Classifications

• • 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
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
  • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • 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
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• • 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
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

• the present invention relates to antisense oligonucleotides with reduced toxicity.
• Nucleic acid drugs are medicines consisting of nucleic acids (oligonucleotides) that form complementary base pairs with target DNA or RNA, and are expected to be new medicines.
• Various artificial nucleic acid units artificial nucleosides or artificial nucleotides that are phosphate adducts thereof) have been developed as nucleic acid units used in nucleic acid drugs.
• MOE methoxyethylating
• 2',4'-BNA and 2',4'-LNA are compounds in which the 2' and 4' positions of the sugar moiety of the nucleic acid unit are crosslinked, and are known to have high affinity to the target nucleic acid (see, for example, Patent Documents 2 to 5).
• MCE methylcarbamoylethyl
• gapmer-type antisense nucleic acids in which artificial nucleic acid units are introduced into both ends (5'-side region and 3'-side region) of a single-stranded oligodeoxyribonucleotide is progressing. It is known that gapmer-type antisense nucleic acids form a double-stranded complex with a target RNA, and that RNase H in cells recognizes the double-stranded portion between the deoxyribonucleotide portion and the target RNA and cleaves the RNA strand.
• gapmer-type antisense nucleic acids in which 2'-4' bridged nucleotides such as 2',4'-BNA and 2',4'-LNA are introduced into both ends are known to have a strong knockdown effect on the target RNA.
• gapmer-type antisense nucleic acids having 2'-4' bridged nucleotides introduced at both termini can induce cytotoxicity by acting on non-target RNA or by binding to non-specific proteins within cells (see, for example, Non-Patent Document 3).
• a gapmer-type antisense nucleic acid having a nucleotide (2'-O-R-ECE) in which an amino group or a heterocyclic group has been introduced via an alkyl group to the nitrogen atom of a 2'-position modified carbamoylethyl group, in the central region and/or the 5' region and/or the 3' region, can reduce toxicity, and thus completed the present invention. That is, the present invention encompasses the following aspects.
• the 2'-O-R-ECE nucleotide is The following formula (I): (wherein Bx is a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group or a 2-thioxo-pyrimidin-1-yl group (the purin-9-yl group, the 2-oxo-pyrimidin-1-yl group and the 2-thioxo-pyrimidin-1-yl group are each independently unsubstituted or substituted with one or more substituents selected, either singly or differently, from the group consisting of a halogen atom, a C1-6 alkyl group, an amino group, a protected amino group, a hydroxy group, a protected hydroxy group, a sulfanyl group and a protected sulfanyl group), R 1 , R 2 , R 3 and R 4 each independently represent a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group or a C
• the central region consists of 5 to 15 nucleotides, 8.
• the antisense oligonucleotide according to any one of 1 to 7, wherein the 5' region and the 3' region each independently consist of 2 to 7 nucleotides.
• the central region consists of 8 to 12 nucleotides, 9.
• the antisense oligonucleotide according to any one of 1 to 8, wherein the 5' region and the 3' region each independently consist of 2 to 5 nucleotides.
• the antisense oligonucleotide according to any one of 1. to 9., wherein the 2'-4' bridged nucleotide is at least one selected from the group consisting of LNA, cEt-BNA, ENA, BNA NC , AmNA, scpBNA and GuNA.
• the antisense oligonucleotide according to any one of 1. to 9., wherein the 2'-modified nucleotide is at least one selected from the group consisting of a 2'-O-R-ECE nucleotide, a 2'-O-MCE nucleotide, a 2'-O-MOE nucleotide, a 2'-O-NMA nucleotide, and a 2'-O-Me nucleotide.
• each nucleotide constituting the antisense oligonucleotide is independently linked by a phosphodiester bond or a phosphorothioate bond.
• a prodrug of the antisense oligonucleotide according to any one of 1. to 19.
• a double-stranded oligonucleotide complex comprising: (i) the antisense oligonucleotide according to any one of 1. to 19.; and (ii) an oligonucleotide comprising a region that hybridizes to the antisense oligonucleotide.
• An oligonucleotide comprising (i) a group derived from the antisense oligonucleotide according to any one of 1. to 19., and (ii) a group derived from an oligonucleotide comprising a region that hybridizes to the antisense oligonucleotide, wherein the group derived from the antisense oligonucleotide (i) and the group derived from the oligonucleotide (ii) are linked together.
• a pharmaceutical composition comprising the antisense oligonucleotide according to any one of 1. to 19., the prodrug according to 20., the oligonucleotide conjugate according to 21., or the oligonucleotide according to 22., and a pharmacologically acceptable carrier.
• a method for controlling a function of a target RNA comprising the step of contacting a cell with the antisense oligonucleotide according to any one of 1. to 19., the prodrug according to 20., the oligonucleotide conjugate according to 21., or the oligonucleotide according to 22..
• a method for regulating a function of a target RNA in a mammal comprising the step of administering to the mammal the pharmaceutical composition according to 23.
• a method for controlling expression of a target gene comprising the step of contacting a cell with the antisense oligonucleotide according to any one of 1. to 19., the prodrug according to 20., the oligonucleotide conjugate according to 21., or the oligonucleotide according to 22..
• a method for regulating expression of a target gene in a mammal comprising the step of administering to the mammal the pharmaceutical composition according to 23.
• the present invention provides antisense oligonucleotides with reduced toxicity.
• n- stands for normal, "i-” stands for iso, "s-” stands for secondary, “t-” stands for tertiary, “o-” stands for ortho, “m-” stands for meta, and “p-” stands for para.
• Ph stands for phenyl
• Me stands for methyl
• Bu stands for butyl
• Ac stands for acetyl
• DMTr dimethoxytrityl
• Halogen atom means a fluorine atom, chlorine atom, bromine atom or iodine atom.
• C1-6 alkyl group means a monovalent group of linear or branched saturated aliphatic hydrocarbons having 1 to 6 carbon atoms.
• Examples of C1-6 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and isohexyl.
• C1-3 alkyl group means a monovalent group of linear or branched saturated aliphatic hydrocarbons having 1 to 3 carbon atoms.
• Halo C1-6 alkyl group refers to a group in which at least one hydrogen atom at any position of the "C1-6 alkyl group” is replaced with the "halogen atom” described above.
• C2-6 alkenyl group means a monovalent group of a straight-chain or branched unsaturated aliphatic hydrocarbon containing 2 to 6 carbon atoms and containing at least one carbon-carbon double bond.
• Examples of C2-6 alkenyl groups include vinyl groups, allyl groups, propenyl groups, isopropenyl groups, butenyl groups, isobutenyl groups, butadienyl groups, 3-methyl-2-butenyl groups, pentenyl groups, isopentenyl groups, pentadienyl groups, hexenyl groups, isohexenyl groups, and hexadienyl groups.
• C1-6 alkoxy group refers to a group in which the above-mentioned "C1-6 alkyl group” is bonded to an oxy (-O-) group.
• Examples of C1-6 alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, isobutoxy, s-butoxy, n-pentyloxy, isopentyloxy, and n-hexyloxy groups.
• C2-6 alkenyloxy group refers to a group in which the "C2-6 alkenyl group” is bonded to an oxy (-O-) group.
• Examples of C2-6 alkenyloxy groups include vinyloxy groups, allyloxy groups, propenyloxy groups, isopropenyloxy groups, butenyloxy groups, isobutenyloxy groups, butadienyloxy groups, 3-methyl-2-butenyloxy groups, pentenyloxy groups, isopentenyloxy groups, pentadienyloxy groups, hexenyloxy groups, isohexenyloxy groups, and hexadienyloxy groups.
• C2-20 alkylene group means a divalent linear or branched saturated aliphatic hydrocarbon group having 2 to 20 carbon atoms.
• C8-12 alkylene group means a divalent linear or branched saturated aliphatic hydrocarbon group having 8 to 12 carbon atoms, among the above-mentioned “C2-20 alkylene groups”.
• C2-6 alkylene group refers to a divalent linear or branched saturated aliphatic hydrocarbon group having 2 to 6 carbon atoms, among the above-mentioned "C2-20 alkylene groups”.
• Examples include an ethylene (ethanediyl) group, a propylene group, a propane-1,3-diyl (trimethylene) group, a propane-2,2-diyl (isopropylidene) group, a 2,2-dimethyl-propane-1,3-diyl group, a hexane-1,6-diyl (hexamethylene) group, and a 3-methylbutane-1,2-diyl group.
• C2-20 alkenylene group means a divalent linear or branched unsaturated aliphatic hydrocarbon group containing 2 to 20 carbon atoms and at least one carbon-carbon double bond.
• C1-6 alkylamino group includes mono-C1-6 alkylamino groups and di-C1-6 alkylamino groups.
• mono-C1-6 alkylamino group refers to a group in which one of the above-mentioned "C1-6 alkyl groups” is bonded to an amino group. Examples of mono-C1-6 alkylamino groups include methylamino group, ethylamino group, n-propylamino group, isopropylamino group, n-butylamino group, n-pentylamino group, and n-hexylamino group.
• di-C1-6 alkylamino group refers to a group in which two of the above-mentioned "C1-6 alkyl groups” are bonded to an amino group.
• the two alkyl groups may be the same or different.
• di-C1-6 alkylamino groups include dimethylamino, diethylamino, N,N-diisopropylamino, N-methyl-N-ethylamino, N-isopropyl-N-methylamino, N-n-butyl-N-methylamino, N-tert-butyl-N-methylamino, N-methyl-N-n-pentylamino, N-n-hexyl-N-methylamino, and N-isopropyl-N-ethylamino groups.
• C1-6 alkylaminocarbonyl group refers to a group in which the above-mentioned “C1-6 alkylamino group” is bonded to a carbonyl group.
• C1-6 alkylcarbonyloxy group refers to a group in which the above-mentioned “C1-6 alkylcarbonyl group” is bonded to an oxy group.
• C1-6 alkylcarbonylamino group refers to a group in which one of the above “C1-6 alkylcarbonyl groups” is bonded to an amino group.
• C1-6 alkoxycarbonylamino group refers to a group in which one of the above “C1-6 alkoxycarbonyl groups” is bonded to an amino group.
• Aryl group means a monovalent group formed by removing one hydrogen atom at any position from a monocyclic or bicyclic aromatic hydrocarbon in which all atoms constituting the ring are carbon atoms. Specific examples include “C6-10 aryl groups” such as phenyl and naphthyl groups.
• Alkyl group refers to a monovalent group in which a hydrogen atom at any position of the "C1-6 alkyl group” is replaced by the "C6-10 aryl group.”
• 3-11 membered nitrogen-containing non-aromatic heterocycle refers to a monocyclic, condensed polycyclic (in the condensed polycyclic, the non-aromatic ring may be condensed to a non-aromatic ring or an aromatic ring), bridged or spirocyclic non-aromatic heterocyclic compound containing at least one nitrogen atom and having 3 to 11 atoms constituting the ring, and examples of such heterocyclic compounds include azetidine, pyrrolidine, 2-oxopyrrolidine, piperidine, 3-oxopiperidine, piperazine, morpholine, thiomorpholine, homomorpholine and homopiperazine.
