WO2022181807A1 - ＲＥＳＴ ｍＲＮＡ前駆体のプロセシングにおけるＮエキソンのスキッピングを誘導するオリゴヌクレオチド - Google Patents

ＲＥＳＴ ｍＲＮＡ前駆体のプロセシングにおけるＮエキソンのスキッピングを誘導するオリゴヌクレオチド Download PDF

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WO2022181807A1
WO2022181807A1 PCT/JP2022/008079 JP2022008079W WO2022181807A1 WO 2022181807 A1 WO2022181807 A1 WO 2022181807A1 JP 2022008079 W JP2022008079 W JP 2022008079W WO 2022181807 A1 WO2022181807 A1 WO 2022181807A1
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group
carbon atoms
oligonucleotide
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正仁 下條
聡 小比賀
啓士朗 三島
美紗 吉田
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University of Osaka NUC
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Osaka University NUC
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Priority to JP2023502564A priority patent/JPWO2022181807A1/ja
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C12N2310/3341—5-Methylcytosine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • the present invention provides an oligonucleotide that skips the N exon in pre-mRNA processing of the transcription repressor REST (RE1-silencing transcription factor), or a pharmacologically acceptable salt thereof, and a pharmaceutical comprising the same. Regarding the composition.
  • SCLC small cell lung cancer
  • ASO antisense nucleic acid medicine
  • SRRM4 Serine/Arginine Repetitive Matrix 4
  • the transcriptional repressor REST In the pathology of SCLC, the transcriptional repressor REST, a tumor suppressor, is decreased and the REST isoform (sREST) is abnormally expressed. Splicing of the transcriptional repressor REST is activated by SRRM4. It has been reported that the transcriptional repressor REST is a master molecule of nervous system genes and a tumor suppressor.
  • SRRM4 antisense oligonucleotide suppresses SRRM4 and exhibits anti-tumor effects, while promoting alterations in the splicing of the tumor suppressor REST, and the resulting increase in REST expression is associated with cancer. thought to induce cell death. Furthermore, regarding sREST that is abnormally expressed in SCLC, it has been revealed that SCLC cell death can be induced by directly promoting a splicing change that causes REST to be expressed from sREST (non-patent document 1).
  • the present invention is intended to solve the above problems, and its object is to provide oligonucleotides or pharmacologically acceptable salts thereof capable of appropriately inducing skipping of the N exon of REST, and pharmaceuticals containing the same.
  • the object is to provide a composition.
  • the present invention provides an oligo containing a nucleotide sequence complementary to a continuous sequence of at least 12 bases in the target region consisting of the nucleotide sequence of SEQ ID NO: 1, and inducing N exon skipping in processing of human REST pre-mRNA.
  • a nucleotide or a pharmacologically acceptable salt thereof is provided.
  • the length of the oligonucleotide is 12-35 bases.
  • the oligonucleotide or a pharmacologically acceptable salt thereof has a nucleoside structure represented by the following formula (I):
  • Base is a purin-9-yl group optionally having one or more arbitrary substituents selected from the ⁇ group, or optionally having one or more arbitrary substituents selected from the ⁇ group 2 -oxo-1,2-dihydropyrimidin-1-yl group, wherein the ⁇ group is a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, a linear alkyl group having 1 to 6 carbon atoms, a carbon linear alkoxy group of number 1 to 6, mercapto group, mercapto group protected by protecting group for nucleic acid synthesis, linear alkylthio group of 1 to 6 carbon atoms, amino group, linear alkylamino group of 1 to 6 carbon atoms , an amino group protected by a protective group for nucleic acid synthesis, and a halogen atom, A is:
  • R 1 is selected from a hydrogen atom, an optionally branched or ring-forming alkyl group having 1 to 7 carbon atoms, an optionally branched or ring-forming alkenyl group having 2 to 7 carbon atoms, and the ⁇ group an aryl group having 3 to 12 carbon atoms which may have one or more optional substituents and may contain a heteroatom, and one or more optional substituents selected from the ⁇ group represents an aralkyl group having an aryl moiety of 3 to 12 carbon atoms which may optionally contain a heteroatom, or an amino group-protecting group for nucleic acid synthesis;
  • R 2 and R 3 are each independently a hydrogen atom; optionally substituted with an aryl group having 3 to 12 carbon atoms which may contain a heteroatom, and optionally branched or forming a ring; an alkyl group having 1 to 7 carbon atoms; or an aralkyl group having an aryl moiety having 3 to 12 carbon
  • R 17 , R 18 and R 19 each independently form a hydrogen atom, a branch or a ring an alkyl group having 1 to 7 carbon atoms, an amino group-protecting group, or
  • R 13 and R 14 each independently represents a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms which may be branched or forming a ring; A group selected from the group consisting of an alkoxy group of 7; an amino group; and an amino group protected by a protecting group for nucleic acid synthesis; m is an integer from 0 to 2; n is an integer from 0 to 1; R 10 is a hydrogen atom, an optionally branched or ring-forming alkyl group having 1 to 7 carbon atoms, an amino-protecting group, or
  • the linkages between nucleotides that make up the oligonucleotide contain phosphorothioate.
  • the base sequence of the above oligonucleotide comprises any of the base sequences of SEQ ID NOs: 3-11 and SEQ ID NOs: 40-49.
  • nucleoside structure represented by formula (I) above is
  • the present invention is further a pharmaceutical composition comprising the oligonucleotide or a pharmacologically acceptable salt thereof.
  • the pharmaceutical composition is a cancer therapeutic.
  • the cancer therapeutic agent is used to treat at least one cancer selected from the group consisting of small cell lung cancer, prostate cancer and breast cancer.
  • the pharmaceutical composition is a therapeutic agent for heart failure.
  • the present invention further provides a method for treating cancer or heart failure in a mammal, which comprises administering to the mammal an effective amount of the oligonucleotide or a pharmacologically acceptable salt thereof.
  • the present invention is further the above oligonucleotide or a pharmacologically acceptable salt thereof for use in treating cancer or heart failure.
  • the present invention is further the oligonucleotide or a pharmacologically acceptable salt thereof for the production of a therapeutic drug for cancer or heart failure.
  • N exon skipping can be induced in the processing of REST pre-mRNA.
  • it can exhibit anti-tumor effects in vivo. This will be useful for the development of pharmaceutical compositions for the treatment or prevention of diseases involving REST processing (splicing) (for example, cancer such as small cell lung cancer, or heart failure).
  • FIG. 2 is a graph showing the expression ratio of sREST to REST (sREST/REST) when added to SCLC cells in primary screening for various antisense oligonucleotides.
  • FIG. 2 is a graph showing the expression ratio of sREST to REST (sREST/REST) for various antisense oligonucleotides when added to prostate cancer cells.
  • A is a photograph of the results of RT-PCR showing altered splicing (N exon skipping) by an oligonucleotide (SSO) targeting REST
  • B is a graph showing the results of evaluation of cell viability.
  • FIG. 10 is a graph showing that N exon skipping of REST-targeting oligonucleotides (SSO) is concentration dependent.
  • FIG. 2 shows the positional relationship of REST-targeting oligonucleotides (SSO) in REST pre-mRNA (upper panel) and the concentration dependence of N exon skipping of SSO (lower panel).
  • the base sequence in the figure is shown as SEQ ID NO:50.
  • REST-targeting oligonucleotides (specifically, AmNA[+23/+40] (hereinafter sometimes referred to as AmNA_2340) and AmNA[+27/+44] (hereinafter referred to as AmNA_2744) ) in REST pre-mRNA (upper diagram), and a graph (lower diagram) showing the concentration dependence of N exon skipping of SSO.
  • SSO SSO
  • AmNA_2340 AmNA[+27/+44]
  • AmNA_2744 AmNA[+27/+44]
  • the base sequence in the figure is SEQ ID NO: 51. show.
  • NCs that do not change splicing are preferable, we decided to use NCs whose relative exon skipping activity is closest to 1 (NC3) as NCs in the future.
  • FIG. 11 is a graph showing the results of quantifying relative exon skipping activity from the photographs of FIG. 10.
  • the results of SSO analysis are summarized based on FIGS. 8, 10 and 11.
  • Photographs of RT-PCR results showing the positional relationship of REST-targeting oligonucleotides (SSO) in REST pre-mRNA (upper diagram), and changes in splicing (N exon skipping) due to SSO, and relative comparisons from the photographs. It is a graph (lower figure) of the results of quantification of exon skipping activity.
