Stable target-editing guide RNA incorporating chemically modified nucleic acids

Abstract

An oligonucleotide that introduces a site-specific edition to a target RNA, said oligonucleotide comprising a first oligonucleotide that specifies the target RNA, a second oligonucleotide that is linked to the 3'-side of the first oligonucleotide, a third oligonucleotide that is capable of forming a complementary pair together with the second oligonucleotide, and a first linkage part that links the second oligonucleotide and the third oligonucleotide. The first oligonucleotide comprises: a target-corresponding nucleotide residue that corresponds to an adenosine residue in the target RNA; an oligonucleotide consisting of 10-30 residues that is linked to the 5'- side of the target-corresponding nucleotide residue and has a base sequence complementary to the target RNA; and an oligonucleotide consisting of 3-6 residues that is linked to the 3'-side of the target-corresponding nucleotide residue and has a base sequence complementary to the target RNA. The second oligonucleotide consists of 5-8 residues and the third oligonucleotide consists of 5-8 residues. At least one residue selected from the target-corresponding nucleotide residue and counter regions, each consisting of one residue, on the 3'- and 5'-sides thereof is a nucleotide residue other than a natural ribonucleotide residue.

Description

[Technical Field]

[0001] The present invention relates to a stable target-editing guide RNA into which a chemically modified nucleic acid has been introduced. [Background technology]

[0002] With the development of genome editing technology, methods for controlling life phenomena by modifying the genetic information that serves as the blueprint for living organisms, i.e., the DNA information within cells, are beginning to be used as a disease treatment approach in the medical and drug discovery fields. Because DNA is a constant and unchanging molecule within cells, the effects of DNA modification remain permanently in the target cell or organism. On the other hand, RNA is a nucleic acid molecule that contains copied DNA information, and unlike DNA, it is a transient genetic information molecule that undergoes repeated synthesis and degradation. Therefore, modifying RNA information can produce temporary, non-permanent genetic information modifications in the target organism. In other words, while RNA modification technology is a genetic modification technology like DNA modification, its properties are significantly different.

[0003] As an RNA modification technique, for example, Patent Document 1 describes an oligonucleotide construct for site-specific editing of nucleotides in a target RNA sequence, which includes a targeting portion containing an antisense sequence complementary to a portion of the target RNA and a recruitment portion that can bind to and recruit an RNA editing entity present in cells and capable of editing nucleotides. Furthermore, for example, Patent Document 2 describes a site-specific RNA mutagenesis method in which double-strand-specific adenosine deaminase (ADAR) acts on a complex between a target RNA and a target-editing guide RNA. Furthermore, Non-Patent Document 1 describes the introduction of modified nucleic acids into antisense oligonucleotides that recruit ADAR. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2016 / 097212 [Patent Document 2] International Publication No. 2017 / 010556 [Non-patent literature]

[0005] [Non-Patent Document 1] Nature Biotechnology 37,133-138(2019) Summary of the Invention [Problem to be solved by the invention]

[0006] The oligonucleotide construct described in Patent Document 1 has a targeting moiety and a stem-loop structure with a specific repeat sequence as a recruitment moiety, and the recruitment moiety requires 16 or more oligonucleotides. The target-editing guide RNA described in Patent Document 2 requires an ADAR-binding region with a stem-loop structure consisting of a specific sequence of 40 or 49 residues in addition to an antisense region. The oligonucleotides used in the RNA modification techniques described in Patent Documents 1 and 2 all require an intramolecular double-stranded region of a certain length for binding to ADAR in addition to a region that forms a duplex with the target RNA, which inevitably tends to result in a long overall length for the oligonucleotide.

[0007] One aspect of the present invention aims to provide a target-editing guide RNA that adds a small number of nucleotides to a target recognition site, has excellent stability in vivo, and is capable of inducing site-specific editing in cells. [Means for solving the problem]

[0008] Specific means for solving the above problems are as follows, and the present invention includes the following aspects. (1-1) An oligonucleotide that induces site-specific editing of the target RNA, or a pharmaceutically acceptable salt thereof, comprising: a first oligonucleotide that identifies a target RNA; a second oligonucleotide linked to the 3' side of the first oligonucleotide; a third oligonucleotide that can form a complementary pair with the second oligonucleotide; and a first linking portion that links the second oligonucleotide and the third oligonucleotide, wherein the first oligonucleotide consists of a target-corresponding nucleotide residue corresponding to an adenosine residue in the target RNA; an oligonucleotide of 10 to 30 residues that is linked to the 5' side of the target-corresponding nucleotide residue and has a base sequence complementary to the target RNA; and an oligonucleotide of 3 to 6 residues that is linked to the 3' side of the target-corresponding nucleotide residue and has a base sequence complementary to the target RNA; the second oligonucleotide has 5 to 8 residues, and the third oligonucleotide has 5 to 8 residues; and at least one residue selected from the target-corresponding nucleotide residue and counter regions consisting of one residue each on the 3' side and the 5' side of the target-corresponding nucleotide residue is a nucleotide residue other than a naturally occurring ribonucleotide residue.

[0009] (1-2) The oligonucleotide according to (1-1), or a pharmaceutically acceptable salt thereof, wherein the first linking portion comprises at least one selected from the group consisting of an oligonucleotide of 4 or 5 residues and a polyalkyleneoxy group consisting of 1 to 8 alkyleneoxy units.

[0010] (1-3) The oligonucleotide according to (1-1) or (1-2), or a pharmaceutically acceptable salt thereof, comprising a second linking moiety containing an alkyleneoxy unit between the first oligonucleotide and the second oligonucleotide.

[0011] (1-4) The oligonucleotide according to any one of (1-1) to (1-3), or a pharmaceutically acceptable salt thereof, wherein the first oligonucleotide contains a phosphorothioate bond.

[0012] (1-5) The oligonucleotide according to any one of (1-1) to (1-4), wherein the first oligonucleotide comprises at least one modified nucleotide residue selected from the group consisting of 2'-O-alkylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, bridged nucleotide residues, and 2'-deoxyribonucleotides, or a pharmaceutically acceptable salt thereof.

[0013] (1-6) The oligonucleotide according to any one of (1-1) to (1-5), or a pharmaceutically acceptable salt thereof, wherein the counter region contains a phosphorothioate bond.

[0014] (1-7) The oligonucleotide according to any one of (1-1) to (1-6), or a pharmaceutically acceptable salt thereof, wherein the target-corresponding nucleotide residue comprises a phosphorothioate bond.

[0015] (1-8) The oligonucleotide according to any one of (1-1) to (1-7), or a pharmaceutically acceptable salt thereof, wherein at least one of the residues on the 3' and 5' sides of the target-corresponding nucleotide residue is at least one modified nucleotide residue selected from the group consisting of 2'-O-alkylribonucleotide residues and 2'-deoxy-2'-fluororibonucleotide residues.

[0016] (1-9) The oligonucleotide according to any one of (1-1) to (1-8), or a pharmaceutically acceptable salt thereof, wherein at least one of the second oligonucleotide and the third oligonucleotide contains at least one modified nucleotide residue selected from the group consisting of 2'-O-alkylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, and 2'-deoxyribonucleotide residues.

[0017] (1-10) The oligonucleotide according to any one of (1-1) to (1-9), or a pharmaceutically acceptable salt thereof, wherein the site-specific editing is caused by an enzymatic reaction catalyzed by adenosine deaminase.

[0018] (1-11) A medicine containing the oligonucleotide according to any one of (1-1) to (1-10) or a pharmaceutically acceptable salt thereof.

[0019] (1-12) A therapeutic agent for a genetic disease, comprising the oligonucleotide according to any one of (1-1) to (1-10) or a pharmaceutically acceptable salt thereof.

[0020] (1-13) A pharmaceutical composition comprising, as an active ingredient, the oligonucleotide according to any one of (1-1) to (1-10) or a pharmaceutically acceptable salt thereof.

[0021] (1-14) The pharmaceutical composition according to (1-13) for preventing or treating a genetic disease.

[0022] (1-15) The pharmaceutical composition according to (1-14), wherein the genetic disease is a disease that can be treated by converting an adenosine residue in the target RNA to an inosine residue.

[0023] (1-16) The pharmaceutical composition according to (1-14), wherein the hereditary disease is a hereditary disease caused by a mutation from a guanosine residue to an adenosine residue in a gene.

[0024] (1-17) Use of the oligonucleotide according to any one of (1-1) to (1-10) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of a disease.

[0025] (1-18) The oligonucleotide according to any one of (1-1) to (1-10), or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of a disease.

[0026] (2-1) An oligonucleotide that induces site-specific editing of the target RNA, or a pharmaceutically acceptable salt thereof, comprising: a first oligonucleotide that identifies a target RNA; a second oligonucleotide linked to the 3' side of the first oligonucleotide; a third oligonucleotide that can form a complementary pair with the second oligonucleotide; and a first linking portion that links the second oligonucleotide and the third oligonucleotide, wherein the first oligonucleotide consists of a target-corresponding nucleotide residue corresponding to an adenosine residue in the target RNA; an oligonucleotide of 10 to 30 residues that is linked to the 5' side of the target-corresponding nucleotide residue and has a base sequence complementary to the target RNA; and an oligonucleotide of 3 to 6 residues that is linked to the 3' side of the target-corresponding nucleotide residue and has a base sequence complementary to the target RNA; the second oligonucleotide has 5 to 8 residues, and the third oligonucleotide has 5 to 8 residues; and at least one residue selected from the target-corresponding nucleotide residue and counter regions consisting of one residue each on the 3' side and the 5' side of the target-corresponding nucleotide residue is a nucleotide residue other than a naturally occurring ribonucleotide residue.

[0027] (2-2) The oligonucleotide according to (2-1), or a pharmaceutically acceptable salt thereof, wherein the first linking portion comprises at least one selected from the group consisting of an oligonucleotide having 4 or 5 residues and a polyalkyleneoxy group having 1 to 8 alkyleneoxy units.

[0028] (2-3) The oligonucleotide according to (2-2), or a pharmaceutically acceptable salt thereof, wherein the first linking portion comprises an oligonucleotide having a base sequence selected from the group consisting of GCUAA, UUCG, UACG, UGCG, UCCG, GAAA, GUAA, GCAA, GGAA, GAGA, GUGA, GCGA, and GGGA.

[0029] (2-4) The oligonucleotide according to (2-1) or (2-3), or a pharmaceutically acceptable salt thereof, wherein the first linking moiety contains a polyethyleneoxy group consisting of 1 to 8 ethyleneoxy units.