• C2-9 aromatic heterocyclic group means a monovalent group formed by removing one hydrogen atom at any position from an aromatic monocyclic or fused ring compound having one or more identical or different heteroatoms selected from oxygen atoms, sulfur atoms, and nitrogen atoms in the ring and having 2 to 9 carbon atoms constituting the ring.
• 5-10 membered heterocyclic group refers to a monovalent group obtained by removing one hydrogen atom at any position from a monocyclic or condensed polycyclic aromatic heterocyclic compound having 5 to 10 atoms constituting the ring and containing 1 to 5 heteroatoms (heteroatoms refer to nitrogen atoms, oxygen atoms, or sulfur atoms, and when there are two or more heteroatoms, they may be the same or different) among the atoms constituting the ring.
• Examples of monocyclic "5- to 10-membered heterocyclic groups” include 2-thienyl, 3-thienyl, 2-furyl, 3-furyl, 2-pyranyl, 3-pyranyl, 4-pyranyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 1-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl, and 5-pyrazolyl groups.
• the aryl group include 1,3,4-oxazolyl group, 2-oxazolyl group, 4-oxazolyl group, 5-oxazolyl group, 3-isoxazolyl group, 4-isoxazolyl group, 5-isoxazolyl group, 2-pyridyl group, 3-pyridyl group, 4-pyridyl group, 2-pyrazinyl group, 2-pyrimidinyl group, 4-pyrimidinyl group, 5-pyrimidinyl group, 3-pyrid
• Fused polycyclic "5- to 10-membered heterocyclic group” includes 2-benzofuranyl group, 3-benzofuranyl group, 4-benzofuranyl group, 5-benzofuranyl group, 6-benzofuranyl group, 7-benzofuranyl group, 1-isobenzofuranyl group, 4-isobenzofuranyl group, 5-isobenzofuranyl group, 2-benzothienyl group, 3-benzothienyl group, 4-benzothienyl group, 5-benzothienyl group, 6- Benzothienyl group, 7-benzothienyl group, 1-isobenzothienyl group, 4-isobenzothienyl group, 5-isobenzothienyl group, 2-benzothiazolyl group, 4-benzothiazolyl group, 5-benzothiazolyl group, 6-benzothiazolyl group, 7-benzothiazolyl group, 2-chromenyl group, 3-chromenyl group, 4-chromenyl group,
• protected hydroxy group refers to a hydroxy group protected by a hydroxy-protecting group, an amino group protected by an amino-protecting group and a sulfanyl group protected by a sulfanyl-protecting group, respectively.
• the hydroxy-protecting group, the amino-protecting group and the sulfanyl-protecting group are not particularly limited as long as they are stable during the synthesis of antisense oligonucleotides, and examples of the protecting groups include those described in Protective Groups in Organic Synthesis, 4th Edition, T. W. Greene, P. G. M. Wuts, John Wiley & Sons Inc. (2006), which are well known to those skilled in the art.
• the "amino protecting group” may be an amide-based protecting group such as acyl (e.g., formyl, acetyl, propionyl, pivaloyl (Pv), tigloyl, etc.), haloacyl (e.g., fluoroacetyl, difluoroacetyl, trifluoroacetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, etc.), arylcarbonyl (e.g., benzoyl, p-bromobenzoyl, p-nitrobenzoyl, 2,4-dinitrobenzoyl, etc.); C1-6 alkoxycarbonyl (e.g., methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, etc.); nyl, i-propoxycarbonyl, n-butoxycarbonyl, i-butoxycarbonyl, t
• iMds 2,6-dimethyl-4-methoxybenzenesulfonyl
• Mds 2,4,6-trimethoxybenzenesulfonyl
• the "antisense effect” means that the function of a target RNA is controlled by hybridization of a target RNA selected corresponding to a target gene with, for example, an oligonucleotide having a sequence complementary to a partial sequence of the target RNA.
• a target RNA selected corresponding to a target gene
• an oligonucleotide having a sequence complementary to a partial sequence of the target RNA for example, when the target RNA is an mRNA, this means inhibition of translation of the target RNA by hybridization, a splicing function conversion effect such as exon skipping, or degradation of the target RNA by recognition of the hybridized portion.
• an "antisense oligonucleotide” is an oligonucleotide that produces the antisense effect. Examples include, but are not limited to, DNA and oligodeoxyribonucleotides, and may also be RNA, oligoribonucleotides, or oligonucleotides designed to normally produce an antisense effect. The same applies to antisense nucleic acids.
• Hybridize refers to the act of forming a double strand between oligonucleotides or groups derived from oligonucleotides that contain complementary sequences, and the phenomenon in which oligonucleotides or groups derived from oligonucleotides that contain complementary sequences form a double strand.
• “Complementary” means that two nucleobases can form Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen base pairs, etc.) through hydrogen bonds.
• Two oligonucleotides or groups derived from oligonucleotides can "hybridize” if their sequences are complementary.
• the complementarity for two oligonucleotides or groups derived from oligonucleotides to hybridize is preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more (e.g., 95%, 96%, 97%, 98%, or 99% or more).
• Sequence complementarity can be determined by using a computer program that automatically identifies partial sequences of oligonucleotides.
• OligoAnalyzer is one such software and is provided by Integrated DNA Technologies. This program is also available on the website.
• Those skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which two oligonucleotides or groups derived from oligonucleotides can hybridize.
• Those skilled in the art can also easily design an antisense oligonucleotide complementary to a target RNA, for example, by using a BLAST program based on the nucleotide sequence information of the target RNA. For the BLAST program, see Proceedings of the National Academy of Sciences of the United States of America, 1990, 87, pp. 2264-2268; ibid., 1993, 90, pp. 5873-5877; and Journal of Molecular Biology, 1990, 215, pp. 403-410, etc.
• Nucleotide refers to a molecule that can be a building block of a nucleic acid (oligonucleotide), and typically has a base as a component.
• a nucleotide is composed of, for example, a sugar, a base, and a phosphate.
• Nucleotides include ribonucleotides, deoxyribonucleotides, and sugar-modified nucleotides, which are described below.
• Oligonucleotide refers to a molecule having a polymerized structure of one or more of the above-mentioned nucleotides.
• the oligonucleotide can be referred to as a "nucleotide.”
• the nucleotides contained in the "antisense oligonucleotide” molecule of the present invention are each independently linked to each other by a phosphodiester bond, a modified phosphodiester bond as described below, or a linking group containing a non-nucleotide structure as described below.
• the 3'-terminal nucleotide of the antisense oligonucleotide molecule of the present invention preferably has a hydroxy group or a phosphate group, more preferably a hydroxy group, and usually a hydroxy group, at its 3' position.
• the 5'-terminal nucleotide of the antisense oligonucleotide molecule preferably has a hydroxy group or a phosphate group, more preferably a hydroxy group, and usually a hydroxy group, at its 5' position.
• a group derived from an oligonucleotide means a group in which a hydrogen atom, a hydroxyl group, etc. has been removed from at least one of the hydroxyl groups at the 3' end and the 5' end of the oligonucleotide, and is indirectly covalently linked to another group (e.g., a group derived from another oligonucleotide) by forming a phosphodiester bond or a modified phosphodiester bond.
• the hydroxyl group at the 3' end or the 5' end includes a hydroxyl group possessed by a phosphate group.
• a group in which a hydrogen atom has been removed from the hydroxyl group at the 3' end of an oligonucleotide and a group in which a hydroxyl group has been removed from the phosphate group at the 5' end of another oligonucleotide form a phosphodiester bond or a modified phosphodiester bond.
• Nucleotide sequence means the base sequence of the nucleotides that make up an oligonucleotide.
• the 3' position of the deoxyribonucleotide is usually linked to another nucleotide, etc., via a phosphodiester bond or a modified phosphodiester bond (e.g., phosphorothioate bond), and the 5' position of the deoxyribonucleotide is linked to another nucleotide, etc., via a phosphodiester bond or a modified phosphodiester bond (e.g., phosphorothioate bond).
• the deoxyribonucleotide at the 3' end of the antisense oligonucleotide molecule of the present invention preferably has a hydroxyl group or a phosphate group at its 3' position, and the 5' position is as described above.
• the deoxyribonucleotide at the 5' end of the antisense oligonucleotide molecule preferably has a hydroxyl group or a phosphate group at its 5' position, and the 3' position is as described above.
• Oligonucleotide means an oligonucleotide composed of the above-mentioned deoxyribonucleotides.
• the deoxyribonucleotides that compose the oligodeoxyribonucleotide may be the same or different.
• DNA refers to an oligonucleotide composed of natural deoxyribonucleotides.
• the natural deoxyribonucleotides that make up DNA may be identical or different.
• “Ribonucleotide” refers to a molecule in which the sugar is ribose, a base is bound to the carbon atom at position 1 of the ribose, and a phosphate group is at position 2, 3, or 5.
• the ribonucleotide in the present invention may be a naturally occurring ribonucleotide, or a ribonucleotide in which the base portion or the phosphodiester bond portion of a naturally occurring ribonucleotide has been modified. Modifications of the base portion or the phosphodiester bond portion may be combined and performed on one ribonucleotide.
• modified ribonucleotides are described, for example, in Journal of Medicinal Chemistry, 2016, 59, pp. 9645-9667, Medicinal Chemistry Communication, 2014, 5, pp. 1454-1471, Future Medicinal Chemistry, 2011, 3, pp. 339-365, etc.
• the 3' position of the ribonucleotide is typically linked to another nucleotide via a phosphodiester bond or a modified phosphodiester bond (e.g., phosphorothioate bond), and the 5' position of the ribonucleotide is linked to another nucleotide, etc. via a phosphodiester bond or a modified phosphodiester bond (e.g., phosphorothioate bond).
• the 3'-terminal ribonucleotide of the antisense oligonucleotide molecule of the present invention preferably has a hydroxyl group or a phosphate group at its 3' position, and the 5' position is as described above.
• the 5'-terminal ribonucleotide of the antisense oligonucleotide molecule preferably has a hydroxyl group or a phosphate group at its 5' position, and the 3' position is as described above.
• Oligonucleotide means an oligonucleotide composed of the above-mentioned ribonucleotides.
• the ribonucleotides that compose the oligoribonucleotide may be the same or different.
• RNA means an oligonucleotide composed of natural ribonucleotides.
• the natural ribonucleotides that make up the RNA may be identical or different.
• Modified sugar refers to (Z1) A molecule in which ribose or 2-deoxyribose is partially replaced by one or more substituents; (Z2) a pentose or hexose other than ribose and 2-deoxyribose (e.g., hexitol, threose, etc.); (Z3) A molecule in which the entire ribose or 2-deoxyribose, or the tetrahydrofuran ring thereof, is replaced with a 5- to 7-membered saturated or unsaturated ring (e.g., cyclohexane, cyclohexene, morpholine, etc.), or a partial structure capable of forming a 5- to 7-membered ring by hydrogen bonding (e.g., a peptide structure), Or (Z4) means a molecule in which ribose or 2-deoxyribose is replaced with C2-6 alkylene glycol (e
• Modified sugars include "2-modified sugars,””2-4 linked sugars,” and “5-modified sugars,” as described below.