  • FIG. 2 shows the positional relationship of REST-targeting oligonucleotides (SSO) in REST pre-mRNA (top), and a graph of the results of quantifying relative SRRM4 expression levels by SSO (bottom).
  • alkyl group having 1 to 3 carbon atoms includes any alkyl group having 1 to 3 carbon atoms. Specific examples include a methyl group, an ethyl group, an n-propyl group, and an isopropyl group.
  • linear alkyl group having 1 to 6 carbon atoms includes any linear alkyl group having 1 to 6 carbon atoms. Specific examples include methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group and n-hexyl group.
  • linear alkoxy group having 1 to 6 carbon atoms includes an alkoxy group having any linear alkyl group having 1 to 6 carbon atoms. Examples include methyloxy group, ethyloxy group, n-propyloxy group and the like.
  • a straight or branched chain alkoxy group having 1 to 6 carbon atoms includes an alkoxy group having any straight or branched chain alkyl group having 1 to 6 carbon atoms.
  • Examples include methyloxy group, ethyloxy group, n-propyloxy group, isopropyloxy group, n-butyloxy group, isobutyloxy group, tert-butyloxy group, n-pentyloxy group and isopentyloxy group.
  • linear alkylthio group having 1 to 6 carbon atoms includes an alkylthio group having any linear alkyl group having 1 to 6 carbon atoms. Examples thereof include a methylthio group, an ethylthio group and an n-propylthio group.
  • a straight or branched chain alkylthio group having 1 to 6 carbon atoms includes an alkylthio group having any straight or branched chain alkyl group having 1 to 6 carbon atoms.
  • Examples include methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, tert-butylthio, n-pentylthio and isopentylthio groups.
  • cyanoalkoxy group having 1 to 6 carbon atoms refers to a group in which at least one hydrogen atom constituting the linear alkoxy group having 1 to 6 carbon atoms is substituted with a cyano group.
  • linear alkylamino group having 1 to 6 carbon atoms refers to a group in which one or two hydrogen atoms constituting an amino group are substituted with a linear alkyl group having 1 to 6 carbon atoms. encompasses Examples thereof include methylamino group, dimethylamino group, ethylamino group, methylethylamino group and diethylamino group.
  • linear or branched chain alkylamino group having 1 to 6 carbon atoms means that one or two of the hydrogen atoms constituting the amino group are any linear or branched chain having 1 to 6 carbon atoms. Includes groups substituted with chain alkyl groups.
  • Examples include methylamino group, dimethylamino group, ethylamino group, methylethylamino group, diethylamino group, n-propylamino group, di-n-propylamino group, isopropylamino group and diisopropylamino group.
  • any straight chain alkyl group having 1 to 7 carbon atoms includes methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and n-heptyl groups.
  • Optional branched alkyl groups having 3 to 7 carbon atoms include isopropyl group, isobutyl group, tert-butyl group, isopentyl group and the like, and optional cyclic alkyl groups having 3 to 7 carbon atoms are A cyclobutyl group, a cyclopentyl group, a cyclohexyl group and the like can be mentioned.
  • alkenyl group having 2 to 7 carbon atoms means any linear alkenyl group having 2 to 7 carbon atoms, any branched alkenyl group having 3 to 7 carbon atoms, Chain alkenyl groups and any cyclic alkenyl groups having 3 to 7 carbon atoms are included. It may simply be referred to as a "lower alkenyl group”.
  • any linear alkenyl group having 2 to 7 carbon atoms includes ethenyl group, 1-propenyl group, 2-propenyl group, 1-butenyl group, 2-butenyl group, 1-pentenyl group, 2-pentenyl group, 3-pentenyl group, 4-pentenyl group, 1-hexenyl group and the like
  • arbitrary branched alkenyl groups having 3 to 7 carbon atoms include isopropenyl group, 1-methyl-1-propenyl group, 1-methyl -2-propenyl group, 2-methyl-1-propenyl group, 2-methyl-2-propenyl group, 1-methyl-2-butenyl group and the like
  • any cyclic alkenyl group having 3 to 7 carbon atoms includes a cyclobutenyl group, a cyclopentenyl group, a cyclohexenyl group, and the like.
  • any linear alkoxy group having 1 to 7 carbon atoms includes methoxy, ethoxy, n-propoxy, n-butyroxy, n-pentyloxy, n-hexyloxy, and n-heptyloxy groups.
  • any branched chain alkoxy group having 3 to 7 carbon atoms includes isopropoxy group, isobutyloxy group, tert-butyloxy group, isopentyloxy group and the like, and any cyclic group having 3 to 7 carbon atoms.
  • Alkoxy groups include a cyclobutyloxy group, a cyclopentyloxy group, a cyclohexyloxy group, and the like.
  • an aryl group having 3 to 12 carbon atoms which may contain a heteroatom refers to any aryl group having 6 to 12 carbon atoms, which is composed only of hydrocarbons, and Any heteroaryl group having 3 to 12 carbon atoms in which at least one carbon atom constituting the ring structure is replaced with a heteroatom (e.g., nitrogen atom, oxygen atom, sulfur atom, and combinations thereof) do.
  • a heteroatom e.g., nitrogen atom, oxygen atom, sulfur atom, and combinations thereof
  • the aryl group having 6 to 12 carbon atoms includes phenyl group, naphthyl group, indenyl group, azulenyl group and the like, and the arbitrary heteroaryl group having 3 to 12 carbon atoms includes pyridyl group, pyrrolyl group, quinolyl group, indolyl group, imidazolyl group, furyl group, thienyl group and the like.
  • aralkyl group having an aryl moiety having 3 to 12 carbon atoms which may contain a heteroatom examples include a benzyl group, a phenethyl group, a naphthylmethyl group, a 3-phenylpropyl group, 2 -phenylpropyl group, 4-phenylbutyl group, 2-phenylbutyl group, pyridylmethyl group, indolylmethyl group, furylmethyl group, thienylmethyl group, pyrrolylmethyl group, 2-pyridylethyl group, 1-pyridylethyl group, 3 -thienylpropyl group and the like.
  • acyl group examples include aliphatic acyl groups and aromatic acyl groups.
  • examples of aliphatic acyl groups include formyl, acetyl, propionyl, butyryl, isobutyryl, pentanoyl, pivaloyl, valeryl, isovaleryl, octanoyl, nonanoyl, decanoyl, 3-methylnonanoyl group, 8-methylnonanoyl group, 3-ethyloctanoyl group, 3,7-dimethyloctanoyl group, undecanoyl group, dodecanoyl group, tridecanoyl group, tetradecanoyl group, pentadecanoyl group, hexadecanoyl group, 1-methylpentadecanoyl group, 14-methylpentadecanoyl group, 13,13-dimethyltetradecanoyl group,
  • aromatic acyl groups include arylcarbonyl groups such as benzoyl group, ⁇ -naphthoyl group and ⁇ -naphthoyl group; 2-bromobenzoyl group and halogenoarylcarbonyl group such as 4-chlorobenzoyl group; , 4,6-trimethylbenzoyl group, lower alkylated arylcarbonyl group such as 4-toluoyl group; lower alkoxylated arylcarbonyl group such as 4-anisoyl group; 2-carboxybenzoyl group, 3-carboxybenzoyl group, 4 -carboxylated arylcarbonyl group such as carboxybenzoyl group; nitrated arylcarbonyl group such as 4-nitrobenzoyl group and 2-nitrobenzoyl group; lower alkoxycarbonylated arylcarbonyl group such as 2-(methoxycarbonyl)benzoyl group group; an arylated aryl group
  • sil group examples include a trimethylsilyl group, a triethylsilyl group, an isopropyldimethylsilyl group, a t-butyldimethylsilyl group, a methyldiisopropylsilyl group, a methyldi-t-butylsilyl group, and a triisopropylsilyl group.
  • tri-lower alkylsilyl groups such as; tri-lower alkylsilyl groups substituted with 1 to 2 aryl groups such as diphenylmethylsilyl group, butyldiphenylbutylsilyl group, diphenylisopropylsilyl group and phenyldiisopropylsilyl group; mentioned.
  • Trimethylsilyl group, triethylsilyl group, triisopropylsilyl group, t-butyldimethylsilyl group and t-butyldiphenylsilyl group are preferred, and trimethylsilyl group is more preferred.
  • halogen atom includes, for example, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.
  • a fluorine atom or a chlorine atom is preferred.
