirmed after treatment with FBS for the natural g1p1 pair. Bands of g9p10 pair, which is a miR302 nucleic acid variant, could be confirmed even after treatment with FBS.
　（注記）
　以上のように、本開示の好ましい実施形態を用いて本開示を例示してきたが、本発明は、特許請求の範囲によってのみその範囲が解釈されるべきであることが理解される。本明細書において引用した特許、特許出願および文献は、その内容自体が具体的に本明細書に記載されているのと同様にその内容が本明細書に対する参考として援用されるべきであることが理解される。 (Note)
As described above, the present disclosure has been illustrated using the preferred embodiments of the present disclosure, but it is understood that the scope of the present invention should be interpreted only by the scope of claims. The patents, patent applications and documents cited herein are to be incorporated by reference in their content as they are specifically described herein. Understood.
　本出願は、２０１９年４月２６日に出願された特願２０１９－０８５７８９号に対する優先権の利益を主張し、その内容全体が本明細書に援用される。 This application claims the benefit of priority to Japanese Patent Application No. 2019-085789 filed on April 26, 2019, the entire contents of which are incorporated herein by reference.
　本開示は、医薬品、例えば、がんを治療するための医薬品に利用可能である。 This disclosure is available for pharmaceutical products, such as pharmaceutical products for treating cancer.
・配列番号１：天然ヒトmiR302a配列
CCACCACUUAAACGUGGAUGUACUUGCUUUGAAACUAAAGAAGUAAGUGCUUCCAUGUUUUGGUGAUGG
・配列番号２：天然ヒトmiR302aのガイド配列（ｇ１）
UAAGUGCUUCCAUGUUUUGGUGA
・配列番号３：天然ヒトmiR302aのパッセンジャー配列（ｐ１）
ACUUAAACGUGGAUGUACUUGCU
・配列番号４：miR302核酸改変体（ｇ９）の塩基配列
UAAGUGCTUCCAUGUTUTGGUTGA
・配列番号５：miR302核酸改変体（ｐ１０）の塩基配列
ACCAAAACAUGGAAGCACUUACT
・配列番号６：使用したＨｎＲＮＰＡ２Ｂ１遺伝子の３’ＵＴＲ配列
gcttcttcctatttgccatgggcttcactgtataaataggagaggatgagagcccagaggtaacagaacagcttcaggttatcgaaataacaatgttaaggaaactcttatctcagtcatgcataaatatgcagtgatatggcagaagacaccagagcagatgcagagagccattttgtgaatggattggattatttaataacattaccttactgtggaggaaggattgtaaaaaaaaatgcctttgagacagtttcttagctttttaattgttgtttctttctagtggtctttgtaagagtgtagaagcattccttctttgataatgttaaatttgtaagtttcaggtgacatgtgaaaccttttttaagatttttctcaaagttttgaaaagctattagccaggatcatggtgtaataagacataacgtttttcctttaaaaaaatttaagtgcgtgtgtagagttaagaagctgttgtacatttatgatttaataaaataattctaaaggaaattgtgtaattatagactttttattttaaataagttaaggagtgggtagtataattaaggtccgttgcaaagctgttgttatatttgtataagataaatgctggtcagatgtaagtgtgttgtctgcaattcatcaggattaaattatgtagataacttaagggatatctctgcaaggagaaacacctttttagatcttttagatgctgcttcttcaatgcaaggaaaggaaataaccccagcgaggtactcttcagggacacaggtctagtacaagagaactcttgacggctactaagttcagccagtcttaaaaaactgtgctgtttctacaaaactttaactacagtagtttataaggatgccaacgaaagctgagggtgtagagcaaaatagttctaagcttcagttaaacttctttaggtaagatcttatttacttttcctttcttaattttcctccctaaaagataaactaatactcttaaatggtctttcagtatagtggttcttacgtagtttaacatagctataaattgagtttaacaatttataaactcaagagaataatttttataaaccctgttttccaatctgtcatttacttaaattattttggttgtttttccctttttttccttcttttcccaccccctccccctccatgtgaagatttgggtgcttaacatatcatttttttccctgccggaattttagcattgatatgaaccatggacaagtatattctgctgccacaaagactgtaaagtgcttcatttcaacagctgaggcaagccaagtgatcattaataaagcttttcttggttccttcagtggtgttggtagtaaaatggtaggtaaaagttaggctgcaagttcaataaatcatgagatttcccatcgttacacccttgtgtattcacatttcttggatcaaacattttgagtgaactaggggtttttattaaagacatttgttgtatttatggttgtaactgtacatgcttatcaggatgagactgaaagaaggtagggcaaaaatggttgaatctattttcagatagtagttcatacttgagtgaagtgtcttgtctgcattatgaagcctggtatgtatccagtactaaataggtgggttaaatgtggtaattctagttcagtgtcttaccctgaagagaaagttgtaggttggctgttgaaattcattccttagatatgatcagtttgattgcccggctttattgcctttacaggaatgtgatactcagggcttactctatacaccaatgagtcttctttgatcctaagaccaccactgaagttgtttaggttcttttggacaaacatgataaacttcttcagatactttttttttcctttggcaggaaggtgtcttgctgcaggtaactaatgaagaagtggtcaaccacagagtcttcaagaaataagaaattctgtaccatctgaaagtagttcttgttggtgccttcatttaaaaagcactctttaaaataaaagggaaatgttttctgataaaacaaacatttagttgaggttcttgatataaaacaattacaaaatgagtgttgtttgtaaaacagtaacatcaaattggctagagagataaatgtatcatgttttaaattaggttttgtgagtagacagattacaattctattttaaatataaagtttataaaataaatactttttgtatccaaatacttggtgtaatgtttacacataaaatgtgtgaatcttgttctataaatatttggttgtctaaaagatcaccatcccctaaatttttaaaagcagtttcacaaagctatgcatattttaatattaacaggtaaatgagaagagcattgtggacattattggctgtccccaataaaatgctgttcatta
・配列番号７：例示的な分解配列
UUGAAACUAAAGAAG
  -SEQ ID NO: 1: Natural human miR302a sequence
CCACCACUUAAACGUGGAUGUACUUGCUUUGAAACUAAAGAAGUAAGUGCUUCCAUGUUUUGGUGAUGG
SEQ ID NO: 2: Guide sequence of natural human miR302a (g1)
UAAGUGCUUCCAUGUUUUGGUGA
SEQ ID NO: 3: Passenger sequence of natural human miR302a (p1)
ACUUAAACGUGGAUGUACUUGCU
SEQ ID NO: 4: Nucleotide sequence of miR302 nucleic acid variant (g9)
UAAGUGCTUCCAUGUTUTGGUTGA
SEQ ID NO: 5: Nucleotide sequence of miR302 nucleic acid variant (p10)
ACCAAAACAUGGAAGCACUUACT
SEQ ID NO: 6: 3'UTR sequence of the HnRNPA2B1 gene used

SEQ ID NO: 7: exemplary degraded sequence
UUGAAACUAAAGAAG

Claims (15)

  1. 　ヒトmiR302a核酸改変体。 Human miR302a nucleic acid variant.
  2. UAAGUGCUUCCAUGUUUUGGUGA（ｇ１）(配列番号２)または
    ACUUAAACGUGGAUGUACUUGCU（ｐ１）(配列番号３)
    の塩基配列から０、１、２、３、４または５個の置換、欠失または挿入を含む塩基配列を含む、請求項１に記載の核酸改変体。     UAAGUGCUUCCAUGUUUUGGUGA (g1) (SEQ ID NO: 2) or
    ACUUAAACGUGGAUGUACUUGCU (p1) (SEQ ID NO: 3)
    The nucleic acid variant according to claim 1, which comprises a base sequence containing 0, 1, 2, 3, 4 or 5 substitutions, deletions or insertions from the base sequence of.