• modified sugars and sugar-modified nucleotides described below include sugars and sugar-modified nucleotides disclosed as being suitable for use in the antisense method in JP-A-10-304889, WO 2005/021570, JP-A-10-195098, Published Japanese Translation of PCT International Publication No. 2002-521310, WO 2007/143315, WO 2008/043753, WO 2008/029619, WO 2008/049085, and WO 2017/142054 (hereinafter, these documents are referred to as "documents related to the antisense method" and the like.
• examples of the N-substituted carbamoyl group include an N-methyl-carbamoyl group and an N-ethyl-carbamoyl group, in which the methyl group and the ethyl group of the N-methyl-carbamoyl group and the N-ethyl-carbamoyl group may be substituted with a 5- to 10-membered heterocyclic group or a mono- or di-C 1-6 alkylamino group.
• N-substituted carbamoyl group examples include an N-methylcarbamoyl group, an N-ethylcarbamoyl group, an N-dimethylaminoethyl-carbamoyl group, an N-(morpholinoethyl)carbamoyl group, an N-(2-pyridylethyl)carbamoyl group, an N-((benzimidazol-1-yl)ethyl)carbamoyl group, and the like.
• “Sugar-modified nucleotide” refers to a molecule having the above-mentioned “modified sugar” in place of the sugar portion of a deoxyribonucleotide or ribonucleotide. For example, it includes the "2'-modified nucleotide” and “2'-4' bridged nucleotide” described below, as well as the "5'-modified nucleotide.”
• the modified sugar is (Z3) as defined above, the sugar-modified nucleotide also includes a molecule in which the modified sugar and the nucleic acid base are linked via a methylene chain or the like.
• “Dimodified sugar” means a non-bridged sugar in which the oxygen or carbon atom at position 2 of ribose is modified, and includes "2-O-Me", “2-O-MOE”, “2-O-MCE”, “2-O-NMA”, “2-O-AP”, “2-F”, “2-DMAECE”, “2-MorECE”, “2-PyECE” and “2-BimECE”.
• the 4th and 5th positions of the "dimodified sugar” are preferably not modified.
• a “2'-modified nucleotide” refers to a molecule having a base bound to the carbon atom at position 1 (position 1 of unmodified ribose) of the "2'-modified sugar" and a phosphate group at position 3 or 5, and examples include "2'-O-Me nucleotide,”"2'-O-MOEnucleotide,”"2'-O-MCEnucleotide,”"2'-O-NMAnucleotide,”"2'-O-APnucleotide,”"2'-Fnucleotide,”"2'-DMAECEnucleotide,”"2'-MorECEnucleotide,”"2'-PyECEnucleotide,” and “2'-BimECE nucleotide.”
• 2-O-Me (also called 2-O-methyl) means a sugar in which the hydroxy group at position 2 of ribose is replaced with a methoxy group.
• a "2'-O-Me nucleotide” (also called a 2'-O-methyl nucleotide) means a molecule having a base bound to the carbon atom at position 1 of "2-O-Me” (position 1 of unmodified ribose) and a phosphate group at position 3 or 5.
• 2-O-MOE also known as 2-O-methoxyethyl
• 2-O-methoxyethyl means a sugar in which the hydroxy group at position 2 of ribose is replaced with a 2-methoxyethyloxy group.
• a "2'-O-MOE nucleotide” also called a 2'-O-methoxyethyl nucleotide
• a base is bound to the carbon atom at position 1 of "2-O-MOE” (position 1 of unmodified ribose) and a phosphate group is at position 3 or 5.
• 2-O-MCE also called 2-O-methylcarbamoylethyl
• 2-O-methylcarbamoylethyl means a sugar in which the hydroxy group at position 2 of ribose is replaced with a methylcarbamoylethyloxy group.
• 2'-O-MCE nucleotide also called 2'-O-methylcarbamoylethyl nucleotide
• 2-O-NMA means a sugar in which the hydroxy group at position 2 of ribose is replaced with a [2-(methylamino)-2-oxoethyl]oxy group.
• the term "2'-O-NMA nucleotide” refers to a molecule having a base bound to the carbon atom at position 1 of "2-O-NMA” (position 1 of unmodified ribose) and a phosphate group at position 3 or 5.
• 2-O-AP means a sugar in which the hydroxy group at position 2 of ribose is replaced with a 3-aminopropyloxy group.
• the term "2'-O-AP nucleotide” refers to a molecule having a base bound to the carbon atom at position 1 of "2-O-AP” (position 1 of unmodified ribose) and a phosphate group at position 3 or 5.
• 2-F means a sugar in which the hydroxy group at position 2 of ribose is replaced with a fluorine atom.
• the term "2'-F nucleotide” refers to a molecule having a base bound to the carbon atom at position 1 of "2-F” (position 1 of unmodified ribose) and a phosphate group at position 3 or 5.
• 2'-DMAECE nucleotide refers to molecules in which a base is bound to the carbon atom at position 1 (position 1 of unmodified ribose) of "2-DMAECE”, “2-MorECE”, “2-PyECE” and “2-BimECE”, respectively, and which have a phosphate group at position 3 or 5.
• the "2'-O-R-ECE nucleotide” has the following formula (I):
• Bx is a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group or a 2-thioxo-pyrimidin-1-yl group
• the purin-9-yl group, the 2-oxo-pyrimidin-1-yl group and the 2-thioxo-pyrimidin-1-yl group are each independently unsubstituted or substituted with one or more substituents selected, either singly or differently, from the group consisting of a halogen atom, a C1-6 alkyl group, an amino group, a protected amino group, a hydroxy group, a protected hydroxy group, a sulfanyl group and a protected sulfanyl group)
• R 1 , R 2 , R 3 and R 4 each independently represent a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and the C2-6 alkeny
• the C7-10 aralkyl group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino
• R 1 and R 2 each independently represent a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group, wherein the C1-6 alkyl group and the C2-6 alkenyl group are unsubstituted or substituted with one or more substituents, either singly or differently, selected from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group.
• R 1 is preferably a hydrogen atom or a C1-3 alkyl group, more preferably a hydrogen atom.
• R 2 is preferably a hydrogen atom or a C1-3 alkyl group, more preferably a hydrogen atom.
• R 3 and R 4 each independently represent a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group.
• the C1-6 alkyl group and the C2-6 alkenyl group are unsubstituted or substituted with one or more substituents, either singly or differently, selected from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group.
• R 3 may be the same or different
• R 4 may be the same or different.
• R3 is preferably a hydrogen atom or a C1-3 alkyl group, more preferably a hydrogen atom.
• R4 is preferably a hydrogen atom or a C1-3 alkyl group, more preferably a hydrogen atom.
• R 5 and R 6 each independently represent a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group, or a C7-10 aralkyl group.
• the C1-6 alkyl group and the C2-6 alkenyl group are unsubstituted or substituted with one or more substituents selected independently or differently from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonyloxy group, a C1-6
• the group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group.
• R5 is preferably a hydrogen atom or a C1-3 al
• R 5 and R 6 may together with the nitrogen atom to which they are bonded form a 3-11 membered nitrogen-containing non-aromatic heterocycle, where the 3-11 membered nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkyl
• the 3-11-membered nitrogen-containing non-aromatic heterocycle formed by R5 and R6 together with the nitrogen atom to which they are bonded is preferably a 4-8-membered nitrogen-containing non-aromatic heterocycle containing 4 to 6 methylene groups, such as piperidine, pyrrolidine, morpholine, thiomorpholine, homopiperidine, and homomorpholine. More preferably, it is a ring further containing an oxygen atom or a sulfur atom as an atom constituting the ring, such as morpholine, thiomorpholine, and homomorpholine. Morpholine is particularly preferred. In addition, it is preferable that the 4-8-membered nitrogen-containing non-aromatic heterocycle is unsubstituted.
• Y may be a C2-9 aromatic heterocyclic group. It is preferably a pyridyl group, an imidazolyl group, or a benzimidazolyl group, more preferably 2-pyridyl, imidazol-1-yl, or (benzimidazol)-1-yl, and particularly preferably 2-pyridyl or (benzimidazol)-1-yl.
• the nucleoside structure represented by formula (I) may be contained at the 3'-end or 5'-end of an oligonucleotide. When it is contained at the 3'-end, it has a structure represented by the following formula (II), for example.
• R 1 to R 4 , Bx, Y and n in formula (II) have the same meanings as R 1 to R 4 , Bx, Y and n in formula (I).
• Z 2 is a hydrogen atom or a phosphate group, and is preferably a hydrogen atom. When it is contained at the 5' end, it has a structure represented by the following formula (III), for example.
• R 1 to R 4 , Bx, Y and n have the same meanings as R 1 to R 4 , Bx, Y and n in formula (I).
• Z 1 is a hydrogen atom or a phosphate group, and is preferably a hydrogen atom.
• the "2'-modified nucleotide” includes a "2'-O-R-ECE nucleotide", and the "2'-O-R-ECE nucleotide” includes a "2'-DMAECE nucleotide", a "2'-MorECE nucleotide”, a "2'-PyECE nucleotide”, and a "2'-BimECE nucleotide”.
• 2-4 bridged sugar refers to a sugar in which the bridge unit is substituted at two positions, the 2-position and the 4-position of ribose.
• the bridge unit include a C2-6 alkylene group (the alkylene group is unsubstituted or substituted with one or more substituents selected from the group consisting of a halogen atom, an oxo group, and a thioxo group, and one or two methylene groups of the alkylene group are unsubstituted or independently substituted with a group selected from the group consisting of -O-, -NR'- (R' is a hydrogen atom, a C1-6 alkyl group, or a halo-C1-6 alkyl group), and -S-).
• Position 5 of the "2-4 bridged sugar" is preferably not modified.
• a “2',4' bridged nucleotide”(2',4'-BNA) refers to a molecule having a base bound to the carbon atom at position 1 of the "2,4 bridged sugar" (position 1 of unmodified ribose or 2-deoxyribose) and a nucleobase at position 3 or 5 of a phosphate group.
• 5-modified sugar means a non-bridged sugar in which the 5-position of 2-deoxyribose is modified, and includes "5-CP,””5-methyl,” and “5-aminopropyl.”
• the 2- and 4-positions of the "5-modified sugar” are preferably not modified.
• 5'-modified nucleotide refers to a molecule in which a base is bound to the carbon atom at position 1 (position 1 of 2-deoxyribose before modification) of the "5-modified sugar" and a nucleic acid base is attached to a phosphate group at position 2, 3 or 5, and includes, for example, "5'-CP nucleotide", “5'-methyl nucleotide” and "5'-aminopropyl nucleotide”.
• “5-CP” is a sugar in which the 5-position of 2-deoxyribose is substituted with two methyl groups, which together form a cyclopropane.