[0030] (2-5) The oligonucleotide according to (2-4), or a pharmaceutically acceptable salt thereof, wherein the first linking moiety contains a hexaethyleneoxy group.

[0031] (2-6) The oligonucleotide according to any one of (2-1) to (2-5), which comprises a second linking moiety containing an alkyleneoxy unit between the first oligonucleotide and the second oligonucleotide, or a pharmaceutically acceptable salt thereof.

[0032] (2-7) The oligonucleotide according to (2-6), or a pharmaceutically acceptable salt thereof, wherein the second linking moiety contains an alkyleneoxy group having 3 to 6 carbon atoms.

[0033] (2-8) The oligonucleotide according to (2-6), or a pharmaceutically acceptable salt thereof, wherein the second linking moiety contains a hexaethyleneoxy group consisting of 6 ethyleneoxy units.

[0034] (2-9) The oligonucleotide according to any one of (2-1) to (2-8), or a pharmaceutically acceptable salt thereof, wherein the first oligonucleotide contains a phosphorothioate bond.

[0035] (2-10) The oligonucleotide according to any one of (2-1) to (2-9), wherein the first oligonucleotide comprises at least one modified nucleotide selected from the group consisting of 2'-O-alkylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, bridged nucleotide residues, and 2'-deoxyribonucleotides, or a pharmaceutically acceptable salt thereof.

[0036] (2-11) The oligonucleotide according to any one of (2-1) to (2-9), or a pharmaceutically acceptable salt thereof, wherein the first oligonucleotide comprises at least one modified nucleotide selected from the group consisting of 2'-O-methylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, bridged nucleotide residues, and 2'-deoxyribonucleotides.

[0037] (2-12) The oligonucleotide according to any one of (2-1) to (2-11), or a pharmaceutically acceptable salt thereof, wherein the counter region contains a phosphorothioate bond.

[0038] (2-13) The oligonucleotide according to any one of (2-1) to (2-12), or a pharmaceutically acceptable salt thereof, wherein the target-corresponding nucleotide residue comprises a phosphorothioate bond.

[0039] (2-14) The oligonucleotide according to any one of (2-1) to (2-13), or a pharmaceutically acceptable salt thereof, wherein at least one of the residues on the 3' and 5' sides of the target-corresponding nucleotide residue is at least one modified nucleotide residue selected from the group consisting of 2'-O-alkylribonucleotide residues and 2'-deoxy-2'-fluororibonucleotide residues.

[0040] (2-15) The oligonucleotide according to any one of (2-1) to (2-14), or a pharmaceutically acceptable salt thereof, wherein at least one of the second oligonucleotide and the third oligonucleotide contains at least one modified nucleotide residue selected from the group consisting of 2'-O-alkylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, and 2'-deoxyribonucleotide residues.

[0041] (2-16) The oligonucleotide according to (2-15), or a pharmaceutically acceptable salt thereof, wherein the second oligonucleotide and the third oligonucleotide are all composed of 2'-O-methylribonucleotides.

[0042] (2-17) The oligonucleotide according to (2-15) or (2-16), wherein the second oligonucleotide has a base sequence of GGGUGG and the third oligonucleotide has a base sequence of CCACCU, or a pharmaceutically acceptable salt thereof.

[0043] (2-18) a first oligonucleotide that identifies a target RNA; a second oligonucleotide linked to the 3' side of the first oligonucleotide; a third oligonucleotide capable of forming a complementary pair with the second oligonucleotide; an oligonucleotide that induces site-specific editing of the target RNA, or a pharmaceutically acceptable salt thereof; the oligonucleotide comprises: a first linking portion that links the second oligonucleotide and a third oligonucleotide; the first oligonucleotide comprises a target-corresponding nucleotide residue corresponding to an adenosine residue in the target RNA; an oligonucleotide of 10 to 30 residues that is linked to the 5' side of the target-corresponding nucleotide residue and has a base sequence complementary to the target RNA; and an oligonucleotide of 3 to 6 residues that is linked to the 3' side of the target-corresponding nucleotide residue and has a base sequence complementary to the target RNA; the second oligonucleotide has 5 to 8 residues, and the third oligonucleotide has 5 to 8 residues; the second oligonucleotide and the third oligonucleotide both consist of 2'-O-alkylribonucleotides; at least one residue selected from the target-corresponding nucleotide residue and counter regions consisting of one residue each on the 3' side and the 5' side of the target-corresponding nucleotide residue is a nucleotide residue other than a naturally occurring ribonucleotide residue; and all phosphodiester bonds are phosphorothioate bonds.

[0044] (2-19) The oligonucleotide according to (2-18), or a pharmaceutically acceptable salt thereof, wherein the first linking portion comprises at least one selected from the group consisting of an oligonucleotide having 4 or 5 residues and a polyalkyleneoxy group having 2 to 8 alkyleneoxy units.

[0045] (2-20) The oligonucleotide according to (2-19), or a pharmaceutically acceptable salt thereof, wherein the first linking portion comprises an oligonucleotide having a base sequence selected from the group consisting of GCUAA, UUCG, UACG, UGCG, UCCG, GAAA, GUAA, GCAA, GGAA, GAGA, GUGA, GCGA, and GGGA.

[0046] (2-21) The oligonucleotide according to (2-19), or a pharmaceutically acceptable salt thereof, wherein the first linking moiety contains a polyethyleneoxy group consisting of 1 to 8 ethyleneoxy units.

[0047] (2-22) The oligonucleotide according to (2-21), or a pharmaceutically acceptable salt thereof, wherein the first linking moiety contains a hexaethyleneoxy group.

[0048] (2-23) The oligonucleotide according to any one of (2-18) to (2-22), or a pharmaceutically acceptable salt thereof, comprising a second linking moiety containing an alkyleneoxy unit between the first oligonucleotide and the second oligonucleotide.

[0049] (2-24) The oligonucleotide according to (2-23), or a pharmaceutically acceptable salt thereof, wherein the second linking moiety contains an alkyleneoxy group having 3 to 6 carbon atoms.

[0050] (2-25) The oligonucleotide according to (2-23), or a pharmaceutically acceptable salt thereof, wherein the second linking moiety contains a hexaethyleneoxy group.

[0051] (2-26) The oligonucleotide according to any one of (2-18) to (2-25), or a pharmaceutically acceptable salt thereof, wherein the target-corresponding nucleotide residue is a cytidine residue, a uridine residue, an adenosine residue, or a derivative thereof.

[0052] (2-27) The oligonucleotide according to (2-26), or a pharmaceutically acceptable salt thereof, wherein the target-corresponding nucleotide residue is a cytidine residue or a derivative thereof.

[0053] (2-28) The oligonucleotide according to any one of (2-18) to (2-27), or a pharmaceutically acceptable salt thereof, wherein the first oligonucleotide comprises at least one modified nucleotide selected from the group consisting of 2'-O-alkylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, bridged nucleotide residues, and 2'-deoxyribonucleotides.

[0054] (2-29) The oligonucleotide according to any one of (2-18) to (2-27), or a pharmaceutically acceptable salt thereof, wherein the first oligonucleotide comprises at least one modified nucleotide selected from the group consisting of 2'-O-methylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, bridged nucleotide residues, and 2'-deoxyribonucleotides.

[0055] (2-30) An oligonucleotide according to any one of (2-18) to (2-29), or a pharmaceutically acceptable salt thereof, wherein at least one of the residues on the 3' and 5' sides of the target-corresponding nucleotide residue is at least one modified nucleotide residue selected from the group consisting of 2'-O-alkylribonucleotide residues and 2'-deoxy-2'-fluororibonucleotide residues.

[0056] (2-31) The oligonucleotide according to any one of (2-18) to (2-30), wherein the second oligonucleotide has a base sequence of GGGUGG and the third oligonucleotide has a base sequence of CCACCU, or a pharmaceutically acceptable salt thereof.

[0057] (2-32) The oligonucleotide according to any one of (2-1) to (2-31), or a pharmaceutically acceptable salt thereof, wherein the site-specific editing is caused by an enzymatic reaction catalyzed by adenosine deaminase.

[0058] (2-33) A medicine containing the oligonucleotide according to any one of (2-1) to (2-32) or a pharmaceutically acceptable salt thereof.

[0059] (2-34) A therapeutic agent for a genetic disease, comprising the oligonucleotide according to any one of (2-1) to (2-32) or a pharmaceutically acceptable salt thereof.

[0060] (2-35) A pharmaceutical composition comprising, as an active ingredient, the oligonucleotide according to any one of (2-1) to (2-32) or a pharmaceutically acceptable salt thereof.

[0061] (2-36) Use of the oligonucleotide according to any one of (2-1) to (2-32) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of a disease.

[0062] (2-37) The oligonucleotide according to any one of (2-1) to (2-32), or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of a disease.

[0063] (2-38) A method for preventing or treating a disease by administering a pharmacologically effective amount of the oligonucleotide according to any one of (2-1) to (2-32) or a pharmaceutically acceptable salt thereof to a warm-blooded animal.

[0064] (2-39) The method according to (2-38), wherein the warm-blooded animal is a human.

[0065] (3-1) An oligonucleotide or a pharmaceutically acceptable salt thereof that induces site-specific editing of the target RNA, comprising: a first oligonucleotide that identifies a target RNA; a second oligonucleotide linked to the 3' side of the first oligonucleotide; a third oligonucleotide that can form a complementary pair with the second oligonucleotide; and a first linking portion that links the second oligonucleotide and the third oligonucleotide, wherein the first oligonucleotide consists of a target-corresponding nucleotide residue corresponding to an adenosine residue in the target RNA; an oligonucleotide of 10 to 30 residues that is linked to the 5' side of the target-corresponding nucleotide residue and has a base sequence complementary to the target RNA; and an oligonucleotide of 3 to 6 residues that is linked to the 3' side of the target-corresponding nucleotide residue and has a base sequence complementary to the target RNA; the second oligonucleotide has 5 to 8 residues, and the third oligonucleotide has 5 to 8 residues; and at least one residue selected from the target-corresponding nucleotide residue and counter regions consisting of one residue each on the 3' side and the 5' side thereof is a nucleotide residue other than a naturally occurring ribonucleotide residue.

[0066] (3-2) The oligonucleotide according to (3-1), or a pharmaceutically acceptable salt thereof, wherein the first linking portion comprises at least one selected from the group consisting of an oligonucleotide having 4 or 5 residues and a polyalkyleneoxy group consisting of 1 to 8 alkyleneoxy units.