  • halide ion includes, for example, fluoride ion, chloride ion, bromide ion, or iodide ion. Fluoride ions or chloride ions are preferred.
  • protecting group for amino group for nucleic acid synthesis As used herein, the terms “protecting group for amino group for nucleic acid synthesis”, “protecting group for hydroxyl group for nucleic acid synthesis”, “hydroxyl group protected by protecting group for nucleic acid synthesis”, “protecting group for nucleic acid synthesis”
  • the “protecting group” of "phosphate group” and “mercapto group protected by a protecting group for nucleic acid synthesis” is a group capable of stably protecting an amino group, hydroxyl group, phosphoric acid group or mercapto group during nucleic acid synthesis. If so, it is not particularly limited. Specifically, it refers to protective groups that are stable under acidic or neutral conditions and cleavable by chemical methods such as hydrogenolysis, hydrolysis, electrolysis, and photolysis.
  • Such protecting groups include, for example, lower alkyl groups, lower alkenyl groups, acyl groups, tetrahydropyranyl or tetrahydrothiopyranyl groups, tetrahydrofuranyl or tetrahydrothiofuranyl groups, silyl groups, lower alkoxymethyl groups, lower alkoxy lower alkoxymethyl group, halogeno lower alkoxymethyl group, lower alkoxylated ethyl group, halogenated ethyl group, methyl group substituted with 1 to 3 aryl groups, "lower alkyl group, lower alkoxy group, halogen atom or cyano A methyl group substituted with 1 to 3 aryl groups substituted on an aryl ring with a group", a lower alkoxycarbonyl group, an "aryl group substituted with a halogen atom, a lower alkoxy group or a nitro group", a "halogen atom or A lower alkoxycarbonyl group substitute
  • the tetrahydropyranyl group or tetrahydrothiopyranyl group includes a tetrahydropyran-2-yl group, a 3-bromotetrahydropyran-2-yl group, a 4-methoxytetrahydropyran-4-yl group and a tetrahydropyranyl group.
  • a tetrahydrofuranyl group or a tetrahydrothiofuranyl group includes a tetrahydrofuran-2-yl group and a tetrahydrothiofuran-2-yl group.
  • the lower alkoxymethyl group includes methoxymethyl group, 1,1-dimethyl-1-methoxymethyl group, ethoxymethyl group, propoxymethyl group, isopropoxymethyl group, butoxymethyl group, t-butoxymethyl group and the like.
  • Lower alkoxylated lower alkoxymethyl groups include 2-methoxyethoxymethyl groups and the like.
  • Halogeno lower alkoxymethyl groups include 2,2,2-trichloroethoxymethyl groups and bis(2-chloroethoxy)methyl groups.
  • the lower alkoxylated ethyl group includes 1-ethoxyethyl group, 1-(isopropoxy)ethyl group and the like. Examples of halogenated ethyl groups include 2,2,2-trichloroethyl groups.
  • the methyl group substituted with 1 to 3 aryl groups includes a benzyl group, ⁇ -naphthylmethyl group, ⁇ -naphthylmethyl group, diphenylmethyl group, triphenylmethyl group, ⁇ -naphthyldiphenylmethyl group, 9-an thrylmethyl group and the like.
  • a methyl group substituted with 1 to 3 aryl groups whose aryl ring is substituted with a lower alkyl group, a lower alkoxy group, a halogen atom or a cyano group includes a 4-methylbenzyl group, a 2,4,6- trimethylbenzyl group, 3,4,5-trimethylbenzyl group, 4-methoxybenzyl group, 4-methoxyphenyldiphenylmethyl group, 4,4'-dimethoxytriphenylmethyl group, 2-nitrobenzyl group, 4-nitrobenzyl group , 4-chlorobenzyl group, 4-bromobenzyl group, 4-cyanobenzyl group and the like.
  • lower alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl and isobutoxycarbonyl groups.
  • the "aryl group substituted with a halogen atom, a lower alkoxy group or a nitro group” includes a 4-chlorophenyl group, a 2-fluorophenyl group, a 4-methoxyphenyl group, a 4-nitrophenyl group and a 2,4-dinitrophenyl group. etc.
  • lower alkoxycarbonyl group substituted with a halogen atom or a tri-lower alkylsilyl group includes a 2,2,2-trichloroethoxycarbonyl group and a 2-trimethylsilylethoxycarbonyl group.
  • alkenyloxycarbonyl groups include vinyloxycarbonyl groups and aryloxycarbonyl groups.
  • the "aralkyloxycarbonyl group optionally substituted on the aryl ring with a lower alkoxy or nitro group” includes a benzyloxycarbonyl group, a 4-methoxybenzyloxycarbonyl group, a 3,4-dimethoxybenzyloxycarbonyl group, a 2-nitro benzyloxycarbonyl group, 4-nitrobenzyloxycarbonyl group and the like.
  • Examples of the "lower alkoxycarbonyl group substituted with a cyano group” include a cyanoethoxycarbonyl group and the like.
  • the "benzenesulfonyl group substituted with 1 to 4 nitro groups” includes a 2-nitrobenzenesulfonyl group, a 2,4-dinitrobenzenesulfonyl group and the like.
  • the "hydroxyl-protecting group for nucleic acid synthesis” preferably includes an aliphatic acyl group, an aromatic acyl group, a methyl group substituted with 1 to 3 aryl groups, "lower alkyl, lower alkoxy, halogen, cyano a methyl group substituted with 1 to 3 aryl groups in which the aryl ring is substituted with a group, or a silyl group, more preferably an acetyl group, a benzoyl group, a benzyl group, a p-methoxybenzoyl group, a dimethoxy trityl group, monomethoxytrityl group or tert-butyldiphenylsilyl group.
  • the protecting group for the "hydroxyl group protected by a protecting group for nucleic acid synthesis” is preferably an aliphatic acyl group, an aromatic acyl group, a "methyl group substituted with 1 to 3 aryl groups", a "halogen an aryl group substituted with an atom, a lower alkoxy group or a nitro group, a lower alkyl group or a lower alkenyl group, more preferably a benzoyl group, a benzyl group, a 2-chlorophenyl group, a 4-chlorophenyl group or a 2- It is a propenyl group.
  • the “amino group-protecting group for nucleic acid synthesis” is preferably an acyl group, more preferably a benzoyl group.
  • the "protecting group” of the "phosphate group protected by a protecting group for nucleic acid synthesis” is preferably a lower alkyl group, a lower alkyl group substituted with a cyano group, an aralkyl group, a "nitro group or a halogen atom an aralkyl group substituted with an aryl ring” or an "aryl group substituted with a lower alkyl group, a halogen atom, or a nitro group", more preferably a 2-cyanoethyl group or a 2,2,2-trichloroethyl group , benzyl group, 2-chlorophenyl group or 4-chlorophenyl group.
  • protecting groups constituting "a phosphate group protected by a protecting group for nucleic acid synthesis".
  • the "protecting group” of the "mercapto group protected by a protecting group for nucleic acid synthesis” is preferably an aliphatic acyl group or an aromatic acyl group, more preferably a benzoyl group.
  • the "amino group-protecting group" for the R 10 group includes an acetyl group, a tertiary butoxycarbonyl (Boc) group, a 9-fluorenylmethyloxycarbonyl (Fmoc) group, and the like.
  • —P(R 24 )R 25 [wherein R 24 and R 25 are each independently a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, a mercapto group, a protecting group for nucleic acid synthesis A mercapto group, an amino group, a straight or branched chain alkoxy group having 1 to 6 carbon atoms, a straight or branched chain alkylthio group having 1 to 6 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or carbon representing a linear or branched alkylamino group of numbers 1 to 6], the group in which R 24 can be represented as —OR 24a and R 25 can be represented as —N(R 25a ) 2 is represented by “ phosphoramidite group”.
  • the phosphoramidite group is preferably a group represented by the formula -P(OC 2 H 4 CN)(N(iPr) 2 ), or a group represented by the formula -P(OCH 3 )(N(iPr) 2 ) and the group represented by
  • iPr represents an isopropyl group.
  • nucleoside includes “nucleosides” in which a purine or pyrimidine base and a sugar are attached, as well as aromatic heterocycles and aromatic hydrocarbon rings other than purines and pyrimidines that substitute for a purine or pyrimidine base. Includes “nucleosides” that are possible and sugar-linked. Natural nucleosides are also called “natural nucleosides”. Modified non-natural nucleosides are also referred to as “modified nucleosides”, and nucleotides with modified sugar moieties are particularly referred to as “sugar-modified nucleosides”. "Nucleotide” means a compound in which a phosphate group is attached to the sugar of a nucleoside.