• a “5'-CP nucleotide” is a molecule in which the sugar is the above-mentioned “5-CP,” a base is bound to the carbon atom at position 1 (position 1 of the parent 2-deoxyribose), and a phosphate group is bound to position 3 or 5, and can be represented by the following structural formula.
• Base is a nucleic acid base. The wavy line is understood to represent a bond to an adjacent nucleotide, linker, etc., or a hydrogen atom or a phosphate group.
• 5-methyl is a sugar in which the 5-position of the 2-deoxyribose is substituted with a methyl group.
• 5-aminopropyl is a sugar in which the 5-position of the 2-deoxyribose is substituted with a 3-aminopropyl group.
• the terms "5'-methyl nucleotide” and “5'-aminopropyl nucleotide” refer to molecules in which a base is bound to the 1-position (the 1-position of the parent 2-deoxyribose) carbon atom of a "5-methyl” or “5-aminopropyl” nucleotide, respectively, and which have a nucleobase in a phosphate group at the 3- or 5-position.
• the bond between the carbon atom at the 1' position and the base can be an ⁇ -glycosidic bond or a ⁇ -glycosidic bond, but is usually a ⁇ -glycosidic bond. Therefore, ⁇ -D-methyleneoxy BNA is usually used as LNA.
• the "sugar-modified nucleotide" constitutes the antisense oligonucleotide molecule of the present invention
• the 3' position of the sugar-modified nucleotide is linked to another nucleotide, etc., via a phosphodiester bond or a modified phosphodiester bond (e.g., a phosphorothioate bond)
• the 5' position of the sugar-modified nucleotide is linked to another nucleotide, etc., via a phosphodiester bond or a modified phosphodiester bond (e.g., a phosphorothioate bond).
• the sugar-modified nucleotide at the 3' end of the antisense oligonucleotide molecule of the present invention preferably has a hydroxyl group or a phosphate group at its 3' position, and the 5' position is as described above.
• the sugar-modified nucleotide at the 5' end of the antisense oligonucleotide molecule preferably has a hydroxyl group or a phosphate group at its 5' position, and the 3' position is as described above.
• Examples of modifications of the phosphodiester bond in deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides include phosphorothioation, methylphosphonate (including chiral-methylphosphonate), methylthiophosphonate, phosphorodithioate, phosphoroamidate, phosphorodiamidate, phosphoroamidothioate, and boranophosphate.
• Examples of modifications of the phosphodiester bond in nucleotides are disclosed in Journal of Medicinal Chemistry, 2016, 59, pp. 9645-9667; Medicinal Chemistry Communication, 2014, 5, pp. 1454-1471; Future Medicinal Chemistry, 2011, 3, pp. 339-365, etc., and these can be used for the phosphodiester bond in deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides.
• modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, the pyrimidine base 5-propynyl (-C ⁇ C-CH 3 ) uracil and cytosine and other alkynyl derivatives, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-position substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-bro
• nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).
• tricyclic pyrimidines such as
• the modified nucleic acid base may also include those in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.
• modifications of the base moiety in nucleotides are disclosed in Journal of Medicinal Chemistry, 2016, 59, pp 9645-9667, Medicinal Chemistry Communication, 2014, 5, pp 1454-1471, Future Medicinal Chemistry, 2011, 3, pp 339-365, International Publication No. 2007/090071, etc., and these can be used for the base moiety in deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides.
• the amino and hydroxy of the base moiety may be independently protected.
• the base portion of the deoxyribonucleotide, ribonucleotide, and sugar-modified nucleotide is preferably at least one selected from the group consisting of adenine (A), guanine (G), thymine (T), cytosine (C), uracil (U), and 5-methylcytosine (5-me-C).
• RNase H is generally known as a ribonuclease that recognizes a double strand in which DNA and RNA are hybridized, and cleaves the RNA to produce single-stranded DNA.
• RNase H is not limited to a double strand in which DNA and RNA are hybridized, but can also recognize a double strand in which at least one of the base portion, phosphodiester bond portion, and sugar portion of at least one of DNA and RNA is modified. For example, it can also recognize a double strand in which an oligodeoxyribonucleotide and an oligoribonucleotide are hybridized. Therefore, when DNA hybridizes with RNA, it can be recognized by RNase H.
• RNA hybridizes with DNA, it can be cleaved by RNase H.
• at least one of the base moiety, the phosphodiester bond moiety, and the sugar moiety is modified in at least one of the DNA and the RNA.
• RNase H used in the present invention is preferably mammalian RNase H, more preferably human RNase H, and particularly preferably human RNase H1.
• the "gap region” is a region that contains "at least four consecutive nucleotides that are recognized by RNase H” and is not particularly limited as long as it contains four or more consecutive nucleotides and is recognized by RNase H, but the consecutive nucleotides are preferably independently selected from deoxyribonucleotides and 5'-modified nucleotides.
• the "5' wing region” is a region that is linked to the 5' side of the gap region and contains "at least one nucleotide” without containing the "at least four consecutive nucleotides recognized by RNase H", in which the sugar moiety of the 3'-terminal nucleotide of the 5' wing region is different from the sugar moiety of the 5'-terminal nucleotide of the gap region.
• the difference in sugar moiety identifies the boundary between the 5' wing region and the gap region.
• the 3'-terminal nucleotide of the 5' wing region is generally a 2'-modified nucleotide or a 2'-4' bridged nucleotide.
• the 5' wing region is not particularly limited as long as it satisfies the above definition, but is preferably independently selected from deoxyribonucleotides and sugar-modified nucleotides and contains at least one 2'-modified nucleotide or 2'-4' bridged nucleotide.
• the "3' wing region” is a region that is linked to the 3' side of the gap region and contains "at least one nucleotide” without containing the "at least four consecutive nucleotides recognized by RNase H", in which the sugar moiety of the 5'-terminal nucleotide of the 3' wing region is different from the sugar moiety of the 3'-terminal nucleotide of the gap region.
• the difference in sugar moiety identifies the boundary between the 3' wing region and the gap region.
• the 5'-terminal nucleotide of the 3' wing region is generally a 2'-modified nucleotide or a 2'-4' bridged nucleotide.
• the 3' wing region is not particularly limited as long as it satisfies the above definition, but is preferably independently selected from deoxyribonucleotides and sugar-modified nucleotides and contains at least one 2'-modified nucleotide or 2'-4' bridged nucleotide.
• Antisense oligonucleotides having a gap region, a 5' wing region and a 3' wing region are called gapmer-type antisense oligonucleotides.
• the "central region” is the central region in an oligonucleotide.
• the "5'region” is a region that is linked to the 5' side of the "central region” and contains at least one nucleotide.
• the "3'region” is a region that is linked to the 3' side of the "central region” and contains at least one nucleotide.
• the sugar moiety of the 5'-end nucleotide in the 3'-side region is different from the sugar moiety of the 3'-end nucleotide in the central region.
• the difference in sugar moieties identifies the boundary between the 3'-side region and the central region.
• the sugar moiety of the 3'-end nucleotide in the 5'-side region is different from the sugar moiety of the 5'-end nucleotide in the central region.
• the difference in sugar moieties identifies the boundary between the 5'-side region and the central region.
• At least four consecutive nucleotides recognized by RNase H includes four or more consecutive nucleotides, and is not particularly limited as long as it is recognized by RNase H, but examples thereof include “at least four consecutive deoxyribonucleotides” and "at least four consecutive nucleotides independently selected from the group consisting of deoxyribonucleotides and 5'-modified nucleotides".
• the number of nucleotides is, for example, 5 to 30, preferably 5 to 15, and more preferably 8 to 12.
• Those skilled in the art can determine whether or not a sequence of at least four consecutive nucleotides is "at least four consecutive nucleotides recognized by RNase H" based on the structure of the sugar moieties of the consecutive nucleotides.
• the antisense oligonucleotide of the present invention does not need to hybridize with the entire target RNA, but it is sufficient to hybridize with at least a part of the target RNA, but usually hybridizes with at least a part of the target RNA.
• the expression of the target gene is controlled by hybridizing at least a part of the target RNA with an oligonucleotide (such as DNA, oligodeoxyribonucleotide, or oligonucleotide designed to generate an antisense effect) having an antisense sequence complementary to a partial sequence of the target RNA.
• the entire antisense oligonucleotide does not need to hybridize, and a part of it may not hybridize. The entire antisense sequence part may not hybridize, but it is preferable that it hybridizes.
• antisense sequence refers to the base sequence of nucleotides constituting an oligonucleotide that enables hybridization with a target RNA
• antisense sequence portion refers to the partial structure of the region of an oligonucleotide chain that has the antisense sequence.
• the complementarity between the antisense sequence portion of the antisense oligonucleotide and the partial sequence of the target RNA is preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more (e.g., 95%, 96%, 97%, 98%, 99% or more).
• the number of nucleotides contained in the central region is 5 to 30, preferably 5 to 15, more preferably 8 to 12, and particularly preferably 9 or 10.
• the number of nucleotides contained in the central region is usually selected depending on other factors such as the strength of the antisense effect on the target RNA, low toxicity, cost, and synthesis yield.
• the central region may contain both deoxyribonucleotides and 5'-modified nucleotides.
• the number (total number) of 5'-modified nucleotides contained in the central region is 1 to 30, preferably 1 to 5, more preferably 1 to 3, even more preferably 1 to 2, and particularly preferably 1.
• the number of 5'-modified nucleotides contained in the central region is usually selected depending on other factors such as the strength of the antisense effect on the target RNA, low toxicity, cost, and synthesis yield.
• the 5'-modified nucleotide in the central region may be present at any position in the central region, but is preferably present between the third nucleotide counting from the 5' end and the 5' end of the central region.
• the position in which the 5'-modified nucleotide is present is usually selected depending on other factors such as the strength of the antisense effect on the target RNA and low toxicity.
• the nucleotides contained in the central region may all be deoxyribonucleotides.
• the 5' region is composed of at least two nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides, and includes at least one 2'-4' bridged nucleotide, and its 3' end is a 2'-4' bridged nucleotide or a 2'-modified nucleotide, wherein the 3' end sugar-modified nucleotide is linked to the central region and does not include an oligonucleotide chain composed of at least four consecutive nucleotides independently selected from the group consisting of at least four consecutive deoxyribonucleotides and 5'-modified nucleotides.
• the 3' region is composed of at least two nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides, and includes at least one 2'-4' bridged nucleotide, and its 5' end is a 2'-4' bridged nucleotide or a 2'-modified nucleotide, wherein the 5' end sugar-modified nucleotide is linked to the central region and does not include an oligonucleotide chain composed of at least four consecutive nucleotides independently selected from the group consisting of at least four consecutive deoxyribonucleotides and 5'-modified nucleotides.
• At least one 2'-O-R-ECE nucleotide is included in at least one of the central region, 5' region, and 3' region.
• at least one 2'-O-R-ECE nucleotide is included in at least one of the 5' region and 3' region. More preferably, at least one 2'-O-R-ECE nucleotide is included in the 5' region.
• the sugar-modified nucleotide contained in the 5'-region is preferably a nucleotide having increased affinity for a partial sequence of a target RNA or increased resistance to a nuclease due to substitution, etc. More preferably, it is independently selected from a 2'-modified nucleotide and a 2',4'-BNA.