[0067] (3-3) The oligonucleotide according to (3-1) or (3-2), or a pharmaceutically acceptable salt thereof, comprising a second linking moiety containing an alkyleneoxy unit between the first oligonucleotide and the second oligonucleotide.

[0068] (3-4) The oligonucleotide according to any one of (3-1) to (3-3), or a pharmaceutically acceptable salt thereof, wherein the first oligonucleotide contains a phosphorothioate bond.

[0069] (3-5) The oligonucleotide according to any one of (3-1) to (3-4), or a pharmaceutically acceptable salt thereof, wherein the first oligonucleotide comprises at least one modified nucleotide residue selected from the group consisting of 2'-O-alkylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, bridged nucleotide residues, and 2'-deoxyribonucleotides.

[0070] (3-6) The oligonucleotide according to any one of (3-1) to (3-5), or a pharmaceutically acceptable salt thereof, wherein the counter region contains a phosphorothioate bond.

[0071] (3-7) The oligonucleotide according to any one of (3-1) to (3-6), or a pharmaceutically acceptable salt thereof, wherein the target-corresponding nucleotide residue comprises a phosphorothioate bond.

[0072] (3-8) The oligonucleotide according to any one of (3-1) to (3-7), or a pharmaceutically acceptable salt thereof, wherein at least one of the residues on the 3' and 5' sides of the target-corresponding nucleotide residue is at least one modified nucleotide residue selected from the group consisting of 2'-O-alkylribonucleotide residues and 2'-deoxy-2'-fluororibonucleotide residues.

[0073] (3-9) The oligonucleotide according to any one of (3-1) to (3-8), or a pharmaceutically acceptable salt thereof, wherein at least one of the second oligonucleotide and the third oligonucleotide contains at least one modified nucleotide residue selected from the group consisting of 2'-O-alkylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, and 2'-deoxyribonucleotide residues.

[0074] (3-10) An oligonucleotide according to any one of (3-1) to (3-9), wherein the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue has a base sequence in which 2'-deoxy-2'-fluoronucleotide residues and 2'-O-alkylribonucleotide residues are alternately linked, or a pharmaceutically acceptable salt thereof.

[0075] (3-11) The oligonucleotide according to (3-10), or a pharmaceutically acceptable salt thereof, wherein in the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue, the third nucleotide residue counting in the 5' direction from the target-corresponding nucleotide residue is a 2'-deoxy-2'-fluoronucleotide residue.

[0076] (3-12) An oligonucleotide according to any one of (3-1) to (3-9), wherein the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue has a base sequence in which bridged nucleotide residues and 2'-O-alkylribonucleotide residues are alternately linked, or a pharmaceutically acceptable salt thereof.

[0077] (3-13) The oligonucleotide according to (3-12), or a pharmaceutically acceptable salt thereof, wherein in the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue, the third nucleotide residue counting in the 5' direction from the target-corresponding nucleotide residue is a bridged nucleotide residue.

[0078] (3-14) An oligonucleotide according to any one of (3-1) to (3-9), wherein the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue has a base sequence in which 2'-deoxy-2'-fluoronucleotide residues and bridged nucleotide residues are alternately linked, or a pharmaceutically acceptable salt thereof.

[0079] (3-15) The oligonucleotide according to (3-14), or a pharmaceutically acceptable salt thereof, wherein in the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue, the third nucleotide residue counting in the 5' direction from the target-corresponding nucleotide is a 2'-deoxy-2'-fluoronucleotide residue.

[0080] (3-16) An oligonucleotide according to any one of (3-1) to (3-15), wherein in the first oligonucleotide, the oligonucleotide linked to the 3' side of the target-corresponding nucleotide residue has a base sequence in which a 2'-O-alkylribonucleotide residue is linked, or a pharmaceutically acceptable salt thereof.

[0081] (3-17) An oligonucleotide according to any one of (3-1) to (3-15), wherein in the first oligonucleotide, the oligonucleotide linked to the 3' side of the target-corresponding nucleotide residue has a base sequence in which 2'-O-alkylribonucleotide residues and bridged nucleotide residues are alternately linked, or a pharmaceutically acceptable salt thereof.

[0082] (3-18) The oligonucleotide according to any one of (3-1) to (3-17), wherein the oligonucleotide linked to the 3' side of the target-corresponding nucleotide residue in the first oligonucleotide consists of 4 to 6 residues, or a pharmaceutically acceptable salt thereof.

[0083] (3-19) The oligonucleotide according to any one of (3-1) to (3-18), wherein the second oligonucleotide and the third oligonucleotide have a base sequence in which 2'-O-alkylribonucleotide residues are linked, or a pharmaceutically acceptable salt thereof.

[0084] (3-20) The oligonucleotide according to any one of (3-1) to (3-19), or a pharmaceutically acceptable salt thereof, wherein each of the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide has nucleotide residues linked via phosphorothioate bonds.

[0085] (3-21) The oligonucleotide according to any one of (3-1) to (3-20), or a pharmaceutically acceptable salt thereof, wherein the site-specific editing is caused by an enzymatic reaction catalyzed by adenosine deaminase.

[0086] (3-22) A medicine containing the oligonucleotide according to any one of (3-1) to (3-21) or a pharmaceutically acceptable salt thereof.

[0087] (3-23) A therapeutic agent for a genetic disease, comprising the oligonucleotide according to any one of (3-1) to (3-21) or a pharmaceutically acceptable salt thereof.

[0088] (3-24) A pharmaceutical composition comprising, as an active ingredient, the oligonucleotide according to any one of (3-1) to (3-21) or a pharmaceutically acceptable salt thereof.

[0089] (3-25) The pharmaceutical composition according to (3-24) for preventing or treating a genetic disease.

[0090] (3-26) The pharmaceutical composition according to (3-25), wherein the genetic disease is a disease that can be treated by converting an adenosine residue in the target RNA to an inosine residue.

[0091] (3-27) The pharmaceutical composition according to (3-25), wherein the hereditary disease is a hereditary disease caused by a mutation of a guanosine residue to an adenosine residue in a gene.

[0092] (3-28) Use of the oligonucleotide according to any one of (3-1) to (3-21) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of a disease.

[0093] (3-29) The oligonucleotide according to any one of (3-1) to (3-21), or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of a disease.

[0094] (3-30) A method for preventing or treating a disease, which comprises administering a pharmacologically effective amount of the oligonucleotide according to any one of (3-1) to (3-29) or a pharmacologically acceptable salt thereof to a warm-blooded animal.

[0095] (3-31) The method according to (3-30), wherein the disease is a genetic disease.

[0096] (3-32) The method according to (3-31), wherein the genetic disease is a disease that can be treated by converting adenosine residues in the target RNA to inosine residues.

[0097] (3-33) The method according to (3-31), wherein the hereditary disease is a hereditary disease caused by a mutation from a guanosine residue to an adenosine residue in a gene.

[0098] (3-34) The method according to any one of (3-30) to (3-33), wherein the warm-blooded animal is a human.

[0099] (4-1) An oligonucleotide that induces site-specific editing of the target RNA, or a pharmaceutically acceptable salt thereof, comprising: a first oligonucleotide that identifies a target RNA; a second oligonucleotide linked to the 3' side of the first oligonucleotide; a third oligonucleotide that can form a complementary pair with the second oligonucleotide; and a first linking portion that links the second oligonucleotide and the third oligonucleotide, wherein the first oligonucleotide consists of a target-corresponding nucleotide residue corresponding to an adenosine residue in the target RNA; an oligonucleotide of 10 to 30 residues that is linked to the 5' side of the target-corresponding nucleotide residue and has a base sequence complementary to the target RNA; and an oligonucleotide of 3 to 6 residues that is linked to the 3' side of the target-corresponding nucleotide residue and has a base sequence complementary to the target RNA; the second oligonucleotide has 5 to 8 residues, and the third oligonucleotide has 5 to 8 residues; and at least one residue selected from the target-corresponding nucleotide residue and counter regions consisting of one residue each on the 3' side and the 5' side of the target-corresponding nucleotide residue is a nucleotide residue other than a naturally occurring ribonucleotide residue.

[0100] (4-2) The oligonucleotide according to (4-1), or a pharmaceutically acceptable salt thereof, wherein the first linking portion comprises at least one selected from the group consisting of an oligonucleotide of 4 or 5 residues and a polyalkyleneoxy group consisting of 1 to 8 alkyleneoxy units.

[0101] (4-3) The oligonucleotide according to (4-2), or a pharmaceutically acceptable salt thereof, wherein the first linking portion comprises an oligonucleotide having a base sequence selected from the group consisting of GCUAA, UUCG, UACG, UGCG, UCCG, GAAA, GUAA, GCAA, GGAA, GAGA, GUGA, GCGA, and GGGA.

[0102] (4-4) The oligonucleotide according to (4-1) or (4-3), or a pharmaceutically acceptable salt thereof, wherein the first linking moiety contains a polyethyleneoxy group consisting of 1 to 8 ethyleneoxy units.

[0103] (4-5) The oligonucleotide according to (4-4), or a pharmaceutically acceptable salt thereof, wherein the first linking moiety contains a hexaethyleneoxy group.

[0104] (4-6) The oligonucleotide according to any one of (4-1) to (4-5), which comprises a second linking moiety containing an alkyleneoxy unit between the first oligonucleotide and the second oligonucleotide, or a pharmaceutically acceptable salt thereof.

[0105] (4-7) The oligonucleotide according to (4-6), or a pharmaceutically acceptable salt thereof, wherein the second linking moiety contains an alkyleneoxy group having 3 to 6 carbon atoms.

[0106] (4-8) The oligonucleotide according to (4-6), or a pharmaceutically acceptable salt thereof, wherein the second linking moiety contains a hexaethyleneoxy group consisting of 6 ethyleneoxy units.

[0107] (4-9) The oligonucleotide according to any one of (4-1) to (4-8), wherein the first oligonucleotide contains a phosphorothioate bond, or a pharmaceutically acceptable salt thereof.

[0108] (4-10) The oligonucleotide according to any one of (4-1) to (4-9), wherein the first oligonucleotide comprises at least one modified nucleotide selected from the group consisting of 2'-O-alkylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, bridged nucleotide residues, and 2'-deoxyribonucleotides, or a pharmaceutically acceptable salt thereof.

[0109] (4-11) The oligonucleotide according to any one of (4-1) to (4-9), or a pharmaceutically acceptable salt thereof, wherein the first oligonucleotide comprises at least one modified nucleotide selected from the group consisting of 2'-O-methylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, bridged nucleotide residues, and 2'-deoxyribonucleotides.