  • oligonucleotide refers to a polymer of “nucleotides” in which from 2 to 50 identical or different “nucleosides” are linked by phosphodiester or other linkages, naturally occurring and non-naturally occurring. Including those of type.
  • the non-natural "oligonucleotide” preferably includes a sugar derivative with a modified sugar moiety; a thioate derivative with a thioated phosphodiester moiety; an ester with an esterified terminal phosphate moiety; Examples include amides in which the amino group on the base is amidated, and more preferably sugar derivatives in which the sugar moiety is modified.
  • a salt thereof refers to a salt of the compound represented by formula (II) described below.
  • Such salts include, for example, alkali metal salts such as sodium salts, potassium salts and lithium salts, alkaline earth metal salts such as calcium salts and magnesium salts, aluminum salts, iron salts, zinc salts, copper salts, Metal salts such as nickel salts and cobalt salts; inorganic salts such as ammonium salts, t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts , guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benz
  • the term "pharmacologically acceptable salt thereof” refers to a salt of an oligonucleotide containing at least one nucleoside structure represented by formula (I) shown below, wherein the oligonucleotide according to the present invention ie, salts that retain the desired biological activity of the oligonucleotide and do not impart undesired toxicological effects therein.
  • Such salts include, for example, alkali metal salts such as sodium salts, potassium salts and lithium salts, alkaline earth metal salts such as calcium salts and magnesium salts, aluminum salts, iron salts, zinc salts, copper salts, Metal salts such as nickel salts and cobalt salts; inorganic salts such as ammonium salts, t-octylamine salts, dibenzylamine salts, morpholine salts, glucosamine salts, phenylglycine alkyl ester salts, ethylenediamine salts, N-methylglucamine salts , guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, tris Amine
  • oligonucleotide oligonucleotide
  • the oligonucleotide of the present invention has the activity of inducing N exon skipping in the processing of REST pre-mRNA (REST pre-mRNA).
  • REST pre-mRNA REST pre-mRNA
  • Such oligonucleotides may be pharmacologically acceptable salts thereof.
  • the nucleotide sequence information of the human REST gene is available from NCBI Reference Sequence: NG_029447.1 Homo sapiens RE1 silencing transcription factor (REST), RefSeqGene on chromosome 4.
  • the base sequence of SEQ ID NO: 1 in the Sequence Listing corresponds to positions 24740 to 24789 of the human REST gene and spans the entire length of the N exon.
  • the N exon is located between exons 3 and 4.
  • the term "activity to induce N exon skipping in processing of REST pre-mRNA” means, for example, that an oligonucleotide binds to REST pre-mRNA, and then during processing of the pre-mRNA, Inducing skipping of the N exon, which promotes expression of REST and/or suppresses expression of the mutant sREST.
  • the oligonucleotide is an antisense oligonucleotide (ASO) to the REST mRNA or pre-mRNA.
  • This oligonucleotide is also called “splicing control oligonucleotide (SSO)" because it has the activity of inducing N exon skipping and thereby controls REST and sREST expression by pre-mRNA splicing.
  • SSO splicing control oligonucleotide
  • the activity of inducing N exon skipping can be determined by measuring the expression levels and expression ratios of REST and sREST by known methods (eg, reverse transcription-polymerase chain reaction (RT-PCR)). Oligonucleotides with N exon skipping activity can be concentration dependent, e.g. Activity can be determined by measuring the ratio (sREST/REST), or the ratio of REST to the "sum of REST and sREST" ("% exclusion") based on band intensity. The higher the skipping induction activity, the lower the "sREST/REST” and the higher the "%exclusion".
  • RT-PCR reverse transcription-polymerase chain reaction
  • “sREST/REST” is lower than when no antisense oligonucleotide is added (control), and when “sREST/REST” in the control is set to 1 (in other words, oligonucleotide "sREST/REST” is divided by the “sREST/REST” of the control), for example, 0.8 or less, preferably 0.7 or less, more preferably 0.6 or less, even more preferably 0.4 or less, and 0.2 It may be below.
  • the "relative exon skipping activity” can be calculated by dividing the "% exclusion” of the oligonucleotide by the "% exclusion” of the control.
  • the “relative exon skipping activity” is, for example, 1.2 or higher, preferably 1.4 or higher, more preferably 1.6 or higher, still more preferably 1.8 or higher, even if it is 2.0 or higher. good.
  • an oligonucleotide "binds" to a REST pre-mRNA means that multiple different single-stranded oligonucleotides or nucleic acids can form two or more strands of nucleic acid due to nucleobase complementarity. Say. Preferably, it refers to the ability to form a double-stranded nucleic acid.
  • the melting temperature (T m ) of a double-stranded or higher-stranded nucleic acid which is an index of thermal stability of binding, is not particularly limited.
  • the melting temperature ( Tm ) of double - stranded nucleic acids can be determined, for example, as follows: , The oligonucleotide and the target RNA are mixed in equimolar amounts, heated at 95° C. for 5 minutes, then slowly cooled to room temperature for annealing to form a double-stranded nucleic acid.
  • the change in absorbance (A) at 260 nm with temperature (T) was measured when the double-stranded nucleic acid was heated from 20°C to 95°C at a heating rate of 0.5°C/min.
  • T is prepared, and the temperature at which the value of dA/dT in this graph becomes the largest, that is, the temperature at which the change in A due to T is the largest is taken as the Tm of the double-stranded nucleic acid.
  • the melting temperature (T m ) is, for example, 40° C. or higher, preferably 50° C. or higher.
  • complementary means that two different single-stranded oligonucleotides or nucleic acids are in a pairing relationship that allows them to form a double-stranded nucleic acid.
  • the base sequences of the double-strand forming regions are completely complementary, but have one or several mismatches as long as they can form the double-stranded nucleic acid and have N exon skipping action.
  • One or several mismatches means 1 to 4, preferably 1 to 3, more preferably 1 or 2 mismatches, depending on the length of the oligonucleotide.
  • the oligonucleotide of the present invention is preferably 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the base sequence of the region forming the double strand or complete (100%) complementarity.
  • Antisense oligonucleotides (ASO) that target REST genes are examples of oligonucleotides that have the activity of inducing N exon skipping in the processing (splicing) of REST mRNA precursors.
  • Antisense oligonucleotides (ASOs) are capable of binding to target gene RNA (e.g., mRNA or pre-mRNA)/DNA, and have the activity of regulating the expression of the target gene (including splicing regulation), Refers to a single-stranded oligonucleotide that is complementary to the RNA (eg, mRNA or pre-mRNA)/DNA sequence of its target gene.
  • Antisense oligonucleotides (ASO) in the present invention are "splicing control oligonucleotides (SSO)" in that they can regulate splicing of target genes.
  • the oligonucleotide of the present invention can bind to a target region that is at least part of SEQ ID NO:1. Oligonucleotides of the invention may also bind to a target region that is at least part of SEQ ID NO:38 or 39.
  • the sequences represented by SEQ ID NOs: 38 and 39 are both partial regions of SEQ ID NO: 1.
  • a given "target region” refers to a region of the pre-mRNA shown in any of SEQ ID NOs: 1, 38 and 39, for example.
  • the target region is preferably a region associated with the activity of inducing N exon skipping in REST.
  • the target region is, for example, at least 12 bases long, e.g.
  • a nucleic acid molecule or oligonucleotide "binds to a target region” means that the nucleic acid molecule or oligonucleotide does not necessarily form two or more strands (preferably double strands) with the entire target region, and the REST mRNA precursor It may form two or more strands (preferably two strands) with a region that is part of the target region, as long as it exhibits the activity of inducing skipping of the N exon in the processing of .
  • the oligonucleotide having the activity of inducing skipping of the N exon of the REST pre-mRNA is, for example, complementary to the nucleotide sequence of the target region, which is at least part of SEQ ID NO: 1, and preferably has complete complementarity.
  • Oligonucleotides e.g., antisense oligonucleotides
  • Oligonucleotides of the invention are at least 12 bases long, e.g. 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 bases long. When the oligonucleotide has the above length, it can more effectively bind to the REST pre-mRNA and induce skipping of the N exon.