• the 2'-modified nucleotide is preferably independently selected from the group consisting of 2'-O-methylated nucleotides, 2'-O-methoxyethyl (MOE)-modified nucleotides, 2'-O-aminopropyl (AP)-modified nucleotides, 2'-fluorinated nucleotides, 2'-O-(N-methylacetamide) (NMA)-modified nucleotides, 2'-O-methylcarbamoylethyl (MCE)-modified nucleotides, and 2'-O-R-ECE nucleotides, more preferably independently selected from 2'-O-methoxyethyl (MOE)-modified nucleotides, 2'-O-methylcarbamoylethyl (MCE)-modified nucleotides, and 2'-O-R-ECE nucleotides, even more preferably 2'-O-R-ECE
• the type, number and position of sugar-modified nucleotides, deoxyribonucleotides and ribonucleotides in the 5'-side region may affect the antisense effect of the antisense oligonucleotide disclosed herein.
• the type, number and position of the sugar-modified nucleotides vary depending on the sequence of the target RNA, and therefore cannot be generalized. However, a person skilled in the art can determine a preferred embodiment by taking into consideration the description in the literature on the antisense method.
• the antisense effect of the oligonucleotide after modification of the base portion, sugar portion or phosphodiester bond portion is measured, and if the measured value is not significantly lower than that of the oligonucleotide before modification (for example, if the measured value of the oligonucleotide after modification is 30% or more of the measured value of the oligonucleotide before modification), the modification can be evaluated as a preferred embodiment.
• the antisense effect can be measured, for example, by introducing a test oligonucleotide into cells, etc., as shown in the examples described below, and measuring the expression level of the target RNA, the expression level of cDNA related to the target RNA, the amount of protein related to the target RNA, etc., controlled by the antisense effect of the test oligonucleotide, by appropriately using known techniques such as Northern blotting, quantitative PCR, and Western blotting.
• the 3' region is the same as the 5' region.
• the 5'-side region preferably consists of 2 to 5 nucleotides independently selected from the group consisting of 2'-modified nucleotides, 2',4'-BNA, and deoxyribonucleotides, and includes at least one 2'-O-R-ECE nucleotide and at least one 2',4'-BNA. More preferably, it consists of 2 to 5 nucleotides independently selected from the group consisting of 2'-O-R-ECE nucleotides and 2',4'-BNA, and even more preferably, it consists of 3 to 5 nucleotides independently selected from the group consisting of 2'-O-R-ECE nucleotides and 2',4'-BNA.
• it consists of 4 to 5 nucleotides independently selected from LNA and 2'-O-R-ECE nucleotides. Even more preferably, it consists of 1 to 3 LNA and 1 to 3 nucleotides independently selected from 2'-DMAECE nucleotides, 2'-MorECE nucleotides, 2'-PyECE nucleotides, and 2'-BimECE nucleotides.
• the 3' region is similar to the 5' region.
• the 3' region preferably consists of 2 to 5 2',4'-BNAs, and particularly preferably consists of 3 LNAs.
• nucleotides contained in the 5' region it is preferred that at least one nucleotide is phosphorothioated, more preferably that 50% of the nucleotides are phosphorothioated, even more preferably that 80% of the nucleotides are phosphorothioated, and particularly preferably that all are phosphorothioated. In another embodiment, it is preferred that all of the nucleotides contained in the 5' region are linked by phosphodiester bonds.
• the 3' region is the same as the 5' region.
• the 3' end of the 5' side region and the 5' end of the central region are linked by forming a phosphodiester bond or a modified phosphodiester bond, and the 5' end of the 3' side region and the 3' end of the central region are linked by forming a phosphodiester bond or a modified phosphodiester bond.
• the 3' end of the 5' side region and the 5' end of the central region are linked by forming a modified phosphodiester bond, and the 5' end of the 3' side region and the 3' end of the central region are linked by forming a modified phosphodiester bond.
• the 3' end of the 5' side region and the 5' end of the central region are linked by a phosphorothioate bond, and the 5' end of the 3' side region and the 3' end of the central region are linked by a phosphorothioate bond.
• the 3'-terminal nucleotide of the 5'-region is preferably an LNA or 2'-O-R-ECE nucleotide, more preferably a 2'-O-R-ECE nucleotide.
• the 5'-terminal nucleotide of the 3'-region is preferably an LNA or 2'-O-R-ECE nucleotide, more preferably an LNA.
• the antisense oligonucleotide of the present invention may be bound directly or indirectly to a functional molecule.
• the binding between the functional molecule and the antisense oligonucleotide may be direct or indirect via another substance, but it is preferable that the oligonucleotide and the functional molecule are bound by a covalent bond, an ionic bond, or a hydrogen bond. From the viewpoint of high stability of the bond, it is more preferable that they are bound directly by a covalent bond, or bound covalently via a linker (linking group).
• the functional molecule When the functional molecule is covalently linked to the antisense oligonucleotide, the functional molecule is preferably linked directly or indirectly to the 3'-end or 5'-end of the antisense oligonucleotide molecule.
• the bond between the linker or functional molecule and the terminal nucleotide of the antisense oligonucleotide molecule is selected depending on the functional molecule.
• the linker or functional molecule and the terminal nucleotide of the antisense oligonucleotide molecule are preferably linked via a phosphodiester bond or a modified phosphodiester bond, more preferably via a phosphodiester bond.
• the linker or functional molecule may be directly linked to the 3' oxygen atom of the 3' terminal nucleotide or the 5' oxygen atom of the 5' terminal nucleotide of the antisense oligonucleotide molecule.
• the structure of the "functional molecule” is not particularly limited, and the binding of the functional molecule confers a desired function to the antisense oligonucleotide. Desired functions include labeling, purification, and delivery to a target site. Examples of molecules that confer a labeling function include compounds such as fluorescent proteins and luciferase. Examples of molecules that confer a purification function include compounds such as biotin, avidin, His tag peptide, GST tag peptide, and FLAG tag peptide.
• a molecule having a function of delivering the antisense oligonucleotide to the target site is bound as a functional molecule.
• a functional molecule see, for example, European Journal of Pharmaceutics and Biopharmaceutics, 2016, 107, pp. 321-340; Advanced Drug Delivery Reviews, 2016, 104, pp. 78-92; Expert Opinion on Drug Delivery, 2014, 11, pp. 791-822, etc.
• lipids and sugars can be mentioned from the viewpoint of being able to deliver antisense oligonucleotides with high specificity and efficiency to the liver, etc., for example.
• lipids include cholesterol; fatty acids; fat-soluble vitamins such as vitamin E (tocopherols, tocotrienols), vitamin A, vitamin D, and vitamin K; intermediate metabolites such as acylcarnitine and acyl CoA; glycolipids; glycerides; and derivatives thereof.
• vitamin E tocopherols, tocotrienols
• sugars sugar derivatives that interact with the asialoglycoprotein receptor can be mentioned.
• the "asialoglycoprotein receptor" is present on the surface of liver cells, and recognizes the galactose residue of asialoglycoprotein, and takes up the molecule into the cell and degrades it.
• the "sugar derivative that interacts with the asialoglycoprotein receptor” is preferably a compound that has a structure similar to that of the galactose residue and is taken up into the cell by interacting with the asialoglycoprotein receptor, and examples of such a compound include GalNAc (N-acetylgalactosamine) derivatives, galactose derivatives, and lactose derivatives.
• examples of the "functional molecule” include sugars (e.g., glucose, sucrose, etc.).
• examples of the "functional molecule” include receptor ligands, antibodies, and peptides or proteins of their fragments.
• the linker that mediates the bond between the functional molecule and the antisense oligonucleotide is not particularly limited as long as it can exert the function of the functional molecule as an antisense oligonucleotide molecule, and can stably bond the functional molecule and the oligonucleotide.
• Examples of the linker include a group derived from an oligonucleotide having 1 to 20 nucleotides, a group derived from a polypeptide having 2 to 20 amino acids, an alkylene having 2 to 20 carbon atoms, and an alkenylene having 2 to 20 carbon atoms.
• the group derived from an oligonucleotide having 2 to 20 nucleotides is a group obtained by removing a hydroxyl, a hydrogen atom, or the like from an oligonucleotide having 2 to 20 nucleotides.
• For the group derived from an oligonucleotide having 1 to 20 nucleotides see, for example, International Publication No. WO 2017/053995.
• International Publication No. WO 2017/053995 describes, for example, a 3-base linker having a TCA motif and a 1-5-base linker not having a TCA motif.
• the group derived from a polypeptide having 2 to 20 amino acids is a group obtained by removing a hydroxyl, hydrogen atom, amino, or the like from a polypeptide having 2 to 20 amino acids.
• the linker is preferably a C2-20 alkylene or a C2-20 alkenylene (methylene contained in the alkylene and alkenylene is each independently unsubstituted or substituted with 1 or 2 substituents selected from the group consisting of a halogen atom, hydroxy, protected hydroxy, oxo and thioxo.
• the left side [C2-6 alkylene] binds to the functional molecule
• the linker is more preferably a C2-20 alkylene (wherein each methylene of the alkylene is independently unsubstituted or replaced by -O-. Each unsubstituted methylene is independently unsubstituted or substituted by hydroxy or protected hydroxy), and even more preferably a C8-12 alkylene (wherein each methylene of the alkylene is independently unsubstituted or replaced by -O-. Each unsubstituted methylene is independently unsubstituted or substituted by hydroxy).
• the linker is more preferably a C2-20 alkylene (wherein the methylenes of the alkylene are each independently unsubstituted or replaced by -O- or -NR B - (wherein R B is a hydrogen atom or a C1-6 alkyl).
• the unsubstituted methylenes are each independently unsubstituted or substituted by oxo), and even more preferably a C8-12 alkylene (wherein the methylenes of the alkylene are each independently unsubstituted or replaced by -O- or -NR B - (wherein R B is a hydrogen atom or a C1-6 alkyl).
• the unsubstituted methylenes are each independently unsubstituted or substituted by oxo).
• One to five (preferably one to three, particularly preferably three) 3-11-membered nitrogen-containing non-aromatic heterocycle-diyldimethanol structures may be linked to the 5' or 3' end of the antisense oligonucleotide via phosphodiester or phosphorothioate bonds, and the linker may be linked to the 3-11-membered nitrogen-containing non-aromatic heterocycle.
• the number of functional molecules linked via the linker is also preferably one to five, more preferably one to three, particularly preferably three.
• the 3-11-membered nitrogen-containing non-aromatic heterocycle is preferably pyrrolidine or piperidine.
• the 3-11-membered nitrogen-containing non-aromatic heterocycle-diyldimethanol is particularly preferably piperidine-4,4-diylmethanol.
• the protecting group of the "protected hydroxyl” is not particularly limited as long as it is stable when the functional molecule and the oligonucleotide are bonded.
• the linker is not particularly limited, and may be any protecting group described in, for example, Protective Groups in Organic Synthesis, 4th Edition, T. W. Greene, P. G. M. Wuts, John Wiley & Sons Inc. (2006).