[0110] (4-12) The oligonucleotide according to any one of (4-1) to (4-11), or a pharmaceutically acceptable salt thereof, wherein the counter region contains a phosphorothioate bond.

[0111] (4-13) The oligonucleotide according to any one of (4-1) to (4-12), or a pharmaceutically acceptable salt thereof, wherein the target-corresponding nucleotide residue comprises a phosphorothioate bond.

[0112] (4-14) The oligonucleotide according to any one of (4-1) to (4-13), or a pharmaceutically acceptable salt thereof, wherein at least one of the residues on the 3' and 5' sides of the target-corresponding nucleotide residue is at least one modified nucleotide residue selected from the group consisting of 2'-O-alkylribonucleotide residues and 2'-deoxy-2'-fluororibonucleotide residues.

[0113] (4-15) The oligonucleotide according to any one of (4-1) to (4-14), or a pharmaceutically acceptable salt thereof, wherein at least one of the second oligonucleotide and the third oligonucleotide contains at least one modified nucleotide residue selected from the group consisting of 2'-O-alkylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, and 2'-deoxyribonucleotide residues.

[0114] (4-16) The oligonucleotide according to (4-15), or a pharmaceutically acceptable salt thereof, wherein the second oligonucleotide and the third oligonucleotide are all composed of 2'-O-methylribonucleotides.

[0115] (4-17) The oligonucleotide according to (4-15) or (4-16), wherein the second oligonucleotide has a base sequence of GGGUGG and the third oligonucleotide has a base sequence of CCACCU, or a pharmaceutically acceptable salt thereof.

[0116] (4-18) a first oligonucleotide that identifies a target RNA; a second oligonucleotide linked to the 3' side of the first oligonucleotide; a third oligonucleotide capable of forming a complementary pair with the second oligonucleotide; an oligonucleotide that induces site-specific editing of the target RNA, comprising: a first linking portion linking the second oligonucleotide and a third oligonucleotide, wherein the first oligonucleotide comprises a target-corresponding nucleotide residue corresponding to an adenosine residue in the target RNA; an oligonucleotide of 10 to 30 residues linked to the 5' side of the target-corresponding nucleotide residue and having a base sequence complementary to the target RNA; and an oligonucleotide of 3 to 6 residues linked to the 3' side of the target-corresponding nucleotide residue and having a base sequence complementary to the target RNA; the second oligonucleotide has 5 to 8 residues, and the third oligonucleotide has 5 to 8 residues; the second oligonucleotide and the third oligonucleotide both consist of 2'-O-alkylribonucleotides; at least one residue selected from the target-corresponding nucleotide residue and counter regions consisting of one residue each on the 3' side and the 5' side of the target-corresponding nucleotide residue is a nucleotide residue other than a naturally occurring ribonucleotide residue; and all phosphodiester bond modifications are phosphorothioate bonds; or a pharmaceutically acceptable salt thereof.

[0117] (4-19) The oligonucleotide according to (4-18), or a pharmaceutically acceptable salt thereof, wherein the first linking portion comprises at least one selected from the group consisting of an oligonucleotide of 4 or 5 residues and a polyalkyleneoxy group consisting of 2 to 8 alkyleneoxy units.

[0118] (4-20) The oligonucleotide according to (4-19), or a pharmaceutically acceptable salt thereof, wherein the first linking portion comprises an oligonucleotide having a base sequence selected from the group consisting of GCUAA, UUCG, UACG, UGCG, UCCG, GAAA, GUAA, GCAA, GGAA, GAGA, GUGA, GCGA, and GGGA.

[0119] (4-21) The oligonucleotide according to (4-19), or a pharmaceutically acceptable salt thereof, wherein the first linking moiety contains a polyethyleneoxy group consisting of 1 to 8 ethyleneoxy units.

[0120] (4-22) The oligonucleotide according to (4-21), or a pharmaceutically acceptable salt thereof, wherein the first linking moiety contains a hexaethyleneoxy group.

[0121] (4-23) The oligonucleotide according to any one of (4-18) to (4-22), or a pharmaceutically acceptable salt thereof, comprising a second linking moiety containing an alkyleneoxy unit between the first oligonucleotide and the second oligonucleotide.

[0122] (4-24) The oligonucleotide according to (4-23), or a pharmaceutically acceptable salt thereof, wherein the second linking moiety contains an alkyleneoxy group having 3 to 6 carbon atoms.

[0123] (4-25) The oligonucleotide according to (4-23), or a pharmaceutically acceptable salt thereof, wherein the second linking moiety contains a hexaethyleneoxy group.

[0124] (4-26) The oligonucleotide according to any one of (4-18) to (4-25), or a pharmaceutically acceptable salt thereof, wherein the target-corresponding nucleotide residue is a cytidine residue, a uridine residue, an adenosine residue, or a derivative thereof.

[0125] (4-27) The oligonucleotide according to (4-26), or a pharmaceutically acceptable salt thereof, wherein the target-corresponding nucleotide residue is a cytidine residue or a derivative thereof.

[0126] (4-28) The oligonucleotide according to any one of (4-18) to (4-27), or a pharmaceutically acceptable salt thereof, wherein the first oligonucleotide comprises at least one modified nucleotide selected from the group consisting of 2'-O-alkylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, bridged nucleotide residues, and 2'-deoxyribonucleotides.

[0127] (4-29) The oligonucleotide according to any one of (4-18) to (4-27), or a pharmaceutically acceptable salt thereof, wherein the first oligonucleotide comprises at least one modified nucleotide selected from the group consisting of 2'-O-methylribonucleotide residues, 2'-deoxy-2'-fluororibonucleotide residues, bridged nucleotide residues, and 2'-deoxyribonucleotides.

[0128] (4-30) The oligonucleotide according to any one of (4-18) to (4-29), or a pharmaceutically acceptable salt thereof, wherein at least one of the residues on the 3' and 5' sides of the target-corresponding nucleotide residue is at least one modified nucleotide residue selected from the group consisting of 2'-O-alkylribonucleotide residues and 2'-deoxy-2'-fluororibonucleotide residues.

[0129] (4-31) The oligonucleotide according to any one of (4-18) to (4-30), wherein the second oligonucleotide has a base sequence of GGGUGG and the third oligonucleotide has a base sequence of CCACCU, or a pharmaceutically acceptable salt thereof.

[0130] (4-32) An oligonucleotide according to any one of (4-1) to (4-31), wherein the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue has a base sequence in which 2'-deoxy-2'-fluoronucleotide residues and 2'-O-alkylribonucleotide residues are alternately linked, or a pharmaceutically acceptable salt thereof.

[0131] (4-33) The oligonucleotide according to (4-32), or a pharmaceutically acceptable salt thereof, wherein in the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue, the third nucleotide residue counting in the 5' direction from the target-corresponding nucleotide residue is a 2'-deoxy-2'-fluoronucleotide residue.

[0132] (4-34) An oligonucleotide according to any one of (4-1) to (4-31), wherein the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue has a base sequence in which bridged nucleotide residues and 2'-O-alkylribonucleotide residues are alternately linked, or a pharmaceutically acceptable salt thereof.

[0133] (4-35) The oligonucleotide according to (4-34), or a pharmaceutically acceptable salt thereof, wherein in the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue, the third nucleotide residue counting in the 5' direction from the target-corresponding nucleotide residue is a bridged nucleotide residue.

[0134] (4-36) An oligonucleotide according to any one of (4-1) to (4-31), wherein the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue has a base sequence in which 2'-deoxy-2'-fluoronucleotide residues and bridged nucleotide residues are alternately linked, or a pharmaceutically acceptable salt thereof.

[0135] (4-37) The oligonucleotide according to (4-36), or a pharmaceutically acceptable salt thereof, wherein in the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue, the third nucleotide residue counting in the 5' direction from the target-corresponding nucleotide residue is a 2'-deoxy-2'-fluoronucleotide residue.

[0136] (4-38) An oligonucleotide according to any one of (4-1) to (4-37), wherein in the first oligonucleotide, the oligonucleotide linked to the 3' side of the target-corresponding nucleotide residue has a base sequence in which a 2'-O-alkylribonucleotide residue is linked, or a pharmaceutically acceptable salt thereof.

[0137] (4-39) An oligonucleotide according to any one of (4-1) to (4-37), wherein in the first oligonucleotide, the oligonucleotide linked to the 3' side of the target-corresponding nucleotide residue has a base sequence in which 2'-O-alkylribonucleotide residues and bridged nucleotide residues are alternately linked, or a pharmaceutically acceptable salt thereof.

[0138] (4-40) The oligonucleotide according to any one of (4-1) to (4-39), wherein the oligonucleotide linked to the 3' side of the target-corresponding nucleotide residue in the first oligonucleotide consists of 4 to 6 residues, or a pharmaceutically acceptable salt thereof.

[0139] (4-41) The oligonucleotide according to any one of (4-1) to (4-40), wherein the second oligonucleotide and the third oligonucleotide have a base sequence in which 2'-O-alkylribonucleotide residues are linked, or a pharmaceutically acceptable salt thereof.

[0140] (4-42) The oligonucleotide according to any one of (4-1) to (4-41), or a pharmaceutically acceptable salt thereof, wherein each of the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide is an oligonucleotide in which nucleotide residues are linked via phosphorothioate bonds.

[0141] (4-43) The oligonucleotide according to any one of (2-1) to (4-42), or a pharmaceutically acceptable salt thereof, wherein the site-specific editing is caused by an enzymatic reaction catalyzed by adenosine deaminase.

[0142] (4-45) A medicine containing the oligonucleotide according to any one of (4-1) to (4-44) or a pharmaceutically acceptable salt thereof.

[0143] (4-46) A therapeutic agent for a genetic disease, comprising the oligonucleotide according to any one of (4-1) to (4-44) or a pharmaceutically acceptable salt thereof.

[0144] (4-47) A pharmaceutical composition comprising, as an active ingredient, the oligonucleotide according to any one of (4-1) to (4-44) or a pharmaceutically acceptable salt thereof.

[0145] (4-48) Use of the oligonucleotide according to any one of (4-1) to (4-44) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of a disease.

[0146] (4-49) The oligonucleotide according to any one of (4-1) to (4-44), or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of a disease.