  3. UAAGUGCUUCCAUGUUUUGGUGA（ｇ１）(配列番号２)の８番目および１８番目の塩基の少なくとも一方、または
    ACUUAAACGUGGAUGUACUUGCU（ｐ１）(配列番号３)の４番目および１４番目の塩基の少なくとも一方
    に対応する位置において塩基の置換、欠失または挿入を含む塩基配列を含む、請求項１または２に記載の核酸改変体。     At least one of the 8th and 18th bases of UAAGUGCUUCCAUGUUUUGGUGA (g1) (SEQ ID NO: 2), or
    The nucleic acid modification according to claim 1 or 2, which comprises a base sequence containing a base substitution, deletion or insertion at a position corresponding to at least one of the 4th and 14th bases of ACUUAAACGUGGAUGUACUUGCU (p1) (SEQ ID NO: 3). body.
  4. UAAGUGCUUCCAUGUUUUGGUGA（ｇ１）(配列番号２)の８番目および１８番目以外の塩基において０、１または２個の置換、欠失または挿入を含む塩基配列、または
    ACUUAAACGUGGAUGUACUUGCU（ｐ１）(配列番号３)の４番目および１４番目以外の塩基において０、１または２個の置換、欠失または挿入を含む塩基配列
    を含む、請求項１～３のいずれか一項に記載の核酸改変体。     A base sequence containing 0, 1 or 2 substitutions, deletions or insertions in bases other than the 8th and 18th bases of UAAGUGCUUCCAUGUUUUGGUGA (g1) (SEQ ID NO: 2), or
    ACUUAAACGUGGAUGUACUUGCU (p1) (SEQ ID NO: 3) according to any one of claims 1 to 3, which comprises a base sequence containing 0, 1 or 2 substitutions, deletions or insertions in bases other than the 4th and 14th bases. The nucleic acid variant of the description.
  5. UAAGUGCTUCCAUGUTUTGGUTGA（ｇ９）(配列番号４)の８番目および１８番目以外の塩基が同一である塩基配列、または
    ACCAAAACAUGGAAGCACUUACT（ｐ１０）(配列番号５)の４番目および１４番目以外の塩基が同一である塩基配列
    を含む、請求項１～４のいずれか一項に記載の核酸改変体。     UAAGUGCTUCCAUGUTUTGGUTGA (g9) (SEQ ID NO: 4) has the same base sequence other than the 8th and 18th bases, or
    The nucleic acid variant according to any one of claims 1 to 4, which comprises a base sequence having the same base other than the 4th and 14th bases of ACCAAAACAUGGAAGCACUUACT (p10) (SEQ ID NO: 5).
  6. 　以下の（１）～（７）のいずれかの改変：
    （１）リボースから以下のいずれかの架橋リボースへの置換
    Figure JPOXMLDOC01-appb-C000001
    （式中、Ｒｘは、それぞれ独立して水素、または置換または非置換のＣ１～Ｃ６アルキルである）
    （２）リン酸基の結合に関与しない一つの酸素原子の硫黄原子への置換
    （３）リボースの２’のヒドロキシル基のアルコキシ基への置換
    （４）リボースの２’のヒドロキシル基のハロゲン原子への置換
    （５）リボースからデオキシリボースへの置換
    （６）末端５’のリン酸化
    （７）リボースの２’位の炭素に結合した２つの基の間の結合関係（環の裏または表）が逆転した立体異性体糖への置換を１つまたは複数含む、請求項１～５のいずれか一項に記載の核酸改変体。     Modification of any of the following (1) to (7):
    (1) Substitution of ribose with any of the following cross-linked ribose
    Figure JPOXMLDOC01-appb-C000001
    (In the formula, R x is hydrogen independently, or substituted or unsubstituted C 1 to C 6 alkyl, respectively)
    (2) Substitution of one oxygen atom not involved in the bond of phosphate group to sulfur atom (3) Substitution of ribose 2'hydroxyl group to alkoxy group (4) Halogen atom of ribose 2'hydroxyl group Substitution to (5) Substitution of ribose to deoxyribose (6) Phosphorization of terminal 5'(7) Bonding relationship between two groups bonded to the 2'carbon of ribose (back or front of ring) The nucleic acid variant according to any one of claims 1 to 5, which comprises one or more substitutions with the reversed steric isomer sugar.