• the present invention also includes prodrugs of the antisense oligonucleotides.
• a prodrug is a derivative of a pharmaceutical compound having a group that can be decomposed chemically or metabolically, and is a compound that is derived into a pharmacologically active pharmaceutical compound by solvolysis or decomposition in vivo under physiological conditions. Methods for selecting and preparing suitable prodrug derivatives are described, for example, in Design of Prodrugs (Elsevier, Amsterdam, 1985).
• examples of prodrugs include acyloxy derivatives prepared by reacting the compound with a suitable acyl halide, a suitable acid anhydride, or a suitable alkyloxycarbonyl halide compound.
• examples of the prodrug include those produced by reacting a compound having an amino group with a suitable acid halide, a suitable mixed acid anhydride or a suitable halogenated alkyloxycarbonyl compound.
• oligonucleotides include double-stranded oligonucleotides (e.g., as described in WO 2013/089283, WO 2017/068791, WO 2017/068790, or WO 2018/003739) that are complementary to the antisense oligonucleotide and contain ribonucleotides (e.g., oligoribonucleotides, RNA), oligonucleotides that contain peptide nucleic acids (PNA), or oligonucleotides that contain deoxyribonucleotides (e.g., oligodeoxyribonucleotides, DNA), and single-stranded oligonucleotides in which an RNA oligonucleotide complementary to the antisense oligonucleotide is linked by a linker (e.g., as described in WO 2017/131124).
• ribonucleotides e.g., oligori
• the linker is not limited to those described in WO 2017/131124, and may, for example, contain a non-nucleotide structure. Also included are single-stranded oligonucleotides in which an RNA oligonucleotide complementary to the antisense oligonucleotide is directly linked.
• An oligonucleotide complex comprising: (i) the antisense oligonucleotide; and (ii) an oligonucleotide comprising at least one ribonucleotide and comprising a region that hybridizes to the (i) antisense oligonucleotide.
• An oligonucleotide comprising (i) a group derived from the antisense oligonucleotide, and (ii) a group derived from an oligonucleotide containing at least one ribonucleotide and containing a region that hybridizes to the antisense oligonucleotide (i), wherein the group derived from the antisense oligonucleotide (i) and the group derived from the oligonucleotide (ii) are linked together.
• the group derived from the antisense oligonucleotide and (ii) the group derived from the oligonucleotide may be linked by a group derived from an oligonucleotide that is degraded under physiological conditions, may be linked by a linking group containing a non-nucleotide structure, or may be linked directly.
• the group derived from an oligonucleotide and (iv) the group derived from an oligonucleotide may be linked via a group derived from an oligonucleotide that is degraded under physiological conditions, may be linked via a linking group containing a non-nucleotide structure, or may be linked directly.
• the group derived from the antisense oligonucleotide and the oligonucleotide chain containing at least one ribonucleotide may be linked by a group derived from an oligonucleotide that is degraded under physiological conditions, may be linked by a linking group containing a non-nucleotide structure, or may be linked directly.
• oligonucleotide degraded under physiological conditions may be an oligonucleotide that is degraded by various enzymes such as DNase (deoxyribonuclease) and RNase (ribonuclease) under physiological conditions, and the nucleotides constituting the oligonucleotide may or may not be chemically modified in the base, sugar, or phosphate bond in part or all of them.
• DNase deoxyribonuclease
• RNase ribonuclease
• oligonucleotide degraded under physiological conditions is, for example, an oligonucleotide that contains at least one phosphodiester bond, preferably linked by a phosphodiester bond, more preferably an oligodeoxyribonucleotide or oligoribonucleotide, even more preferably DNA or RNA, and even more preferably RNA.
• the oligonucleotide degraded under physiological conditions may or may not contain a partially complementary sequence within the oligonucleotide degraded under physiological conditions, but preferably does not contain a partially complementary sequence.
• oligonucleotide is (N) k' (wherein N is independently adenosine, uridine, cytidine, guanosine, 2'-deoxyadenosine, thymidine, 2'-deoxycytidine, or 2'-deoxyguanosine, and k is an integer (repetition number) of 1 to 40) linked by a phosphodiester bond.
• k' is preferably 3 to 20, more preferably 4 to 10, even more preferably 4 to 7, even more preferably 4 or 5, and particularly preferably 4.
• the type, number and position of sugar-modified nucleotides, deoxyribonucleotides and ribonucleotides may affect the antisense effect of the prodrug of the antisense oligonucleotide disclosed herein.
• the type, number and position of the sugar-modified nucleotides may vary depending on the sequence of the target RNA, and therefore cannot be generally stated, but a person skilled in the art can determine a preferred embodiment while taking into consideration the description in the literature on the antisense method.
• the antisense effect of the prodrug of the antisense oligonucleotide after modification of the base portion, sugar portion or phosphodiester bond portion is measured, and if the measured value is not significantly lower than that of the prodrug of the antisense oligonucleotide before modification (for example, if the measured value of the prodrug of the antisense oligonucleotide after modification is 30% or more of the measured value of the prodrug before modification), the modification can be evaluated as a preferred embodiment.
• the antisense effect can be measured, for example, as shown in the Examples described below, by introducing a test oligonucleotide into a cell or the like, and measuring the expression level of the target RNA controlled by the antisense effect of the test oligonucleotide, the expression level of cDNA associated with the target RNA, the amount of protein associated with the target RNA, etc., by appropriately using known techniques such as Northern blotting, quantitative PCR, Western blotting, etc.
• the oligonucleotide (ii) in the oligonucleotide complex shown in (A) or the group derived from the oligonucleotide (ii) in the oligonucleotide shown in (B) is independently selected from ribonucleotides, deoxyribonucleotides, and sugar-modified nucleotides, and is preferably selected from ribonucleotides.
• the ribonucleotides are preferably linked to each other via phosphodiester bonds.
• the oligonucleotide or group derived from the oligonucleotide (ii) is selected from ribonucleotides and sugar-modified nucleotides, and the sugar-modified nucleotide is selected from sugar-modified nucleotides excluding 5'-modified nucleotides.
• the terminal of the oligonucleotide is at least one sugar-modified nucleotide.
• This sugar-modified nucleotide is preferably a 2'-O-methylated nucleotide, and is preferably bonded to an adjacent nucleotide via a phosphorothioate bond.
• the oligonucleotide (iii) contains at least one ribonucleotide (the same applies to the portion that hybridizes to an oligonucleotide chain containing at least four consecutive nucleotides recognized by RNase H).
• the 3'- and 5'-terminal nucleotides of the oligonucleotide (ii) are preferably sugar-modified nucleotides.
• the terminal nucleotides of the 3'- and 5'-terminals of the group derived from the oligonucleotide (ii) that are not bonded to the group derived from the antisense oligonucleotide (i) are preferably sugar-modified nucleotides.
• the terminal nucleotides of the 3'- and 5'-terminals of the group derived from the oligonucleotide (iii) that are not bonded to the group derived from the antisense oligonucleotide are preferably sugar-modified nucleotides.
• the number of bases in the oligonucleotide (ii) or the group derived from the oligonucleotide is not particularly limited and may be the same as or different from the number of bases in the antisense oligonucleotide (i) (or the group derived from it).
• the number of bases in the oligonucleotides (i) and (ii) is preferably the same, and it is preferable that the entire oligonucleotides (i) and (ii) hybridize.
• the oligonucleotide (iv) in the oligonucleotide conjugate shown in (C) and the group derived from the oligonucleotide (iv) in the oligonucleotide in (D) are independently selected from ribonucleotides, deoxyribonucleotides, and sugar-modified nucleotides, and are preferably selected from deoxyribonucleotides and sugar-modified nucleotides.
• the sugar-modified nucleotide contained in the oligonucleotide (iv) or group derived from the oligonucleotide is preferably selected from sugar-modified nucleotides excluding 5'-modified nucleotides.
• the 5'-terminus and 3'-terminus of the oligonucleotide or group derived from the oligonucleotide are preferably at least one sugar-modified nucleotide.
• the at least one sugar-modified nucleotide is preferably at least one selected from 2'-modified nucleotides and 2',4'-BNA, and more preferably at least one selected from the group consisting of 2'-O-methylated nucleotides, 2'-O-methoxyethyl (MOE)-modified nucleotides, 2'-O-aminopropyl (AP)-modified nucleotides, 2'-fluorinated nucleotides, 2'-O-(N-methylacetamide) (NMA)-modified nucleotides, 2'-O-methylcarbamoylethyl (MCE)-modified nucleotides, LNA, cEt-BNA, ENA, BNA
• the oligonucleotide chain containing at least one ribonucleotide of (iii) and the oligonucleotide chain containing at least four consecutive nucleotides recognized by RNaseH of (iv) hybridize with each other, and the oligonucleotide chain containing at least one ribonucleotide of (iii) is considered to be recognized by RNaseH, and the oligonucleotide chain containing at least one ribonucleotide of (iii) is considered to be cleaved.
• the antisense oligonucleotide of the present invention is generated in target cells, etc., and the prodrug of (C) is considered to exert a therapeutic effect, etc.
• the oligonucleotide complex shown in (A) has a functional molecule
• the oligonucleotide (ii) contains a functional molecule, and it is preferable that the functional molecule is bound to the end of the oligonucleotide (ii).
• the oligonucleotide shown in (B) has a functional molecule
• the oligonucleotide complex shown in (C) has a functional molecule
• the oligonucleotide (iv) contains a functional molecule, and it is preferable that the functional molecule is bound to the end of the oligonucleotide (iv).
• the oligonucleotide shown in (D) The preferred aspects of the functional molecule and its bond are as described above.
• the oligonucleotide (ii) or the group derived from the oligonucleotide may further have a group derived from the antisense oligonucleotide.
• the group derived from the antisense oligonucleotide (ii) may be the same as or different from the group derived from the antisense oligonucleotide (i). In addition, it may or may not be the group derived from the antisense oligonucleotide of the present invention.
• the group derived from the antisense oligonucleotide (ii) does not hybridize with the antisense oligonucleotide (i) or the group derived from the antisense oligonucleotide.
• the oligonucleotide (iv) or the group derived from the oligonucleotide may be an antisense oligonucleotide or a group derived from the antisense oligonucleotide.