[0147] (4-50) A method for preventing or treating a disease by administering a pharmacologically effective amount of the oligonucleotide according to any one of (4-1) to (4-44) or a pharmaceutically acceptable salt thereof to a warm-blooded animal. (4-51) The method according to (4-50), wherein the disease is a genetic disease. (4-52) The method according to (4-51), wherein the genetic disease is a disease that can be treated by converting adenosine residues in the target RNA to inosine residues. (4-53) The method according to (4-51), wherein the genetic disease is a genetic disease caused by a mutation from a guanosine residue to an adenosine residue in a gene. (4-54) The method according to any one of (4-50) to (4-53), wherein the warm-blooded animal is a human.

[0148] (4-51) The method according to (4-50), wherein the warm-blooded animal is a human. [Effects of the Invention]

[0149] According to one aspect of the present invention, a target editing guide RNA can be provided that requires a small number of nucleotides to be added to a target recognition site, has excellent stability in vivo, and is capable of inducing site-specific editing. [Brief explanation of the drawings]

[0150] [Figure 1A] 1 is a chromatograph showing the rate of editing of target RNA by oligonucleotides. [Figure 1B] 1 is a graph showing the percentage of target RNA edited by oligonucleotides. [Figure 2] 1 is a graph showing the percentage of target RNA edited by oligonucleotides. [Figure 3A] 1 is a chromatograph showing the rate of editing of target RNA by oligonucleotides. [Figure 3B] 1 is a graph showing the percentage of target RNA edited by oligonucleotides. [Figure 4A] 1 is a chromatograph showing the rate of editing of target RNA by oligonucleotides. [Figure 4B] 1 is a graph showing the percentage of target RNA edited by oligonucleotides. [Figure 5A] 1 is a chromatograph showing the rate of editing of target RNA by oligonucleotides. [Figure 5B] 1 is a graph showing the percentage of target RNA edited by oligonucleotides. [Figure 6A] 1 is a graph showing the percentage of target RNA edited by oligonucleotides in cells. [Figure 6B] 1 is a graph showing changes in luminescence intensity due to RNA editing of oligonucleotides in cells. [Figure 6C] 1 is a graph showing the percentage of target RNA edited by oligonucleotides in cells. [Figure 6D]1 is a graph showing changes in luminescence intensity due to RNA editing of oligonucleotides in cells. [Figure 7A] 1 is a graph showing the percentage of target RNA edited by oligonucleotides in cells. [Figure 7B] 1 is a graph showing changes in luminescence intensity due to RNA editing of oligonucleotides in cells. [Figure 8A] 1 is a graph showing the percentage of target RNA edited by oligonucleotides in cells. [Figure 8B] 1 is a graph showing changes in luminescence intensity due to RNA editing of oligonucleotides in cells. [Figure 9A] 1 is a chromatograph showing the rate of editing of ATCB and GAPDH RNA by oligonucleotides. [Figure 9B] 1 is a graph showing the rate of editing of ATCB and GAPDH RNA by oligonucleotides. [Figure 10] 1 is a graph showing the percentage of target RNA edited by oligonucleotides in the presence of hADAR1. [Figure 11A] 1 is a graph showing the percentage of target RNA edited by oligonucleotides in the presence of ADAR2. [Figure 11B] 1 is a graph showing the percentage of target RNA edited by oligonucleotides in the presence of ADAR1. [Figure 12A] 1 is a graph showing the percentage of target RNA edited by oligonucleotides in the presence of ADAR2. [Figure 12B] 1 is a graph showing the percentage of target RNA edited by oligonucleotides in the presence of ADAR1. [Figure 13A] 1 is a graph showing the percentage of target RNA edited by oligonucleotides in cells. [Figure 13B] 1 is a graph showing changes in luminescence intensity due to RNA editing of oligonucleotides in cells. [Figure 14A]1 is a graph showing the percentage of target RNA edited by oligonucleotides in cells. [Figure 14B] 1 is a graph showing changes in luminescence intensity due to RNA editing of oligonucleotides in cells. [Figure 15A] 1 is a graph showing the percentage of target RNA edited by oligonucleotides in cells. [Figure 15B] 1 is a graph showing changes in luminescence intensity due to RNA editing of oligonucleotides in cells. [Figure 16A] 1 is a graph showing the percentage of target RNA edited by oligonucleotides in the presence of ADAR2. [Figure 16B] 1 is a graph showing the percentage of target RNA edited by oligonucleotides in the presence of ADAR1. [Figure 16C] 1 is a graph showing the percentage of target RNA edited by oligonucleotides in cells. [Figure 17A] 1 is a graph showing the percentage of target RNA edited by oligonucleotides in the presence of ADAR2. [Figure 17B] 1 is a graph showing the percentage of target RNA edited by oligonucleotides in the presence of ADAR1. [Figure 18] 1 is a graph showing the percentage of target RNA edited by oligonucleotides in cells. DETAILED DESCRIPTION OF THE INVENTION

[0151] As used herein, the term "step" refers not only to an independent step, but also to a step that cannot be clearly distinguished from other steps, as long as the intended purpose of the step is achieved. Furthermore, the content of each component in a composition refers to the total amount of each component in the composition, unless otherwise specified, when multiple substances corresponding to each component are present in the composition. Hereinafter, embodiments of the present invention will be described in detail. However, the embodiments shown below are examples of oligonucleotides for embodying the technical concept of the present invention, and the present invention is not limited to the oligonucleotides shown below.

[0152] An oligonucleotide that induces site-specific editing of a target RNA (hereinafter also referred to as a target editing guide RNA) includes a first oligonucleotide that identifies the target RNA, a second oligonucleotide linked to the 3' side of the first oligonucleotide, a third oligonucleotide that can form a complementary pair with the second oligonucleotide, and a first linking portion that links the second oligonucleotide to the third oligonucleotide. The first oligonucleotide consists of a target-corresponding nucleotide residue corresponding to an adenosine residue in the target RNA, an oligonucleotide of 10 to 30 residues that is linked to the 5' side of the target-corresponding nucleotide residue and has a base sequence complementary to the target RNA, and an oligonucleotide of 3 to 6 residues that is linked to the 3' side of the target-corresponding nucleotide residue and has a base sequence complementary to the target RNA. The second oligonucleotide has 5 to 8 residues, and the third oligonucleotide has 5 to 8 residues. At least one residue selected from the target-corresponding nucleotide residue and counter regions consisting of one residue each on the 3' and 5' sides thereof is a nucleotide residue other than a naturally occurring ribonucleotide residue.

[0153] The target editing guide RNA has a second oligonucleotide, a first linking portion, and a third oligonucleotide on the 3' side of the first oligonucleotide that specifies the target RNA, and has a nucleotide residue other than a natural ribonucleotide residue in the counter region, which results in excellent stability in cells and can induce excellent site-specific editing. This is thought to be because, for example, ADAR, which catalyzes target editing, recognizes a double-stranded region consisting of the target RNA and the first oligonucleotide, and its editing activity is enhanced by at least a portion of the second oligonucleotide, the first linking portion, and the third oligonucleotide, which do not form a double strand with the target RNA and can exist in a state free from the target RNA. That is, in the target editing guide RNA, the first oligonucleotide functions as a region complementary to the target RNA (antisense region; ASR), and at least a portion of the second oligonucleotide, the first linking portion, and the third oligonucleotide function as an editing-enhancing region, an ADAR-binding region (ADAR-recruiting region; ARR), etc.

[0154] Target-editing guide RNAs induce site-specific editing of target RNAs, for example, by recruiting ADARs, which catalyze target editing, to the target RNA. ADARs are enzymes that convert adenosine residues in double-stranded RNA to inosine residues through hydrolytic deamination and are widely present in mammalian cells. Because inosine residues are structurally similar to guanosine residues, they are translated as guanosine residues during translation of RNA information, resulting in editing of the RNA information. When such RNA editing occurs in a region encoding an amino acid, amino acid substitutions occur even in the absence of DNA mutations in the genome. In mammals, ADARs are known to be ADAR1, ADAR2, and ADAR3, which are distinct genes. Target-editing guide RNAs enhance the target-editing activity of at least ADAR1 or ADAR2.

[0155] When introduced into mammalian cells, the target-editing guide RNA can recruit ADARs present in the cells to the target RNA and induce site-specific editing of the target RNA.

[0156] The nucleotides constituting the target editing guide RNA may be composed of natural ribonucleotides (RNA), natural deoxyribonucleotides (DNA), RNA / DNA chimeras, or modified nucleotides, which are modifications of these. The nucleotides constituting the target editing guide RNA may be linked to each other by a phosphodiester bond bound to the hydroxyl group of the sugar moiety of the nucleoside. The phosphodiester bond may be formed using the 2'-, 3'-, or 5'-hydroxyl group of the sugar moiety. The nucleotides constituting the target editing guide RNA may form a natural 3'-5' phosphodiester bond. At least one of the nucleotides constituting the target editing guide RNA may be a modified nucleotide. Examples of modified nucleotides include those with modified sugar moieties, those with modified phosphodiester bonds, those with modified bases, and combinations thereof.

[0157] Examples of ribonucleotides modified in the sugar moiety include 2'-O-alkylated ribonucleotides of D-ribofuranose (e.g., 2'-O-methylated, 2'-O-aminoethylated, 2'-O-propylated, 2'-O-allylated, 2'-O-methoxyethylated, 2'-O-butylated, 2'-O-pentylated, 2'-O-propargylated, etc.); Bridged ribonucleotides in which the 2' and 4' positions of D-ribofuranose are bridged (e.g., 2'-O,4'-C-ethylenated, 2'-O,4'-C-methylenated, 2'-O,4'-C-propylenated, 2'-O,4'-C-tetramethylenated, 2'-O,4'-C-pentamethyleneated, 2'-S,4'-C-methylenated, 2'-deoxy-2'-C,4'-C-methyleneoxymethylenated D-ribofuranose, S-cEt (2',4'-constrained ethyl), AmNA, etc.); Examples include 3'-deoxy-3'-amino-2'-deoxy-D-ribofuranose; 3'-deoxy-3'-amino-2'-deoxy-2'-fluoro-D-ribofuranose; and 2'-deoxy-2'-fluoro-D-ribofuranose.

[0158] Examples of modified phosphodiester bonds include phosphorothioate bonds (including optically active forms derived from the asymmetry of the phosphorus atom), methylphosphonate bonds, methylthiophosphonate bonds, phosphorodithioate bonds, and phosphoramidate bonds.