  7. 　Ｒｘが、それぞれ独立して水素、メチル、エチル、ｎ－プロピル、イソプロピル、またはベンジルである、請求項６に記載の核酸改変体。 The nucleic acid variant of claim 6, wherein R x is hydrogen, methyl, ethyl, n-propyl, isopropyl, or benzyl, respectively.
  8. U^A(M)A^G(M)U^G(M)C^T(M)U^C(M)C^A(M)U^G(M)U^T(M)U^T(M)G^G(M)U^T(M)G(L)^A(L)または
    A(L)^C(L)^C^A^A^A^A^C^A^U^G^G^A^A^G^C^A^C^U^U^A^C(L)^T(L)
    の構造
    （式中、
    　Uはウラシル、Tはチミン、Aはアデニン、Cはシトシンの各塩基を表し、
    　(M)は、この記号の左のリボヌクレオチド単位中のリボースの２’のヒドロキシル基がメトキシ基に置き換えられている改変を表し、
    　(L)は、この記号の左のリボヌクレオチド単位中のリボースが
    Figure JPOXMLDOC01-appb-C000002
    に置き換えられている改変を表し、
    　^は、両隣のリボヌクレオチド単位の間のリン酸基の結合に関与しない一つの酸素原子が硫黄原子に置き換えられている改変を表す）
    から、(M)、(L)および^の改変が、０、１、２、３、４または５個追加および／または削除されている構造を含む、請求項１～７のいずれか一項に記載の核酸改変体。     U ^ A (M) A ^ G (M) U ^ G (M) C ^ T (M) U ^ C (M) C ^ A (M) U ^ G (M) U ^ T (M) U ^ T (M) G ^ G (M) U ^ T (M) G (L) ^ A (L) or
    A (L) ^ C (L) ^ C ^ A ^ A ^ A ^ A ^ C ^ A ^ U ^ G ^ G ^ A ^ A ^ G ^ C ^ A ^ C ^ U ^ U ^ A ^ C ( L) ^ T (L)
    Structure (in the formula,
    U stands for uracil, T stands for thymine, A stands for adenine, C stands for cytosine,
    (M) represents a modification in which the 2'hydroxyl group of ribose in the ribonucleotide unit to the left of this symbol is replaced with a methoxy group.
    (L) is the ribose in the ribonucleotide unit to the left of this symbol
    Figure JPOXMLDOC01-appb-C000002
    Represents a modification that has been replaced by
    ^ Represents a modification in which one oxygen atom that is not involved in the bond of the phosphate group between the adjacent ribonucleotide units is replaced with a sulfur atom)
    To any one of claims 1-7, comprising a structure in which modifications of (M), (L) and ^ are added and / or deleted by 0, 1, 2, 3, 4 or 5 The nucleic acid variant of the description.
  9. U^A(M)A^G(M)U^G(M)C^T(M)U^C(M)C^A(M)U^G(M)U^T(M)U^T(M)G^G(M)U^T(M)G(L)^A(L)または
    A(L)^C(L)^C^A^A^A^A^C^A^U^G^G^A^A^G^C^A^C^U^U^A^C(L)^T(L)
    の構造
    （式中、U、T、A、C、(M)、(L)、および^の定義は前記と同じ）
    を含む、請求項８に記載の核酸改変体。     U ^ A (M) A ^ G (M) U ^ G (M) C ^ T (M) U ^ C (M) C ^ A (M) U ^ G (M) U ^ T (M) U ^ T (M) G ^ G (M) U ^ T (M) G (L) ^ A (L) or
    A (L) ^ C (L) ^ C ^ A ^ A ^ A ^ A ^ C ^ A ^ U ^ G ^ G ^ A ^ A ^ G ^ C ^ A ^ C ^ U ^ U ^ A ^ C ( L) ^ T (L)
    (In the formula, the definitions of U, T, A, C, (M), (L), and ^ are the same as above)