• the antisense oligonucleotide (iv) or the group derived from the antisense oligonucleotide may be the same as or different from the group derived from the antisense oligonucleotide contained in the oligonucleotide (iii). In addition, it may or may not be a group derived from the antisense oligonucleotide of the present invention. It is preferable that the antisense oligonucleotide (iv) or the group derived from the antisense oligonucleotide does not hybridize with the antisense oligonucleotide (iii) or the group derived from the antisense oligonucleotide. Examples of antisense oligonucleotides that are not the antisense oligonucleotides of the present invention include the following antisense oligonucleotides:
• An antisense oligonucleotide having a central region, a 5' region, and a 3' region, - The central area is at least five nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides, said sugar-modified nucleotides being selected from sugar-modified nucleotides excluding 5'-modified nucleotides; at least one oligonucleotide chain whose 3' and 5' ends are each independently a deoxyribonucleotide or a ribonucleotide, and which is comprised of at least four consecutive nucleotides independently selected from deoxyribonucleotides and 5'-modified nucleotides;
• the 5' region is at least one nucleotide independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleot
• antisense oligonucleotide (2) An antisense oligonucleotide having a central region, a 5' region, and a 3' region, - The central area is consisting of at least five nucleotides independently selected from deoxyribonucleotides;
• the 5' region is at least one nucleotide independently selected from the group consisting of deoxyribonucleotides and sugar-modified nucleotides, the 3' terminus of which is a sugar-modified nucleotide, wherein the sugar-modified nucleotide at the 3' terminus is linked to a central region and is selected from sugar-modified nucleotides excluding 5'-modified nucleotides; and does not include an oligonucleotide chain composed of at least four consecutive nucleotides independently selected from the group consisting of deoxyribonucleotides and 5'-modified nucleotides;
• the 3' region is at
• the central region is preferably a gap region
• the 5' region is preferably a 5' wing region
• the 3' region is preferably a 3' wing region.
• preferred aspects of the 5' region and the 3' region are the same as the 5' region and the 3' region in the antisense oligonucleotide of the present invention.
• Preferred aspects of the central region are the same as the central region in the antisense oligonucleotide of the present invention.
• antisense oligonucleotides consisting of at least five nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides;
• An antisense oligonucleotide comprising an oligonucleotide strand consisting of 1 to 3 nucleotides independently selected from deoxyribonucleotides and 5'-modified nucleotides, and an oligonucleotide strand consisting of 1 to 3 nucleotides independently selected from 2'-modified nucleotides and 2',4'-BNA, alternating therewith.
• a linking group containing a non-nucleotide structure can be bonded to an oligonucleotide by the general amidite method or H-phosphonate method.
• an amidite reagent e.g., 2-cyanoethyl chloro(diisopropylamino)phosphinite, 2-cyanoethyl bis(diisopropylamino)phosphinite, etc.
• an H-phosphonate reagent e.g., diphenyl phosphite, phosphorous acid, etc.
• the protected hydroxyl group can be deprotected and the nucleotide can be further extended using a commercially available automated nucleic acid synthesizer.
• the compound having the two hydroxyl groups can be synthesized from raw materials such as amino acids, carboxylic acids, diol compounds, etc., by combining protection/deprotection reactions known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, 4th Edition), oxidation reactions, reduction reactions, condensation reactions (see, for example, Comprehensive Organic Transformations, 2nd Edition, R.C. Larock, Wiley-VCH (1999) for oxidation reactions, reduction reactions, and condensation reactions).
• the linking group containing a non-nucleotide structure has a functional group other than the two hydroxyl groups (e.g., an amino group, a hydroxyl group, or a thiol group), it can be efficiently elongated by protecting it with a protecting group known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, 4th Edition).
• a protecting group known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, 4th Edition).
• two oligonucleotides can be synthesized separately and then linked to a linking group containing a non-nucleotide structure.
• An example of a synthesis method is shown below.
• a partial structure having a functional group such as an amino group is linked to the 5' end of an oligonucleotide by a method known to those skilled in the art (for example, 6-(trifluoroacetylamino)hexyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, etc.
• a partial structure having a functional group such as an amino group is linked to the 3' end of another oligonucleotide by a method known to those skilled in the art (for example, 2-((4,4'-dimethoxytrityl)oxymethyl)-6-fluorenylmethoxycarbonylamino-hexane-1-succinoyl-long chain alkylamino-CPG (Glen RESEARCH, product number 20-2958), etc. is used).
• the two functional groups of the linking group containing a non-nucleotide structure can be converted to the desired functional groups that react with the amino group, etc., to link the two oligonucleotides.
• the two functional groups of the linking group containing a non-nucleotide structure can be linked by converting them to a carboxylic acid, ester, activated ester (e.g., N-hydroxysuccinimidation), acid chloride, activated carbonate diester (e.g., 4-nitrophenyl carbonate diester), isocyanate, or the like, and then reacting them under known N-carbonylation conditions.
• activated ester e.g., N-hydroxysuccinimidation
• acid chloride e.g., activated carbonate diester (e.g., 4-nitrophenyl carbonate diester), isocyanate, or the like
• activated carbonate diester e.g., 4-nitrophenyl carbonate diester
• isocyanate or the like
• Those skilled in the art can protect one of the two functional groups as necessary, bind one oligonucleotide to the linking group containing a non-nucleotide structure, deprotect it, and then similarly bind another oligonucleotide to the linking group containing a non-nucleotide structure.
• antisense oligonucleotides or prodrugs thereof also include those that exist as mixtures thereof or mixtures of the respective isomers.
• an asymmetric center exists or an asymmetric center is generated as a result of isomerization, it also includes those that exist as the respective optical isomers and mixtures in any ratio.
• diastereomers due to the respective optical isomers also exist.
• the present invention also includes those that include all of these forms in any ratio.
• optically active forms can be obtained by methods well known for this purpose.
• the antisense oligonucleotide or prodrug thereof of the present invention contains a modified phosphodiester bond (e.g., a phosphorothioate bond) and the phosphorus atom is an asymmetric atom
• a modified phosphodiester bond e.g., a phosphorothioate bond
• the phosphorus atom is an asymmetric atom
• both oligonucleotides in which the phosphorus atom is stereoregulated and oligonucleotides in which the phosphorus atom is not stereoregulated are included within the scope of the present invention.
• the antisense oligonucleotides, their prodrugs, or their pharma- ceutically acceptable salts of the present invention can exist in any crystalline form or any hydrate depending on the manufacturing conditions, and these crystalline forms, hydrates, and mixtures thereof are also included in the scope of the present invention. They may also exist as solvates containing organic solvents such as acetone, ethanol, 1-propanol, and 2-propanol, and all of these forms are included in the scope of the present invention.
• the antisense oligonucleotide or its prodrug of the present invention can be converted into a pharmaceutically acceptable salt or released from the resulting salt, if necessary.
• pharmaceutically acceptable salts of the antisense oligonucleotide or its prodrug include salts with alkali metals (lithium, sodium, potassium, etc.), alkaline earth metals (magnesium, calcium, etc.), ammonium, organic bases (triethylamine, trimethylamine, etc.), amino acids (glycine, lysine, glutamic acid, etc.), inorganic acids (hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, etc.) or organic acids (acetic acid, citric acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, etc.).
• may be converted to an anionic partial structure represented by -P( O)(S - )-, which similarly forms a salt with an alkali metal, an alkaline earth metal, ammonium, etc.
• the antisense oligonucleotide or its prodrug of the present invention can be prepared by a person skilled in the art by appropriately selecting a known method.
• a person skilled in the art can design the nucleotide sequence of the antisense oligonucleotide based on the information of the nucleotide sequence of the target RNA, and synthesize it using a commercially available automated nucleic acid synthesizer (Applied Biosystems, Beckman, GeneDesign, etc.). It can also be synthesized by a reaction using an enzyme. Examples of the enzyme include, but are not limited to, polymerase, ligase, and restriction enzymes.
• the method for producing the antisense oligonucleotide or its prodrug according to this embodiment can include a step of extending the nucleotide chain at the 3' end or 5' end.
• the functional molecule and the linker can be bound by a known method, and then the linker can be converted into an amidite form using an amidite reagent or into an H-phosphonate form using an H-phosphonate reagent, and then bound to the oligonucleotide.
• the resulting oligonucleotide can be purified by reverse phase column chromatography or the like to prepare an antisense oligonucleotide or its prodrug.
• the antisense oligonucleotide or prodrug thereof of the present invention can effectively control the expression of a target gene. Therefore, the present invention can provide a composition that contains the antisense oligonucleotide of the present invention as an active ingredient, for example, for controlling the expression of a target gene by the antisense effect.
• the antisense oligonucleotide or prodrug thereof of the present invention can obtain high efficacy by administration at low concentrations, and in some embodiments, a pharmaceutical composition can be provided for treating, preventing, or ameliorating diseases associated with increased expression of a target gene, such as metabolic diseases, tumors, and infectious diseases.
• composition containing the antisense oligonucleotide or its prodrug of the present invention can be formulated by known pharmaceutical methods.
• it can be used enterally (orally, etc.) or parenterally as capsules, tablets, pills, liquids, powders, granules, fine granules, film coatings, pellets, lozenges, sublinguals, chewable tablets, buccal tablets, pastes, syrups, suspensions, elixirs, emulsions, liniments, ointments, plasters, poultices, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories, etc.
• these formulations can be appropriately combined with carriers that are pharmacologically or food and beverage acceptable, specifically, sterile water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, pH adjuster, stabilizer, flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, isotonicity agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, colorant, sweetener, thickener, flavoring agent, dissolution aid, or other additives.
• carriers that are pharmacologically or food and beverage acceptable, specifically, sterile water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, pH adjuster, stabilizer, flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, isotonicity agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, colorant, sweet
• the administration form of the composition containing the antisense oligonucleotide or its prodrug of the present invention is not particularly limited, but may be enteral (oral, etc.) or parenteral administration. More preferably, it may be intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intratracheal administration, rectal administration, intramuscular administration, intrathecal administration, intraventricular administration, intranasal administration, intravitreal administration, etc., or administration by infusion.
• Diseases that can be treated, prevented, or ameliorated by the nucleic acid pharmaceutical using the antisense oligonucleotide or its prodrug of the present invention are not particularly limited, and examples include metabolic diseases, cardiovascular diseases, tumors, infectious diseases, eye diseases, inflammatory diseases, autoimmune diseases, rare genetic diseases, and other diseases caused by gene expression.
• hypercholesterolemia hypertriglyceridemia, spinal muscular atrophy, muscular dystrophy (Duchenne muscular dystrophy, myotonic dystrophy, congenital muscular dystrophy (Fukuyama congenital muscular dystrophy, Ullrich congenital muscular dystrophy, merosin-deficient congenital muscular dystrophy, integrin deficiency, Walker-Warburg syndrome, etc.), Becker muscular dystrophy, limb-girdle muscular dystrophy, Miyoshi muscular dystrophy, facioscapulohumeral muscular dystrophy, etc.), Huntington's disease, Alzheimer's disease, and other conditions.
• rheumatoid arthritis transthyretin amyloidosis, familial amyloidotic cardiomyopathy, multiple sclerosis, Crohn's disease, inflammatory bowel disease, acromegaly, type 2 diabetes, chronic nephropathy, respiratory syncytial virus infection, Ebola hemorrhagic fever, Marburg fever, HIV, influenza, hepatitis B, hepatitis C, cirrhosis, chronic heart failure, myocardial fibrosis, atrial fibrillation, prostate cancer, melanoma, breast cancer, pancreatic cancer, colon cancer, renal cell carcinoma, bile duct cancer, cervical cancer, liver cancer, lung cancer, leukemia, non-Hodgkin's lymphoma, atopic dermatitis, glaucoma, and age-related macular degeneration.