[0159] Modifications of bases include halogenation; alkylations having 1 to 6 or 1 to 4 carbon atoms, such as methylation, ethylation, propylation, isopropylation, cyclopropylation, butylation, isobutylation, s-butylation, t-butylation, and cyclobutylation; hydroxylation; amination; deamination; demethylation; etc. Specific examples include 5-methylation, 5-fluorolation, 5-bromination, 5-iodination, N4-methylation, etc. of cytosine; 5-demethylation (uracil), 5-fluorolation, 5-bromination, 5-iodination, etc. of thymine; N6-methylation, 8-bromination, etc. of adenine; and N2-methylation, 8-bromination, etc. of guanine.

[0160] The first oligonucleotide contained in the target editing guide RNA identifies the target RNA. The target RNA is not particularly limited as long as it contains an adenosine residue to be edited, and may be either cellular RNA or viral RNA, and is usually a protein-encoding mRNA or pre-mRNA. The editing site in the target RNA may be located in an untranslated region, splice region, exon, intron, or any region that affects the stability, structure, or function of the RNA. The target RNA may also contain a mutation to be corrected or altered. Alternatively, the target RNA may be mutated so that its sequence encodes a phenotype different from the native form.

[0161] The target RNA is preferably RNA that encodes a protein, and specific examples of the encoded protein include serotonin receptors, glutamate receptors, membrane potential-dependent potassium channels, and phosphorylated proteins involved in signal transduction such as STAT3, NFkBIA, and MAPK14.

[0162] The target-editing guide RNA can be applied to, for example, the treatment of genetic diseases. The genetic disease can be, for example, a disease that can be treated by converting adenosine residues in the target RNA to inosine residues. The genetic disease can also be, for example, a genetic disease caused by a mutation from guanosine residues to adenosine residues in a gene. Examples of genetic disorders include cystic fibrosis, albinism, alpha-1 antitrypsin deficiency, Alzheimer's disease, amyotrophic lateral sclerosis, asthma, beta-thalassemia, CADASIL syndrome, Charcot-Marie-Tooth disease, chronic obstructive pulmonary disease (COPD), distal spinal muscular atrophy (DSMA), Duchenne / Becker muscular dystrophy, dystrophic epidermolysis bullosa, epidermylosis bullosa, Fabry disease, factor V Leiden-related disorders, familial adenomatous polyposis, galactosemia, Gaucher disease, glucose-6-phosphate dehydrogenase deficiency, hemophilia, hereditary hemacromatosis, Hunter syndrome, Huntington's disease, Hurler syndrome, inflammatory bowel disease (IBD), hereditary polyaggregation syndrome, Leba syndrome, and others. These include congenital amaurosis, Leschnihan syndrome, Lynch syndrome, Marfan syndrome, mucopolysaccharidoses, muscular dystrophies, myotonic dystrophy types I and II, neurofibromatosis, Niemann-Pick disease types A, B, and C, NY-eso1-associated pancreatic cancer, Parkinson's disease, Peutz-Jeghers syndrome, phenylketonuria, Pompe disease, primary ciliary disease, prothrombin mutation-related diseases such as the prothrombin G20210A mutation, pulmonary hypertension, retinitis pigmentosa, Sandhoff disease, severe combined immunodeficiency syndrome (SCID), sickle cell anemia, spinal muscular atrophy, Stargardt disease, Tay-Sachs disease, Usher syndrome, X-linked immunodeficiency, and various forms of cancer (e.g., BRCA1- and 2-associated breast cancer, ovarian cancer, etc.).

[0163] Specific examples of genetic disorders include type I citrullinemia (ASS1), type II citrullinemia (SLC25A13), hemophilia (Factor V Leiden), primary hyperoxaluria (AGXT), distal myopathy (GNE), cystic fibrosis (CFCFTR), homocystinuria (CBS), Hurler syndrome (IDUA (SLC26A1)), Alexander disease (GFAP), retinitis pigmentosa (EYS), phenylketonuria (PAH), hemochromatosis (HFE), and Gilbert syndrome (UGT1A1). The target gene names are in parentheses.

[0164] The first oligonucleotide comprises a target-corresponding nucleotide residue corresponding to the adenosine residue that is the editing target in the target RNA, a 5' oligonucleotide of 10 to 30 residues linked to the 5' side of the target-corresponding nucleotide residue and having a base sequence complementary to the corresponding base sequence in the target RNA, and a 3' oligonucleotide of 3 to 6 residues linked to the 3' side of the target-corresponding nucleotide residue and having a base sequence complementary to the corresponding base sequence in the target RNA. The oligonucleotides linked to the 5' and 3' sides of the target-corresponding nucleotide residue form a duplex with the target RNA, thereby identifying the target RNA and the editing target site in the target RNA. Hereinafter, for convenience, a region consisting of three residues, namely the target-corresponding nucleotide residue, one residue on the 3' side, and one residue on the 5' side, will be referred to as the counter region, and the parts of the first oligonucleotide other than the counter region will sometimes be referred to as the non-counter region.

[0165] The target-corresponding nucleotide residue is a nucleotide residue corresponding to the adenosine residue that is the editing target, such as a cytidine residue, a uridine residue, an adenosine residue, or a derivative thereof. The target-corresponding nucleotide residue is preferably a base that does not form a base pair with the adenosine residue that is the editing target, more preferably a cytidine residue or a derivative thereof, and even more preferably a cytidine residue.

[0166] The base sequence of the oligonucleotide linked to the 5' or 3' side of the target-corresponding nucleotide residue is a base sequence complementary to the corresponding base sequence of the target RNA. The number of residues of the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue is, for example, 10 or more, 12 or more, 14 or more, or 15 or more, and 26 or less, 20 or less, or 16 or less, from the viewpoint of specificity for the target RNA. The number of residues of the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue may be 10 or more and 20 or less, 13 or more and 20 or less, or 13 or more and 15 or less, from the viewpoint of specificity for the target RNA. Furthermore, the number of residues of the oligonucleotide linked to the 3' side of the target-corresponding nucleotide residue may be 3, 3, or 4, from the viewpoint of editing activity. The number of residues of the oligonucleotide linked to the 3' side of the target-corresponding nucleotide residue may be 4 to 6, 4 or 5, or 5 or 6, from the viewpoint of ADAR specificity.

[0167] In the target editing guide RNA, at least one, at least two, or all three residues selected from the counter region may be a nucleotide residue other than a naturally occurring ribonucleotide (RNA) residue; preferably, at least one, at least two, or all three residues are modified nucleotide residues. The modified nucleotide residue in the counter region may have at least one modified sugar moiety and / or phosphodiester bond, at least the sugar moiety may be modified, at least the phosphodiester bond may be modified, or the sugar moiety and / or phosphodiester bond may be modified. For example, the target-corresponding nucleotide residue may be a cytidine residue having a phosphorothioate bond. Furthermore, for example, one residue on the 5' or 3' side of the target-corresponding nucleotide residue may have at least one modified sugar moiety and / or phosphodiester bond, at least the sugar moiety may be modified, at least the phosphodiester bond may be modified, or the sugar moiety and / or phosphodiester bond may be modified. The modification of the sugar moiety in the counter region may be, for example, 2'-O-alkylation, 2'-deoxy-2'-fluorination, or the like.

[0168] At least one, at least three, or all of the residues in the oligonucleotide other than the counter region (non-counter region) of the first oligonucleotide may be modified in at least one sugar moiety or phosphodiester bond, or at least the sugar moiety, the phosphodiester bond, or both the sugar moiety and the phosphodiester bond. Modifications of sugar moieties in the non-counter region may be, for example, 2'-O-alkylation, 2'-deoxy-2'-fluoroation, cross-linking between the 2' and 4' positions, 2'-deoxylation (DNA modification), etc. When the first oligonucleotide contains multiple modified nucleotides, the multiple modified nucleotides may be arranged consecutively or spaced apart. Alternatively, the first oligonucleotide may be configured such that all nucleotide residues are linked by phosphorothioate bonds.

[0169] The oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue of the first oligonucleotide may have a base sequence in which two types of modified nucleotide residues selected from the group consisting of 2'-deoxy-2'-fluoronucleotide residues, 2'-O-alkylribonucleotide residues, and bridged nucleotide residues are alternately linked. That is, the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue may have a base sequence in which 2'-deoxy-2'-fluoronucleotide residues and 2'-O-alkylribonucleotide residues are alternately linked, may have a base sequence in which bridged nucleotide residues and 2'-O-alkylribonucleotide residues are alternately linked, or may have a base sequence in which 2'-deoxy-2'-fluoronucleotide residues and bridged nucleotide residues are alternately linked. As used herein, "having a base sequence in which two types of modified nucleotide residues are alternately linked" means that the target oligonucleotide (here, this refers to the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue of the first oligonucleotide) has a base sequence in which two types of modified nucleotide residues are partially alternately linked, or that the entire target oligonucleotide has a base sequence in which two types of modified nucleotide residues are alternately linked, and this term is used in the same sense in the following explanations of this specification.

[0170] In the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue of the first oligonucleotide, the third nucleotide residue, counting in the 5' direction from the target-corresponding nucleotide residue, may be a modified nucleotide residue selected from the group consisting of 2'-deoxy-2'-fluoronucleotide residues, 2'-O-alkylribonucleotide residues, and bridged nucleotide residues. Furthermore, in the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue, the third nucleotide residue, counting in the 5' direction from the target-corresponding nucleotide residue, may be a modified nucleotide residue selected from the group consisting of 2'-deoxy-2'-fluoronucleotide residues, 2'-O-alkylribonucleotide residues, and bridged nucleotide residues, and the fourth nucleotide residue may be a modified nucleotide residue different from the third nucleotide residue. Here, the third nucleotide residue, counting in the 5' direction from the target-corresponding nucleotide residue, does not include the target-corresponding nucleotide residue itself, and refers to the third nucleotide residue, counting in the 5' direction, from the nucleotide residue adjacent to the target-corresponding nucleotide residue in the 5' direction of the target-corresponding oligonucleotide residue.