  • Oligonucleotides for example, antisense oligonucleotides of the present invention are oligonucleotides that have the activity of inducing N exon skipping of REST pre-mRNA, and are, for example, It includes a sequence that is complementary to at least 12 bases.
  • the oligonucleotide need not be entirely complementary to the target region consisting of the base sequence of SEQ ID NO: 1.
  • a portion of the oligonucleotide may be a peripheral region (e.g., the 5' end and/or 3' end of the target region). 1-5 base extensions) (shown in SEQ ID NO: 2). The same is true for SEQ ID NOs:38 and 39.
  • the design of the sequences of antisense oligonucleotides, such as the oligonucleotides (e.g., antisense oligonucleotides) of the present invention, based on the selected target region can be performed by methods commonly used by those skilled in the art.
  • base sequences of antisense oligonucleotides include the sequences shown below (shown in the 5′ ⁇ 3′ direction) (the numbers in [] indicate the sequence targeted by the oligonucleotide, human REST
  • the position of both ends of the sequence of SEQ ID NO: 1 in the pre-mRNA is represented by the base number at the 5' end of the sequence of SEQ ID NO: 1 being 1 and the number of bases in the direction of the 3' end being + (plus).
  • the number of bases deviating from the end of the sequence of 1 is represented by a - (minus) value, and when simply represented by a numerical value of -, it is the number of bases from the 5' end. is the number of bases from the end.):
  • aatggtatccatacccca (SEQ ID NO: 3) [+1/+18] accaaatggtatccatac (SEQ ID NO: 4) [+5/+22] taaatattaccaaatggt (SEQ ID NO: 5) [+13/+30] agtaaatattaccaaatg (SEQ ID NO: 6) [+15/+32] ctctagtaaatattacca (SEQ ID NO: 7) [+19/+36] cacactctagtaaattatt (SEQ ID NO: 8) [+23/+40] agatcacactctagtaaa (SEQ ID NO: 9) [+27/+44] atctagatcacactctag (SEQ ID NO: 10) [+31/+48] acccatctagatcacact (SEQ ID NO: 11) [+35/-2(3')]
  • Atcacactctagtaaattatt (SEQ ID NO: 40) [+23/+42] cacactctagtaaatattac (SEQ ID NO: 41) [+21/+40] cactctagtaaattatt (SEQ ID NO: 42) [+23/+38] cacactctagtaaata (SEQ ID NO: 43) [+25/+40] ctagatcacactctagtaaa (SEQ ID NO: 44) [+27/+46] agatcacactctagtaaata (SEQ ID NO: 45) [+25/+44] atcacactctagtaaa (SEQ ID NO: 46) [+27/+42] agatcacactctagta (SEQ ID NO: 47) [+29/+44] agatcacactctagtaaattatt (SEQ ID NO: 48) [+23/+44] gatcacactctagtaaat (SEQ ID
  • the added base is complementary to the base of the sequence shown in SEQ ID NO: 2 (showing the base sequence of the target region and the adjacent region) adjacent to the target region (SEQ ID NO: 1) of the sequence to which the base is added. It can be a base.
  • the oligonucleotides of the present invention include both oligonucleotides containing natural DNA (unmodified oligonucleotides) and oligonucleotides containing chemically modified DNA. Such modifications may alter the activity of the oligonucleotide, eg, increase its affinity for a target nucleic acid, or increase its resistance to nucleases. Increasing the affinity of the oligonucleotide for its target may allow the use of shorter oligonucleotides.
  • the oligonucleotide according to the present invention may contain at least one sugar-modified nucleoside at any position.
  • the ratio of the number of sugar-modified nucleotides to all nucleosides constituting the oligonucleotide is, for example, 30% or more, preferably 40% or more, and more preferably 50% or more.
  • the sugar-modified nucleoside has a bridging moiety between the 2' and 4' positions of the sugar ring, eg, as described below.
  • the oligonucleotide of the present invention contains at least one nucleoside structure represented by the following formula (I) as a sugar-modified nucleoside:
  • Base is a purin-9-yl group optionally having one or more arbitrary substituents selected from the ⁇ group, or optionally having one or more arbitrary substituents selected from the ⁇ group 2 -oxo-1,2-dihydropyrimidin-1-yl group, wherein the ⁇ group is a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, a linear alkyl group having 1 to 6 carbon atoms, a carbon linear alkoxy group of number 1 to 6, mercapto group, mercapto group protected by protecting group for nucleic acid synthesis, linear alkylthio group of 1 to 6 carbon atoms, amino group, linear alkylamino group of 1 to 6 carbon atoms , an amino group protected by a protective group for nucleic acid synthesis, and a halogen atom,
  • A is:
  • R 1 is selected from a hydrogen atom, an optionally branched or ring-forming alkyl group having 1 to 7 carbon atoms, an optionally branched or ring-forming alkenyl group having 2 to 7 carbon atoms, and the ⁇ group an aryl group having 3 to 12 carbon atoms which may have one or more optional substituents and may contain a heteroatom, and one or more optional substituents selected from the ⁇ group represents an aralkyl group having an aryl moiety of 3 to 12 carbon atoms which may optionally contain a heteroatom, or an amino group-protecting group for nucleic acid synthesis;
  • R 2 and R 3 are each independently a hydrogen atom; optionally substituted with an aryl group having 3 to 12 carbon atoms which may contain a heteroatom, and optionally branched or forming a ring; an alkyl group having 1 to 7 carbon atoms; or an aralkyl group having an aryl moiety having 3 to 12 carbon
  • R 17 , R 18 and R 19 each independently form a hydrogen atom, a branch or a ring an alkyl group having 1 to 7 carbon atoms, an amino group-protecting group, or
  • R 13 and R 14 each independently represents a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms which may be branched or forming a ring; A group selected from the group consisting of an alkoxy group of 7; an amino group; and an amino group protected by a protecting group for nucleic acid synthesis; m is an integer from 0 to 2; n is an integer from 0 to 1; R 10 is a hydrogen atom, an optionally branched or ring-forming alkyl group having 1 to 7 carbon atoms, an amino-protecting group, or
  • R 15 and R 16 are each independently a hydrogen atom, an optionally branched or ring-forming alkyl group having 1 to 7 carbon atoms, or an amino group-protecting group. ,or
  • X is an oxygen atom, a sulfur atom, or an amino group
  • Y is an oxygen atom or a sulfur atom.
  • nucleoside structure represented by formula (I) above is
  • R 1 is a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may form a branch or a ring, or may form a branch or a ring.
  • It is an aralkyl group having an aryl moiety of 3 to 12 carbon atoms which may have one or more optional substituents selected from the group and which may contain a heteroatom.
  • R 1 is a hydrogen atom, methyl group, ethyl group, n-propyl group, isopropyl group, phenyl group, or benzyl group, and even more preferably R 1 is a hydrogen atom or a methyl group. be.
  • n is an integer from 0 to 1.
  • the ring including the 2'-positions, 3'-positions, 4'-positions, and the bridging moiety may constitute a 5- to 7-membered ring.
  • X is an oxygen atom, a sulfur atom, an amino group, or a methylene group.
  • X is an oxygen atom or an amino group.
  • X when X is an amino group or a methylene group, it may be substituted with a lower alkyl group.
  • the nucleoside structure represented by formula (I) above is a structure represented by formula (I-1) above, and in formula (I-1) m is 0; and R 1 is a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a phenyl group, or a benzyl group.
  • Nucleoside structures represented by formula (I) above include, in addition to formulas (I-1) and (I-2) above, for example, the following (I-3) to (I-7):
  • R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 13 , R 14 , R 15 , R 16 and p are As described above with respect to formula (I).
  • Formula (I-7) is referred to as 2',4'-BNA or LNA (Locked Nucleic Acid) (also referred to herein as "2',4'-BNA/LNA” or "LNA”). (for example, both R 13 and R 14 are hydrogen atoms).
  • Formula (I-3) has a structure in which a spirocyclopropane group is introduced at the 6' position of the 2',4'-BNA/LNA crosslinked portion, and is also referred to as spirocyclopropane crosslinked nucleic acid (spcBNA).
  • Formula (I-4) has a structure in which guanidine is introduced into the crosslinked portion of 2',4'-BNA/LNA, and is also called guanidine-bridged nucleic acid (GuNA).
  • Base is a purine base (ie, purine-9-yl group) or a pyrimidine base (ie, 2-oxo-1,2-dihydropyrimidin-1-yl group).