    8. The nucleic acid variant according to claim 8.
  10. U^A(M)A^G(M)U^G(M)C^T(M)U^C(M)C^A(M)U^G(M)U^T(M)U^T(M)G^G(M)U^T(M)G(L)^A(L)または
    A(L)^C(L)^C^A^A^A^A^C^A^U^G^G^A^A^G^C^A^C^U^U^A^C(L)^T(L)
    の構造
    （式中、U、T、A、C、(M)、(L)、および^の定義は前記と同じ）
    から、(M)および(L)の改変が、０、１、２または３個追加および／または削除されており、^の改変が、０、１、２、３、４、５、６、７、８、９または１０個追加および／または削除されている、請求項８に記載の核酸改変体。     U ^ A (M) A ^ G (M) U ^ G (M) C ^ T (M) U ^ C (M) C ^ A (M) U ^ G (M) U ^ T (M) U ^ T (M) G ^ G (M) U ^ T (M) G (L) ^ A (L) or
    A (L) ^ C (L) ^ C ^ A ^ A ^ A ^ A ^ C ^ A ^ U ^ G ^ G ^ A ^ A ^ G ^ C ^ A ^ C ^ U ^ U ^ A ^ C ( L) ^ T (L)
    (In the formula, the definitions of U, T, A, C, (M), (L), and ^ are the same as above)
    From, the modifications of (M) and (L) are added and / or deleted by 0, 1, 2 or 3, and the modifications of ^ are 0, 1, 2, 3, 4, 5, 6, 7 , 8, 9 or 10 added and / or deleted, the nucleic acid variant of claim 8.
  11. 　ガイド鎖である請求項１～１０のいずれか１項に記載の核酸改変体と、パッセンジャー鎖である請求項１～１０のいずれか１項に記載の核酸改変体とによって形成された二本鎖。 A double strand formed by a nucleic acid variant according to any one of claims 1 to 10 which is a guide strand and a nucleic acid variant according to any one of claims 1 to 10 which is a passenger chain. ..
  12. 　ガイド部分である請求項１～１０のいずれか１項に記載の核酸改変体の部分と、パッセンジャー部分である請求項１～１０のいずれか１項に記載の核酸改変体の部分とを含む、核酸改変体分子。 A portion of the nucleic acid variant according to any one of claims 1 to 10 which is a guide portion and a portion of the nucleic acid variant according to any one of claims 1 to 10 which is a passenger portion. Nucleic acid variant molecule.
  13. 　生体内で分解され得る配列を有する、請求項１２に記載の核酸改変体分子。 The nucleic acid variant molecule according to claim 12, which has a sequence that can be degraded in vivo.
  14. 　ルシフェラーゼ遺伝子と、HnRNPA2B1遺伝子とを結合させた核酸構築物をレンチウイルスにより導入したＨＴ２９細胞と接触させた場合に、天然のヒトmiR302aよりも低いルシフェラーゼ発光をもたらす、かつ/またはインビトロでウシ胎児血清（FBS）と接触させた場合の分解速度が、天然のヒトmiR302aよりも遅い、請求項１～１０のいずれか１項に記載の核酸改変体、請求項１１に記載の二本鎖または請求項１２もしくは１３に記載の核酸改変体分子。 Fetal bovine serum (FBS) results in lower luciferase luminescence than native human miR302a when contacted with HT29 cells introduced by lentivirus with a nucleic acid construct in which the luciferase gene and the HnRNPA2B1 gene are bound, and / or in vitro. ) Is slower than that of the natural human miR302a, the nucleic acid variant according to any one of claims 1 to 10, the double strand according to claim 11, or the double strand according to claim 12 or 13. The nucleic acid variant molecule according to 13.
  15. 　請求項１～１０および１４のいずれか１項に記載の核酸改変体、請求項１１および１４のいずれか１項に記載の二本鎖または請求項１２、１３および１４のいずれか１項に記載の核酸改変体分子を含む、がんを治療するための医薬組成物。 The nucleic acid variant according to any one of claims 1 to 10 and 14, the double strand according to any one of claims 11 and 14, or any one of claims 12, 13 and 14. A pharmaceutical composition for treating cancer, which comprises a nucleic acid variant molecule of.
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