[0171] In the first oligonucleotide, the oligonucleotide linked to the 3' side of the target-corresponding nucleotide residue may have a base sequence in which a 2'-O-alkylribonucleotide residue is linked. Furthermore, in the first oligonucleotide, the oligonucleotide linked to the 3' side of the target-corresponding nucleotide residue may have a base sequence in which two types selected from the group consisting of 2'-deoxy-2'-fluoronucleotide residues, 2'-O-alkylribonucleotide residues, and bridged nucleotide residues are alternately linked, or may have a base sequence in which 2'-deoxy-2'-fluoronucleotide residues and bridged nucleotide residues are alternately linked, or may have a base sequence in which 2'-O-alkylribonucleotide residues and bridged nucleotide residues are alternately linked. Furthermore, in the first oligonucleotide, from the viewpoint of ADAR specificity, the second nucleotide residue counting in the 3' direction from the target-corresponding nucleotide residue may be one type selected from the group consisting of 2'-deoxy-2'-fluoronucleotide residues, 2'-O-alkylribonucleotide residues, and bridged nucleotide residues, or may be a bridged nucleotide residue. Here, the second nucleotide residue counted in the 3' direction from the target-corresponding nucleotide residue does not include the target-corresponding nucleotide residue itself, but refers to the second nucleotide residue counted in the 3' direction, with the nucleotide residue adjacent to the target-corresponding oligonucleotide residue in the 3' direction being the first.

[0172] The second oligonucleotide and the third oligonucleotide each consist of 5 to 8 residues, 5 to 7 residues, or 6 residues, and have a base sequence capable of forming a complementary pair with each other. The number of residues of the second oligonucleotide and the third oligonucleotide may be the same or different. Examples of the base sequence of the second oligonucleotide include base sequences containing GGGUGG, GGGUG, GGUGG, GGGU, GGUG, GUGG, GGG, GGU, GUG, UGG, GG, GC, GA, GU, UC, UG, UA, UU, CG, CA, CU, CC, AG, AA, AC, AU, etc. The second oligonucleotide may have a base sequence containing at least one selected from the group consisting of a sequence consisting of two or three consecutive guanines (GG or GGG), a sequence consisting of consecutive uracil and guanine (UG), and a sequence consisting of consecutive guanine, uracil, and guanine (GUG), or may have a base sequence containing at least two or three selected from this group. The second oligonucleotide may have a sequence consisting of consecutive guanine, uracil, and guanine (GUG) or a sequence consisting of consecutive uracil, guanine, and guanine (UGG) bound to the first linking portion. The base sequence of the third oligonucleotide may be selected so as to form a complementary pair with the base sequence of the second oligonucleotide. Here, the phrase "the second oligonucleotide and the third oligonucleotide can form a complementary pair" means not only that all residues of the second oligonucleotide form Watson-Crick base pairs with all residues of the corresponding third oligonucleotide, but also that the second oligonucleotide and the third oligonucleotide contain one or two mismatch base pairs and one or two wobble base pairs. A mismatch base pair refers to a thermodynamically unstable base pair, and a wobble base pair refers to a thermodynamically stable non-Watson-Crick base pair, such as a GU base pair. A wobble base pair may be formed, for example, at the 3' end of the third oligonucleotide.

[0173] The second and third oligonucleotides may each have at least one modified sugar moiety and / or phosphodiester bond, at least one modified sugar moiety, at least one modified phosphodiester bond, or both modified sugar moieties and phosphodiester bonds in at least one, at least three, or all residues. Modifications of the sugar moiety in the second and third oligonucleotides may be, for example, 2'-O-alkylation, 2'-deoxy-2'-fluoro, 2'-deoxy (DNA) modification, etc. When the second or third oligonucleotide contains multiple modified nucleotides, the multiple modified nucleotides may be arranged consecutively or spaced apart.

[0174] The second and third oligonucleotides may have a base sequence in which 2'-O-alkylribonucleotide residues are linked, and the nucleotide residues in each of the second and third oligonucleotides may all be linked by phosphorothioate bonds.

[0175] The first linking portion may comprise at least one selected from the group consisting of an oligonucleotide having 4 or 5 residues and a polyalkyleneoxy group consisting of 1 to 8 alkyleneoxy units. The first linking portion is linked to the 3' side of the second oligonucleotide and the 5' side of the third oligonucleotide via a phosphodiester bond or a modified phosphodiester bond. That is, the first linking portion may be linked to the hydroxyl group of the sugar moiety at the 3' end of the second oligonucleotide and the hydroxyl group of the sugar moiety at the 5' end of the third oligonucleotide via a phosphodiester bond or a modified phosphodiester bond. This may allow the second and third oligonucleotides to form a complementary pair to form a stem structure, and the first linking portion to form a loop structure.

[0176] When the first linking portion comprises an oligonucleotide of 4 or 5 residues, specific examples of the base sequence include UNCG-fold types such as GCUAA; UUCG, UACG, UGCG, and UCCG; GNRA-fold types such as GAAA, GUAA, GCAA, GGAA, GAGA, GUGA, GCGA, and GGGA; RNYA-fold types such as GUCA, GCCA, GGCA, GACA, AUCA, ACCA, AGCA, AACA, GUUA, GCUA, GGUA, GAUA, AUUA, ACUA, AGUA, and AAUA; GGUG-fold types; CUUG-fold types; and AGNN-fold types such as AGUU, AGUC, AGUG, AGUA, AGCU, AGCC, AGCG, and AGCA. For details of these loop structures, see, for example, Biophys. J., 113, 257-267, 2017. The base sequence of the first linking portion may have the base sequence GCUAA, an UNCG-fold type base sequence, or a GNRA-fold type base sequence, or may have the base sequence UUCG.

[0177] When the first linking part contains an oligonucleotide of 4 or 5 residues, at least one or at least three of the nucleotide residues constituting the first linking part may be modified nucleotide residues. The modified nucleotide in the first linking part may have at least one of a sugar moiety and a phosphodiester bond modified, at least a sugar moiety modified, at least a phosphodiester bond modified, or both a sugar moiety and a phosphodiester bond modified.

[0178] When the first linking moiety contains a polyalkyleneoxy group, the number of carbon atoms in the alkyleneoxy unit may be, for example, 2 to 4, 2 to 3, or 2. That is, the alkyleneoxy unit may be an ethyleneoxy unit, a propyleneoxy unit, or a butyleneoxy unit. The number of alkyleneoxy units constituting the polyalkyleneoxy group may be, for example, 1 to 8, 5 to 7, or 6. The alkyleneoxy units constituting the polyalkyleneoxy group may be the same or different. Furthermore, the first linking moiety may contain a polyethyleneoxy group having 1 to 8 units or may contain a hexaethyleneoxy group. When the first linking moiety contains a polyalkyleneoxy group, the polyalkyleneoxy group may link the hydroxyl group of the sugar moiety at the 3' end of the second oligonucleotide to the hydroxyl group of the sugar moiety at the 5' end of the third oligonucleotide via a phosphodiester bond or a modified phosphodiester bond.

[0179] The first linking portion may be composed of only an oligonucleotide, may contain a nucleotide and an alkyleneoxy unit, or may contain only an alkyleneoxy unit. The target editing guide RNA can exhibit excellent target editing activity even when the first linking portion has a loop structure containing an alkyleneoxy unit.

[0180] The target editing guide RNA may have a second linking portion between the first oligonucleotide and the second oligonucleotide. That is, the first oligonucleotide and the second oligonucleotide may be linked by the second linking portion. The second linking portion may be composed of, for example, an alkyleneoxy unit. The alkylene portion of the alkyleneoxy unit may have 2 to 8 or 2 to 6 carbon atoms. The second linking portion may contain 1 to 8 or 1 to 6 alkyleneoxy units having 2 or 3 carbon atoms, or may contain a polyalkyleneoxy group containing 1 to 8 or 1 to 6 consecutive alkyleneoxy units having 2 or 3 carbon atoms, or may contain a hexaethyleneoxy group. The second linking portion may also contain an alkyleneoxy group having 3 to 6 carbon atoms. The polyalkyleneoxy group constituting the second linking portion may link the hydroxyl group of the sugar moiety at the 3' end of the first oligonucleotide to the hydroxyl group of the sugar moiety at the 5' end of the second oligonucleotide via a phosphodiester bond or a modified phosphodiester bond.

[0181] The target-editing guide RNA can be synthesized using a commercially available synthesizer (e.g., Perkin-Elmer's Model 392 using the phosphoramidite method) according to the method described in known literature (e.g., see Nucleic Acids Research, 12, 4539 (1984)). The phosphoramidite reagent used may be a commercially available reagent, or a reagent appropriately synthesized according to the method described in known literature. After coupling the phosphoramidite reagent, a phosphorothioate bond can be introduced by reacting with a reagent such as sulfur, tetraethylthiuram disulfide (TETD, Applied Biosystems), Beaucage reagent (Glen Research), or xanthan hydride (e.g., see Tetrahedron Letters, 32, 3005 (1991), J. Am. Chem. Soc., 112, 1253 (1990), PCT / WO98 / 54198).

[0182] The oligonucleotide (target editing guide RNA) may be used in the form of a pharmaceutically acceptable salt. A "pharmaceutically acceptable salt" refers to a salt of an oligonucleotide. Examples of such salts include metal salts such as alkali metal salts (e.g., sodium salt, potassium salt, and lithium salt), alkaline earth metal salts (e.g., calcium salt and magnesium salt), aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; inorganic salts (e.g., ammonium salt), t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, 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 ... Examples of suitable pharmaceutically acceptable salts include amine salts such as organic salts such as ethylammonium salts and tris(hydroxymethyl)aminomethane salts; hydrohalides such as hydrofluoride, hydrochloride, hydrobromide, and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonates such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate. Pharmaceutically acceptable salt forms of oligonucleotides are preferably alkali metal salts of oligonucleotides, more preferably sodium salts. These salts can be prepared by known methods.

[0183] Furthermore, oligonucleotides and pharmaceutically acceptable salts thereof may exist as solvates (for example, hydrates), and may be in the form of such solvates.

[0184] Oligonucleotides and pharmaceutically acceptable salts thereof may have optically active forms resulting from the asymmetry of the phosphorus atom, and the oligonucleotides of the present invention also include such optically active forms. Such optically active forms can be synthesized by known methods (e.g., Org. Lett., 114, 967 (2009), Bioorganic & Medicinal Chemistry Letters, 8, 2359 (1998), etc.).

[0185] When an oligonucleotide, a pharmaceutically acceptable salt or solvate thereof is used to treat a disease, it can be administered orally in the form of tablets, capsules, granules, powders or syrups, or parenterally in the form of injections, suppositories, patches or topical preparations, either by itself or mixed with an appropriate pharmaceutically acceptable excipient, diluent or the like.