  • bases include a hydroxyl group, a linear alkyl group having 1 to 6 carbon atoms, a linear alkoxy group having 1 to 6 carbon atoms, a mercapto group, a linear alkylthio group having 1 to 6 carbon atoms, an amino group, and a 6 linear alkylamino groups and one or more optional substituents selected from the ⁇ group consisting of halogen atoms.
  • Base examples include an adenynyl group, a guanynyl group, a cytosinyl group, a uracilyl group, a thyminyl group, a 6-aminopurin-9-yl group, a 2,6-diaminopurin-9-yl group, a 2 -amino-6-chloropurin-9-yl group, 2-amino-6-fluoropurin-9-yl group, 2-amino-6-bromopurin-9-yl group, 2-amino-6-hydroxypurine- 9-yl group, 6-amino-2-methoxypurin-9-yl group, 6-amino-2-chloropurin-9-yl group, 6-amino-2-fluoropurin-9-yl group, 2,6 -dimethoxypurin-9-yl group, 2,6-dichloropurin-9-yl group, 6-mercaptopurin-9-yl group
  • Base has the following structural formula from the perspective of introduction into nucleic acid medicine:
  • thyminyl group cytosinyl group, adenynyl group, guanynyl group, 5-methylcytosinyl group and uracilyl group
  • 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl and thyminyl groups are preferred
  • the oligonucleotide according to the present invention has a nucleoside structure represented by the above formula (I-4), formula (I-4-1) or formula (I-4-2) as a sugar-modified nucleoside.
  • these nucleoside structures represented by formula (I-4) or formula (I-4-1) or formula (I-4-2) are represented by formula (I-4) or formula (I-4- 1) or is held electrically neutral by a pharmaceutically acceptable anion not represented in formula (I-4-2) (which can be represented as, for example, Z 1 ⁇ ).
  • examples of such anions include halide ions (eg, chloride ions), phosphate ions, and the like.
  • a sugar-modified nucleoside compound for example, WO 2011/052436, JP 2014-043462, WO 2014/ 046212 and WO2015/125783.
  • sugar-modified nucleoside compounds include compounds represented by the following formula (II) or salts thereof:
  • Base is a purin-9-yl group optionally having one or more arbitrary substituents selected from the ⁇ group, or optionally having one or more arbitrary substituents selected from the ⁇ group 2 -oxo-1,2-dihydropyrimidin-1-yl group, wherein the ⁇ group is a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, a linear alkyl group having 1 to 6 carbon atoms, a carbon linear alkoxy group of number 1 to 6, mercapto group, mercapto group protected by protecting group for nucleic acid synthesis, linear alkylthio group of 1 to 6 carbon atoms, amino group, linear alkylamino group of 1 to 6 carbon atoms , an amino group protected by a protective group for nucleic acid synthesis, and a halogen atom,
  • A is:
  • R 1 is selected from a hydrogen atom, an optionally branched or ring-forming alkyl group having 1 to 7 carbon atoms, an optionally branched or ring-forming alkenyl group having 2 to 7 carbon atoms, and the ⁇ group an aryl group having 3 to 12 carbon atoms which may have one or more optional substituents and may contain a heteroatom, and one or more optional substituents selected from the ⁇ group represents an aralkyl group having an aryl moiety of 3 to 12 carbon atoms which may optionally contain a heteroatom, or an amino group-protecting group for nucleic acid synthesis;
  • R 2 and R 3 are each independently a hydrogen atom; optionally substituted with an aryl group having 3 to 12 carbon atoms which may contain a heteroatom, and optionally branched or forming a ring; an alkyl group having 1 to 7 carbon atoms; or an aralkyl group having an aryl moiety having 3 to 12 carbon
  • R 17 , R 18 and R 19 each independently form a hydrogen atom, a branch or a ring an alkyl group having 1 to 7 carbon atoms, an amino group-protecting group, or
  • R 13 and R 14 each independently represents a hydrogen atom; a hydroxyl group; an alkyl group having 1 to 7 carbon atoms which may be branched or forming a ring; A group selected from the group consisting of an alkoxy group of 7; an amino group; and an amino group protected by a protecting group for nucleic acid synthesis; m is an integer from 0 to 2; n is an integer from 0 to 1;
  • R 10 is a hydrogen atom, an optionally branched or ring-forming alkyl group having 1 to 7 carbon atoms, an amino-protecting group, or
  • R 15 and R 16 are each independently a hydrogen atom, an optionally branched or ring-forming alkyl group having 1 to 7 carbon atoms, or an amino group-protecting group. ,or
  • R 22 and R 23 are each independently a hydrogen atom, a hydroxyl-protecting group for nucleic acid synthesis, or a C 1-7 group which may form a branch or ring.
  • Sugar-modified nucleotides can be easily prepared from the above sugar-modified nucleosides.
  • triphosphorylation can be readily performed according to the method described in M. Kuwahara et al., Nucleic Acids Res., 2008, vol.36, No.13, pp.4257-65.
  • Phosphate modifications include, for example, phosphodiester bonds of natural nucleic acids, S-oligo (phosphorothioate), D-oligo (phosphodiester), M-oligo (methylphosphonate), boranophosphate, and the like.
  • S-oligo(phosphorothioates) have a PS backbone in which the oxygen atoms in the phosphate groups of the internucleoside phosphodiester linkages are replaced by sulfur atoms. This modification is incorporated into the oligonucleotide according to known methods.
  • An antisense oligonucleotide having one or more such modifications in the oligonucleotide is also called an S-oligo type (phosphorothioate type).
  • Nucleobase modifications include, for example, 5-methylcytosine, 5-hydroxymethylcytosine, 5-propynylcytosine and the like.
  • the position and number of sugar-modified nucleosides are not particularly limited, and can be appropriately designed according to the purpose. Two or more sugar-modified nucleosides include, for example, the same or different from each other. Oligonucleotides can be designed with sugar-modified and unmodified nucleosides alternating. For oligonucleotides of the invention, it is preferred that a region consisting of 1-3 residues of unmodified nucleosides is followed by sugar-modified nucleosides. This makes the RNA to which the oligonucleotide binds difficult to be cleaved via RNase H.
  • nucleoside structure represented by formula (I) above is
  • R 1 is a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a phenyl group, or a benzyl group).
  • the oligonucleotide of the present invention comprises a base sequence represented by any of the base sequences of SEQ ID NOS: 3-11 and SEQ ID NOS: 40-49, and at least one of the bases is the above sugar.
  • Oligonucleotides that are modified nucleosides include, for example, those shown below:
  • the numbers in [] indicate the target sequence of the oligonucleotide, with the 5′ terminal base of the sequence of SEQ ID NO: 1 in human REST pre-mRNA being 1, 3 It is represented by the positions of both ends when the number of bases in the direction of the 'terminal side is regarded as + (plus).
  • the number of bases deviating from the end of the sequence of SEQ ID NO: 1 is represented by a - (minus) value, and when simply represented by a numerical value of -, it is the number of bases from the 5' end, and is described as (3') together with the - value. In case, it is the number of bases from the 3' end.
  • Oligonucleotides in the present invention can be synthesized by conventional methods using sugar-modified nucleosides and natural nucleosides as described above. can be easily synthesized by manufacturing, etc.). Synthetic methods include a solid-phase synthesis method using phosphoramidite, a solid-phase synthesis method using hydrogenphosphonate, and the like. For example, disclosed in Tetrahedron Letters, 1981, vol.
  • the pharmaceutical composition of the present invention contains the above oligonucleotide or a pharmacologically acceptable salt thereof.
  • the pharmaceutical composition of the present invention is useful, for example, for treating or preventing diseases involving REST processing (splicing).
  • Target diseases of the pharmaceutical composition of the present invention include, for example, cancer diseases and heart failure.
  • Cancer diseases associated with REST processing include, for example, SRRM4-mediated cancers (eg, small cell lung cancer, prostate cancer (eg, castration-resistant prostate cancer (CRPC)), breast cancer). Such cancer diseases may originate from neuroendocrine cells.
  • the present invention encompasses a therapeutic agent for cancer containing the above oligonucleotide or a pharmacologically acceptable salt thereof.
  • the present invention also includes a therapeutic agent for heart failure containing the above oligonucleotide or a pharmacologically acceptable salt thereof.
  • the administration method and formulation of the pharmaceutical composition of the present invention or the drug for treating cancer or the drug for treating heart failure may be any administration method and formulation known in the art. Available.