• a gene causing the disease is set as the target gene, and the expression control sequence (e.g., antisense sequence) can be set as the target gene, and
• compositions comprising the antisense oligonucleotides of the present invention or prodrugs thereof can be treated, prevented, or ameliorated with compositions comprising the antisense oligonucleotides of the present invention or prodrugs thereof.
• diseases can be treated in mammalian species including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, or other bovine, ovine, equine, canine, feline, and rodent species such as mice.
• Compositions comprising antisense oligonucleotides can also be used in other species such as birds (e.g., chickens).
• the dosage or intake is appropriately selected depending on the age, weight, symptoms, health condition, type of composition (medicine, food, beverage, etc.) of the subject, and the dosage or intake is preferably 0.0001 mg/kg/day to 100 mg/kg/day in terms of antisense oligonucleotide.
• the antisense oligonucleotide or prodrug thereof of the present invention can effectively control the expression of a target gene while being less toxic than conventional antisense oligonucleotides. Therefore, it is possible to provide a method for more safely controlling the expression of a target gene by the antisense effect by administering the antisense oligonucleotide or prodrug thereof of the present invention to animals, including humans. It is also possible to provide a method for treating, preventing, and ameliorating various diseases accompanied by increased expression of a target gene, which includes administering a composition containing the antisense oligonucleotide or prodrug thereof of the present invention to animals, including humans.
• Preferred methods of using the antisense oligonucleotides of the present invention include the following.
• a method for regulating the function of a target RNA comprising the step of contacting a cell with the antisense oligonucleotide of the present invention or a prodrug thereof.
• a method for regulating the function of a target RNA in a mammal comprising the step of administering to the mammal a pharmaceutical composition containing the antisense oligonucleotide of the present invention or a prodrug thereof.
• a method for regulating expression of a target gene comprising the step of contacting a cell with the antisense oligonucleotide of the present invention or a prodrug thereof.
• a method for regulating expression of a target gene in a mammal comprising the step of administering to the mammal a pharmaceutical composition containing the antisense oligonucleotide of the present invention or a prodrug thereof.
• Use of the antisense oligonucleotide of the present invention or a prodrug thereof to control the expression of a target gene in a mammal Use of the antisense oligonucleotide of the present invention or a prodrug thereof for the manufacture of a drug for controlling the expression of a target gene in a mammal.
• control of the function of the target RNA means that the antisense sequence portion covers a part of the target RNA through hybridization, thereby regulating or converting the splicing function, such as translation inhibition or exon skipping, or that the function of the target RNA is suppressed through degradation of the target RNA, which can occur through recognition of the hybridized part of the antisense sequence portion and a part of the target RNA.
• the mammal is preferably a human.
• the route of administration is preferably enteral. In other embodiments, the route of administration is parenteral.
• the 2'-O-R-ECE nucleotide according to the embodiment of the present invention can be produced by referring to WO2017/142054, etc.
• NMR nuclear magnetic resonance
• (v/v) means (volume/volume).
• ⁇ unit: ppm
• MS was measured using the ESI (electrospray ionization) method under any one of the following analytical conditions 1 to 5.
• ESI + means ESI positive ion mode
• ESI - means ESI negative ion mode.
• Purification by silica gel column chromatography was performed using Purif-Pack (registered trademark)-EX (SI-50 ⁇ m) manufactured by SHOKO SCIENCE or Presep (registered trademark) (Luer Lock) NH 2 (HC) manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd., unless otherwise specified.
• Nucleoside analogue Synthesis of diisopropyl phosphoramidite (2-cyanoethyl) (2R,3R,4R,5R)-5-(6-acetamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(3-((2-morpholinoethyl)amino)-3-oxopropoxy)tetrahydrofuran-3-yl (compound 6)
• the resulting crude product (2.0 g) was dissolved in pyridine (20 mL). To the solution, 4,4'-dimethoxytrityl chloride (2.44 g, 7.2 mmol) was added, and then 4,4'-dimethoxytrityl chloride (0.40 g, 1.2 mmol) was further added, and the mixture was stirred at room temperature for 45 minutes. After the reaction, methanol and a saturated aqueous sodium bicarbonate solution were added, and the mixture was extracted with ethyl acetate. The organic layer was collected, and the solvent was distilled off under reduced pressure.
• the obtained crude product (3.0 g) was azeotroped with toluene, and pyridine (30 mL) was added. 4,4'-dimethoxytrityl chloride (3.5 g, 10.6 mmol) was added to the solution, and the mixture was stirred overnight. After the reaction, methanol and a saturated aqueous solution of sodium bicarbonate were added, and the mixture was extracted with ethyl acetate. The organic layer was collected, and the solvent was distilled off under reduced pressure.
• Nucleoside analogue Synthesis of diisopropyl phosphoramidite (2-cyanoethyl) (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-(((E)-(dimethylamino)methylene)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-(3-((2-morpholinoethyl)amino)-3-oxopropoxy)tetrahydrofuran-3-yl (compound 20)
• N,N-diisopropylethylamine (6.2 mL, 35.6 mmol)
• 4-(2-aminoethyl)morpholine 4 mL, 35.6 mmol
• 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (6.7 g, 17.8 mmol) were added and stirred at room temperature for 1 hour.
• a saturated aqueous solution of sodium bicarbonate was added and extracted with ethyl acetate. The organic layer was collected and the solvent was distilled off under reduced pressure.
• Nucleoside analogue Synthesis of diisopropyl phosphoramidite (2-cyanoethyl) (2R,3R,4R,5R)-5-(6-acetamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-4-(3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propoxy)tetrahydrofuran-3-yl (compound 27)
• the obtained crude product (4.5 g) containing compound 24 was dissolved in tetrahydrofuran (45 mL). Triethylamine (4.1 mL, 29.4 mmol) and triethylamine trihydrofluoride (3.0 mL, 18.5 mmol) were added to the solution, and the mixture was stirred at room temperature for 1 hour.
• Nucleoside analogue Synthesis of diisopropyl phosphoramidite (2-cyanoethyl) (2R,3R,4R,5R)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-(((E)-(dimethylamino)methylene)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)-4-(3-oxo-3-((2-(pyridin-2-yl)ethyl)amino)propoxy)tetrahydrofuran-3-yl (compound 32)
• Nucleoside analogue Synthesis of diisopropyl phosphoramidite (2-cyanoethyl) (2R,3R,4R,5R)-4-(3-((2-(1H-benzo(d)imidazol-1-yl)ethyl)amino)-3-oxopropoxy)-5-(6-benzamido-9H-purin-9-yl)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3-yl (compound 38)
• the obtained crude product (960 mg) containing compound 35 was dissolved in pyridine (34 mL). The solution was cooled to 0° C., trimethylsilyl chloride (1.26 mL, 9.9 mmol) was added, and the mixture was stirred at 0° C. for 30 minutes. Benzoyl chloride (1.1 mL, 9.9 mmol) was added to the obtained reaction solution, and the mixture was stirred at room temperature for 2 hours. After the reaction, the reaction solution was cooled to 0° C., and an aqueous ammonia solution (13.4 mL, 14%, w/w) was added, and the mixture was stirred at room temperature for 30 minutes.
• the solvent was distilled off under reduced pressure, and the obtained residue was purified by silica gel column chromatography (ethyl acetate/chloroform/methanol) to obtain a crude product (1.1 g) containing compound 36.
• the obtained crude product (1.1 g) containing compound 36 was dissolved in pyridine (11 mL). 4,4'-dimethoxytrityl chloride (1.1 g, 3.4 mmol) was added to the solution, and the mixture was stirred at room temperature for 45 minutes. Then, 4,4'-dimethoxytrityl chloride (1.1 g, 3.4 mmol) was added and the mixture was stirred at room temperature for 30 minutes.
• Nucleoside analogue Synthesis of diisopropyl phosphoramidite (2-cyanoethyl) (2R,3R,4R,5R)-4-(3-((2-(1H-benzo(d)imidazol-1-yl)ethyl)amino)-3-oxopropoxy)-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-5-(2-(((E)-(dimethylamino)methylene)amino)-6-oxo-1,6-dihydro-9H-purin-9-yl)tetrahydrofuran-3-yl (compound 43)
• N,N-diisopropylethylamine (380 ⁇ L, 2.2 mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (100 ⁇ L, 0.4 mmol) were added to the reaction solution, and the mixture was stirred for 2 hours.
• Table 1 The columns in Table 1 are denoted as follows: "Compd. No.”: represents the compound number. "Chemical Structure”: represents a chemical structure. "Expected MW”: represents the theoretical molecular weight (Da) of each compound. “Observed MW”: represents the observed molecular weight (Da) of each compound. “Target RNA”: The name of the mouse gene (transcript) targeted by each compound.
• the antisense oligonucleotides shown in Table 1 were synthesized using an automatic nucleic acid synthesizer nS-8II (Gene Design) or an automatic nucleic acid synthesizer M-8-SE (Nihon Techno Service).
• the synthesized antisense oligonucleotides were subjected to mass spectrometry using ESI-MS under the analytical condition 6 shown below, and each was identified as the target compound (the theoretical molecular weight and detected molecular weight of each compound are shown in Table 1).
• RNA was prepared using CellAmp (registered trademark) Direct RNA Prep Kit for RT-PCR (Real Time) (Takara Bio), and then the expression level of mouse Cxcl12 gene was measured by quantitative real-time PCR using One Step PrimeScript (registered trademark) III RT-qPCR Mix (Takara Bio) and TaqMan (registered trademark) Gene Expression Assays (Thermo Fisher Scientific).
• real-time PCR the amount of mRNA of the housekeeping gene Gapdh was also quantified at the same time, and the amount of Cxcl12 mRNA relative to the amount of Gapdh mRNA was evaluated as the expression level of Cxcl12.
• Table 2 The results are presented in Table 2 as percentage inhibition at 10 nM relative to untreated control cells and 50% inhibitory concentration (IC 50 ) calculated from the percentage expression of Cxcl12 using GraphPad PRISM 9.0 (Dotmatics).
• RNA was prepared using CellAmp (registered trademark) Direct RNA Prep Kit for RT-PCR (Real Time) (Takara Bio), and then the expression level of mouse F11 gene was measured by quantitative real-time PCR using One Step PrimeScript (registered trademark) III RT-qPCR Mix (Takara Bio) and TaqMan (registered trademark) Gene Expression Assays (Thermo Fisher Scientific).
• real-time PCR the amount of mRNA of the housekeeping gene Gapdh was also quantified at the same time, and the amount of F11 mRNA relative to the amount of Gapdh mRNA was evaluated as the expression level of F11.
• the results are presented in Table 3 as percentage inhibition at 10 nM relative to untreated control cells and 50% inhibitory concentration (IC 50 ) calculated from the percentage expression of F11 using GraphPad PRISM 9.0 (Dotmatics).
• oligonucleotides of the present invention are believed to be able to suppress off-target effects and therefore reduce toxicity, and are useful as pharmaceutical compositions for treating and preventing diseases associated with increased target RNA function and/or target gene expression, such as metabolic diseases, tumors, and infectious diseases.

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