  • compositions, etc. of the present invention can be administered locally or systemically or by various methods depending on the region to be treated.
  • Methods of administration may be, for example, topical (including, for example, eye drops, intravaginal, intrarectal, intranasal and transdermal), oral, or parenteral.
  • Parenteral administration includes intravenous injection or infusion, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration through the respiratory tract by inhalation or inhalation, and the like.
  • formulations such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders can be used.
  • compositions for oral administration include powders, granules, suspensions or solutions dissolved in water or non-aqueous media, capsules, powders, tablets and the like.
  • compositions for parenteral administration include sterile aqueous solutions containing buffers, diluents and other suitable additives.
  • the pharmaceutical composition, etc. of the present invention contains an effective amount of the above-mentioned oligonucleotide or a pharmacologically acceptable salt thereof and excipients, binders, wetting agents, disintegrants, lubricants, and diluents suitable for the dosage form. It can be obtained by mixing various pharmaceutical additives such as, if necessary. In the case of an injection, it may be sterilized with an appropriate carrier to form a preparation.
  • the present invention provides a method of inducing N exon skipping during REST processing.
  • the present invention also provides methods for the treatment or prevention of diseases involving REST processing. In one embodiment, these methods are used to treat or prevent the target diseases described above. These methods comprise administering to an individual the oligonucleotides described above or pharmacologically acceptable salts thereof.
  • "Individuals" are preferably mammals, more preferably humans, monkeys, dogs, cats, rats and mice, and even more preferably humans. In these methods, the administration method and dosage form are not limited as long as an effective dose of the oligonucleotide of the present invention is administered.
  • the effective dose depends on the individual to be administered, it can be arbitrarily determined according to the individual's sex, age, body weight, symptoms, etc., as well as the administration method, route, frequency, and the like.
  • the administration method and the like are as described above.
  • Example 1 Antisense oligonucleotide synthesis
  • Oligonucleotides related to the present invention were synthesized by the method described in Tetrahedron Letters 22, 1859-1862 (1981), International Publication No. 2011/052436, and the like. Based on the design in Example 1, an oligonucleotide containing an amide BNA (AmNA) represented by the following formula was synthesized with reference to the method described in WO 2011/052436:
  • AmNA amide BNA
  • Base is a 5-methylcytosinyl group, thyminyl group, adenynyl group or guanynyl group, and Me is a methyl group.
  • An 18-mer oligonucleotide containing amide BNA (AmNA) was synthesized on a 0.2 ⁇ mol scale using an automatic nucleic acid synthesizer (nS-8 type, manufactured by Gene Design Co., Ltd.). Chain length extension was performed using standard phosphoramidite protocols (solid support: CPG resin, sulfurization for phosphorothioated (PS) backbone formation was performed using DDT (3H-1,2-benzodisiol-3-one, 1, 1-dioxide), etc.) to obtain an oligonucleotide in which the hydroxyl group at the terminal 5'-position was protected with a DMTr (dimethoxytrityl) group and the 3'-position was supported on a solid phase.
  • DMTr dimethyl methoxytrityl
  • the desired product was cleaved from the solid phase carrier by base treatment. After neutralization with dilute acid, the solvent was distilled off, and the resulting crude product was purified by gel filtration column chromatography and reversed-phase HPLC to obtain the desired product.
  • Antisense oligonucleotides were designed to target the pre-mRNA of the human REST gene (NCBI Reference Sequence: NG_029447.1 Homo sapiens RE1 silencing transcription factor (REST), RefSeqGene on chromosome 4.).
  • the reverse sequences (CG, GGA, GCA) of CG, TCC, and TGC that express toxicity in antisense were excluded.
  • regions such as loop structures that are easily accessible to antisense oligonucleotides were selected.
  • Blast was used to allow human application based on the results evaluated in mice.
  • the regions of REST pre-mRNA that are highly homologous to humans and mice were selected.
  • 18 candidate sequences were selected.
  • An oligonucleotide consisting of a base sequence complementary to the candidate sequence selected as described above was designed as an antisense oligonucleotide.
  • the antisense oligonucleotides were 18-mers in length, and the 5' end was designed to alternate artificial nucleic acids and DNA as artificial nucleic acids (AmNA) containing sugar-modified nucleosides.
  • AmNA artificial nucleic acids
  • the numerical value in [] indicates the target sequence of the oligonucleotide, and the 5' terminal base of the sequence of SEQ ID NO: 1 in human REST pre-mRNA is 1, and the 3' It is represented by the position of both ends when the number of bases in the terminal side direction is regarded as + (plus).
  • the number of bases deviating from the end of the sequence of SEQ ID NO: 1 is represented by a - (minus) value, and when simply represented by a numerical value of -, it is the number of bases from the 5' end, and is described as (3') together with the - value. In case, it is the number of bases from the 3' end.
  • Phosphorothioate refers to a structure in which the oxygen atom of the phosphate group in the phosphodiester bond is substituted with a sulfur atom (the group corresponding to the phosphate group is called a phosphorothioate group).
  • an oligonucleotide in which all phosphate groups of the oligonucleotide are replaced with phosphorothioate groups is particularly referred to as an S-oligonucleotide.
  • Example 3 Evaluation of antisense oligonucleotide splicing control (N exon skipping) effect in SCLC cells
  • STC1 human SCLC cells
  • test oligonucleotide 1.0 nmol
  • STC1 cells 1.0 ⁇ 10 6 cells
  • Sterile water was used as a control (mock).
  • SRRM4 which is aberrantly expressed in SCLC cells, is also expressed in endocrine prostate cancer (NEPC). Therefore, in order to further expand the indication, we evaluated a prostate cancer cell line (VCaP) that has been confirmed to express SRRM4.
  • test oligonucleotides were introduced by the lipofection method. After 48 hours, the cells were collected and analyzed according to standard methods. The sREST/REST expression ratio was determined in the same manner as in Example 3, and the splicing control (N exon skipping) effect of these antisense oligonucleotides was evaluated.
  • splicing control (N exon skipping) effect by oligonucleotides was confirmed not only in SCLC cells but also in VCaP cells.
  • Antisense oligonucleotides for which splicing control effects have been confirmed are referred to as splicing control oligonucleotides (SSO).
  • SSO splicing control oligonucleotide
  • AmNA_[+31/+48] 0.1, 1.0, 2.0 nmol
  • control AmNA_26 were introduced into H146 cells (1.0 ⁇ 10 6 cells) by electroporation, and the cells were harvested after 96 hours.
  • control AmNA_26 (0.1, 1.0 nmol) did not change the REST band in H146 cells (1.0 ⁇ 10 6 cells), but AmNA_[+31/+48] reduced sREST. and the intensity of the REST band increased (Fig. 3A).
  • Example 6 Concentration-dependent evaluation of REST splicing control of splicing control oligonucleotides (SSO)
  • SSO splicing control oligonucleotides
  • test oligonucleotides 2.5 nM, 5 nM, 10 nM
  • VCaP cells 1.0 ⁇ 10 6 cells
  • Example 7 Concentration-dependent evaluation of REST splicing control of splicing control oligonucleotide (SSO) 2
  • SSO splicing control oligonucleotide
  • test oligonucleotides 2.5 nM, 5 nM, 10 nM
  • VCaP cells 1.0 ⁇ 10 5 cells
  • Example 8 Concentration-dependent evaluation of REST splicing regulation of splicing control oligonucleotides (AmNA[+23/+40] and AmNA[+27/+44])
  • AmNA[+23/+40] and AmNA[+27/+44] which were shown to have high relative exon skipping activity in Example 7, the splicing control effect was verified by finely varying the concentrations.
  • the above SSO was added to prostate cancer cells (VCaP) at various concentrations, and the expression of REST and sREST in vitro was examined by RT-PCR to determine "% exclusion" and determine the splicing control (N exon skipping) effect. evaluated.
  • Test oligonucleotides (0.1 nM, 0.2 nM, 0.5 nM, 1 nM, 2 nM, 5 nM, 10 nM) were introduced into VCaP cells (1.0 x 10 5 cells) by the lipofection method. Analysis was performed based on Sterile water was used as a control (mock).
  • Example 9 EC50 analysis of splicing control oligonucleotides (AmNA[+23/+40] and AmNA[+27/+44])
  • AmNA_2340 AmNA[+27/+44]
  • AmNA_2744 AmNA[+27/+44]
  • Test oligonucleotides (0.1 nM, 0.2 nM, 0.5 nM, 1 nM, 2 nM, 5 nM, 10 nM) were introduced into VCaP cells (1.0 x 10 5 cells) by the lipofection method. Analysis was performed based on The 'relative exon skipping activity' at each concentration was calculated, plotted on a graph and curve regression was performed.
  • Example 10 Optimization study of splicing control oligonucleotide (SSO)) Based on AmNA[+23/+40] (AmNA_2340) and AmNA[+27/+44] (AmNA_2744), which were shown to have high relative exon skipping activity in Example 7, the sequence length was changed, AmNA We examined the optimization of SSO by exchanging the positions of natural nucleic acids and/or changing the number of introduced AmNAs.
  • Fig. 8 shows an overview of the sequence length of the SSO that was studied. In addition, SSO as a negative control was also examined. Four scrambled sequences were designed (Table 1) without changing the ATGC ratio of AmNA [+35/-2] and the rate of artificial nucleic acid introduction (for each base).
  • the above SSO was added to prostate cancer cells (VCaP) at a concentration of 1.0 nM, and the expression of REST and sREST in vitro was examined by RT-PCR to determine "% exclusion” and splicing control (N exon skipping) effect. evaluated. Sterilized water was used as a control (mock), and NC3 was used as a control (NC).
  • Example 11 REST splicing control evaluation of splicing control oligonucleotides (SSO) in 22Rv1 cells
  • AmNA[+23/+40] AmNA_2340
  • AmNA[+27/+44] AmNA_2744
  • AmNA[+21/+40] AmNA_2140
  • AmNA[ +23/+44] was verified whether it has similar splicing regulatory effects in different cell types.
  • the cells used in this example are 22Rv1, a human prostate cancer epithelial cell line derived from serially grown xenografts in mice.
  • the above SSO was added to prostate cancer cells (22Rv1) at a concentration of 1.0 nM, and the expression of REST and sREST in vitro was examined by RT-PCR to determine "% exclusion” and splicing control (N exon skipping) effect. evaluated.
  • 22Rv1 cells (1.0 ⁇ 10 5 cells) were transfected with the test oligonucleotide (1.0 nM) by lipofection, and 48 hours later, the cells were collected and analyzed according to standard methods. Sterilized water was used as a control (mock), and NC3 was used as a control (NC).
  • Example 12 Verification of the effect of splicing control oligonucleotide (SSO) on SRRM4 gene expression
  • SSO splicing control oligonucleotide
  • the SRRM4 gene has an RE1 sequence whose expression is controlled by REST, and high expression of REST suppresses SRRM4 gene expression.
  • the SRRM4 protein is a protein that controls the splicing of REST pre-mRNA, and it is known that the SRRM4 protein highly expresses sREST in SCLC (Fig. 15).
  • test oligonucleotide 1.0 nM
  • 22Rv1 cells 1.0 ⁇ 10 5 cells
  • a control no SSO added
  • the expression level of SRRM4 mRNA was measured. Sterilized water was used as a control (mock), and NC3 was used as a control (NC).
  • Example 13 Evaluation of antitumor effect in vivo
  • AmNA_2140 which has a high splicing effect and an SRRM4 mRNA expression-suppressing effect, exerts an antitumor effect in vivo.
  • Physiological saline was used as a control.
  • SCLC cells (cell culture) Cell lines were purchased from the American Type Culture Collection (ATCC). SCLC cells (H146, H209) were cultured as floating cells, and prostate cancer cells (VCaP, 22Rv1) as adherent cells.
  • the medium was RPMI-1640 (containing 4,500 mg/l glucose, L-glutamine, phenol red, HEPES, sodium pyruvate: Ref 187-02705: LOT ESH7003) and FBS (Gibco TM fetal bovine serum, qualified, Brazil: Ref 10270106: LOT 42F0288K) was added to 10%.
  • the inside of the incubator was 37°C and 5% CO 2 .
  • the cell lines used are as follows. SCLC cell lines: H146 (HTB-173), H209 (HTB-172), prostate cancer cell lines: VCaP (CRL-2876), 22Rv1 (CRL-2505)
  • SRRM4 expression analysis by qRT-PCR 0.5 ⁇ 10 6 cells were seeded in 6-well plates with 1750 ⁇ l of RPMI-1640 24 hours before transfection. 7.5 ⁇ l/well of Lipofectamine TM 3000 (manufactured by Thermo Fisher Scientific) and 250 ⁇ l of a complex solution of SRRM4-ASO and Lipofectamine TM 3000 Reagent prepared according to the package insert for other items were added to each well. For 24-well plates, 0.1 ⁇ 10 6 cells were seeded with 450 ⁇ l of RPMI-1640 24 hours before transfection.
  • Lipofectamine TM 3000 (manufactured by Thermo Fisher Scientific) was added to each well at 1.5 ⁇ L/well, and 50 ⁇ L of the complex solution obtained according to the package insert for other items was added to each well.
  • 5.0 ⁇ 10 4 cells were seeded in 100 ⁇ l of RPMI-1640 in 96-well plates 24 hours before transfection.
  • 0.3 ⁇ l/well of Lipofectamine TM 3000 manufactured by Thermo Fisher Scientific
  • 10 ⁇ l of a complex solution of SRRM4-ASO and Lipofectamine TM 3000 prepared according to the manufacturer's instructions were added to each well.
  • Total RNA extraction/cDNA synthesis Total RNA was extracted using RNeasy Plus Micro Kit (Qiagen) according to the package insert. Total RNA was quantified spectrophotometrically at 260 nm, adjusted with RNase-free water to 100 ng/16 ⁇ l, then 4 ⁇ l of SuperScript VILO (manufactured by Thermo Fischer Scientific) was added for reverse transcription (42°C, 60 minutes). ) was implemented.
  • Quantitative PCR was performed using StepOnePlus TM Real-Time PCR Systems (Thermo Fisher Scientific). A master mix was prepared using TaqMan TM Fast Advanced Master Mix (manufactured by Thermo Fisher Scientific). For SRRM4, each primer was prepared at 10 pmol in a 20 ⁇ l reaction. ⁇ -actin was prepared according to the package insert of TaqMan TM ⁇ -Actin Detection Reagents (manufactured by Thermo Fisher Scientific). 1/100 of the cDNA obtained from the reverse transcription reaction was analyzed by qRT-PCR. The reaction conditions were 40 cycles of 95° C. for 20 seconds, 95° C. for 3 seconds, and 60° C. for 30 seconds.
  • RNA expression analysis by RT-PCR method 1/10 of the cDNA obtained from the RT reaction was amplified using Hot Start Taq DNA polymerase (New England Biolabs). The reaction conditions were 95°C for 30 seconds, 95°C for 15 seconds, 58°C for 15 seconds, and 68°C for 30 seconds for 20 cycles for ⁇ -actin, 35 cycles for REST and sREST, followed by 2 cycles at 68°C. minutes. Each primer was prepared at 5 pmol each in a 25 ⁇ L reaction solution. PCR products were subjected to electrophoresis using 5% Mini-PROTEAN TBE Precast Gels (BIO RAD). As the electrophoresis buffer, 10 ⁇ Tris-Borate-EDTA Buffer (manufactured by Nacalai Tesque, Inc.) was diluted 0.5 times and used.
  • Mini-PROTEAN Tetra System (BIO RAD) was used as the electrophoresis apparatus.
  • the I Bright FL1500 Imaging System was used for gel photography and band quantification. A value representing the brightness of the band per unit area was quantified from the image using the function of the I Bright FL1500 Imaging System, and the REST value with respect to the sum of the sREST and REST values was used as an evaluation index. This value is shown in Figure 3B as %exclusion.
  • the primer sequences were as follows: REST For. Primer: 5'-gaacgcccatataaatgtgaa-3' (SEQ ID NO: 33); REST Rev.
  • Primer 5'-tttgaagttgcttctatctgctgt-3' (SEQ ID NO: 34); ⁇ -actin For.Primer: 5′-ggccgtcttccctccatcg-3′ (SEQ ID NO: 35); ⁇ -actin Rev. Primer: 5′-ccagttggtgacgatgccgtgc-3′ (SEQ ID NO: 36)
  • Example 9 The data obtained in Example 9 were introduced into graphing software GraphPad Prism, curve regression was performed by 4 Parameter Logistic ( 4PL ), and EC50 was calculated.
  • the present invention is useful, for example, in the production of pharmaceuticals for treating cancer.

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