[0186] These preparations contain excipients (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, bayleaf starch, alpha starch, and dextrin; cellulose derivatives such as crystalline cellulose; gum arabic; dextran; organic excipients such as pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminometasilicate; phosphates such as calcium hydrogen phosphate; carbonates such as calcium carbonate; and sulfates such as calcium sulfate), lubricants, and the like. lubricants (e.g., stearic acid; metal stearates such as calcium stearate and magnesium stearate; talc; colloidal silica; waxes such as beeswax and Gay wax; boric acid; adipic acid; sulfates such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL-leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; the above-mentioned starch derivatives, etc.), binders (e.g., hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, lydone, macrogol, compounds similar to the above-mentioned excipients, etc.), disintegrants (e.g., cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, and internally cross-linked sodium carboxymethyl cellulose; chemically modified starches and celluloses such as carboxymethyl starch, carboxymethyl starch sodium, and cross-linked polyvinylpyrrolidone, etc.), emulsifiers (e.g., colloidal clays such as bentonite and Veegum; metal hydroxides such as magnesium hydroxide and aluminum hydroxide; anionic surfactants such as sodium lauryl sulfate and calcium stearate; cationic surfactants such as benzalkonium chloride; nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, and sucrose fatty acid esters, etc.), stabilizers (parahydroxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal;It is produced by a well-known method using additives such as dehydroacetic acid, sorbic acid, flavoring agents (e.g., commonly used sweeteners, acidulants, flavoring agents), diluents, etc.;

[0187] Therapeutic agents containing oligonucleotides or pharmaceutically acceptable salts or solvates thereof may contain 0.1 to 250 μmoles / ml of oligonucleotide, preferably 1 to 50 μmoles / ml, and may also contain a predetermined amount of oligonucleotides or pharmaceutically acceptable salts or solvates thereof, 0.02 to 10% w / v of carbohydrates or polyhydric alcohols, and 0.01 to 0.4% w / v of a pharmaceutically acceptable surfactant.

[0188] The carbohydrate is preferably at least one of monosaccharides and disaccharides. Examples of these carbohydrates and polyhydric alcohols include glucose, galactose, mannose, lactose, maltose, mannitol, and sorbitol. These may be used alone or in combination.

[0189] Preferred examples of surfactants include polyoxyethylene sorbitan mono- to tri-esters, alkylphenyl polyoxyethylene, sodium taurocholate, sodium cholate, and polyhydric alcohol esters. Among these, polyoxyethylene sorbitan mono- to tri-esters are particularly preferred, and oleate, laurate, stearate, and palmitate are particularly preferred esters. These may be used alone or in combination.

[0190] Therapeutic agents containing oligonucleotides, pharmaceutically acceptable salts or solvates thereof may further preferably contain 0.03M to 0.09M of a pharmaceutically acceptable neutral salt, such as sodium chloride, potassium chloride and / or calcium chloride.

[0191] Therapeutic agents containing oligonucleotides, pharmaceutically acceptable salts, or solvates thereof may further preferably contain 0.002 to 0.05 M of a pharmaceutically acceptable buffer. Examples of preferred buffers include sodium citrate, sodium glycinate, sodium phosphate, and tris(hydroxymethyl)aminomethane. These buffers may be used alone or in combination.

[0192] Furthermore, the above-mentioned therapeutic agent may be supplied in a solution state. However, when storage for a certain period of time is required, it is usually preferable to freeze-dry the oligonucleotide in order to stabilize it and prevent a decrease in its therapeutic effect. In this case, it is sufficient to reconstitute it with a solvent (such as distilled water for injection) at the time of use, i.e., to prepare it in a liquid form for administration. Therefore, the therapeutic agent of the present invention also includes a freeze-dried form that is to be reconstituted with a solvent so that each component has a predetermined concentration range. In order to promote the solubility of the freeze-dried product, an amino acid such as albumin or glycine may be further contained.

[0193] When an oligonucleotide, a pharmaceutically acceptable salt, or a solvate thereof is administered to a human, it may be administered, for example, at a dose of about 0.01 mg / kg to 100 mg / kg (body weight), preferably 0.1 mg / kg to 20 mg / kg (body weight), per day for an adult, by subcutaneous injection, intravenous drip injection, or intravenous injection, either once or in several divided doses; however, the dose and number of administrations may be varied as appropriate depending on the type of disease, symptoms, age, administration method, etc.

[0194] Methods for treating diseases The method for treating a disease includes a step of administering the therapeutic agent to a subject. As used herein, "treatment" refers to any treatment administered to a disease, including, for example, cure, amelioration, inhibition of progression (prevention of aggravation), prevention, etc. (preferably, cure or prevention) of a disease. The subject of treatment may be a warm-blooded animal, including a human, or a non-human warm-blooded animal.

[0195] Method for site-specific editing of target RNA The method for site-specific editing of a target RNA includes a step of contacting the target RNA with a target-editing guide RNA, which is an oligonucleotide that induces site-specific editing of the target RNA, in the presence of adenosine deaminase. The target-editing guide RNA partially forms a double strand with the target RNA and recruits adenosine deaminase, thereby converting adenosine residues contained in the target RNA to inosine residues in a site-specific manner. The method for site-specific editing of a target RNA may further include a step of preparing the target-editing guide RNA.

[0196] The site-specific editing method for target RNA can be carried out, for example, by introducing the above-mentioned target-editing guide RNA into a eukaryotic cell having the target RNA. The method for introducing the target-editing guide RNA into a eukaryotic cell can be appropriately selected and applied from various techniques used in nucleic acid medicines. The site-specific editing method for target RNA can be carried out in vitro or in vivo.

[0197] In other aspects, the present invention also includes the use of a target-edited guide RNA in the manufacture of a pharmaceutical composition for use in treating a disease (e.g., a genetic disease), the use of a target-edited guide RNA in the treatment of a disease (e.g., a genetic disease), and a target-edited guide RNA for use in the treatment of a disease (e.g., a genetic disease). [Example]

[0198] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0199] (Reference example 1) The oligonucleotide (hereinafter referred to as del03_01) consisting entirely of natural RNA residues and having the sequence shown in Table 1 was synthesized using the phosphoramidite method (see, for example, Nucleic Acids Research, 12, 4539 (1984), Nature Communications 6, Article number: 6317 (2015)). The resulting compound was identified by negative ion ESI mass spectrometry (measured value: 11194.40).

[0200] In Table 1, the underlined part corresponds to the first oligonucleotide (ASR), and the target RNA is Rluc_sRNA, which will be described later. The oligonucleotide of Reference Example 1 is thought to be able to take, for example, the following stem-loop structure.

[0201] [Table 1]

[0202] [ka]

[0203] Examples 1 to 50 The oligonucleotide compounds of Examples 1 to 46 were obtained by introducing modified nucleotides as shown below based on the sequence of the oligonucleotide of Reference Example 1. The oligonucleotide compounds of Examples 47 to 50 have a first oligonucleotide (ASR) with the sequence shown below and were obtained by introducing modified nucleotides as shown below. These oligonucleotide compounds were synthesized by the phosphoramidite method as in Reference Example 1. In the sequences, DMT Hexaethylene Glycol phosphoramidite (ChemGene, catalog number: CLP-9765) was used to synthesize the portion "18." DMT-Triethoxy-glycol phosphoramidite (ChemGene, catalog number: CLP-1113) was used to synthesize the portion "9." DMT-hexane-Diol phosphoramidite (ChemGene, catalog number: CLP-1120) was used to synthesize the portion "6." DMT-propane-Diol phosphoramidite (ChemGene, catalog number: CLP-9908) was used to synthesize the portion "3." The "2'-O-methylnucleoside" portion was synthesized using the phosphoramidite described in Nucleic Acids Research 17, 3373 (1989). The "DNA" portion was synthesized using the phosphoramidite described in Nucleic Acids Research 11, 4539 (1984). The "2'-O,4'-C-methylenenucleoside" portion was synthesized using the phosphoramidite described in International Publication No. 99 / 14226. The "2'-deoxy-2'-fluoronucleoside" portion was synthesized using the phosphoramidite described in J. Med. Chem., 36, 831 (1993).

[0204] [Table 2] [Table 3] [Table 4]

[0205] The "molecular weight" in the table indicates the actual value measured by negative ion electrospray ionization mass spectrometry. In the "sequence" section, uppercase letters indicate RNA, lowercase letters indicate DNA, N(M) indicates 2'-O-methylation of D-ribofuranose, N(F) indicates 2'-deoxy-2'-fluorination of D-ribofuranose, N(L) indicates 2'-O,4'-C-methylenation of D-ribofuranose, and N(E) indicates 2'-O,4'-C-ethylenation of D-ribofuranose. "3" indicates -O(CH2)3O-, "6" indicates -O(CH2)6O-, "9" indicates -O(CH2CHO)3-, and "18" indicates a linker represented by -O(CH2CHO)6-. "^" indicates a -P(=S)(OH)- bond between nucleoside units. Unless otherwise specified, the bonds between nucleoside units or between nucleoside units and linkers are shown as -P(=O)(OH)-. The 5' and 3' ends of the oligonucleotide are hydroxyl groups, with hydrogen atoms bonded to oxygen atoms in the chemical formula. The structure of a nucleoside unit is shown below. The dashed lines in the chemical formula indicate the bond sites.

[0206] [ka]

[0207] [ka]

[0208] [ka]

[0209] [ka]

[0210] [ka]

[0211] [ka]

[0212] [ka]

[0213] (Reference example 2) The oligonucleotide compounds whose base sequences and chemical modification states are shown in Table 5 were synthesized by the phosphoramidite method in the same manner as in Reference Example 1. The symbols in the table are the same as above.

[0214] [Table 5]

[0215] (Test Example 1) Evaluation of editing induction ability for Rluc_sRNA (A) Synthesis of Rluc_sRNA psiCHECK TM Rluc_WT RNA (SEQ ID NO: 7) was prepared by standard transcription from the -2 Vector (Promega) to a concentration of 0.2 nM. Recombinant hADAR2 (synthesized and purified using a yeast expression system; Macbeth, M.R. and Bass, B.L. Methods Enzymol. 424, 319-331 (2007); Fukuda, M. et al. Sci. Rep. srep41478 (2017)) was added to a final concentration of 1 μM. The in vitro editing reaction was performed by incubating the mixture at 37°C for 2 hours in editing reaction buffer (20 mM HEPES-KOH (pH 7.5), 2 mM MgCl2, 100 mM NaCl, 0.5 mM DTT, 0.01% Triton X-100, 5% glycerol).

[0216] After the editing reaction, the Rluc_WT RNA was purified by phenol / chloroform extraction and ethanol precipitation. Next, cDNA was synthesized using Primescript Reverse Transcriptase II (TaKaRa) with the Rluc_WT_BamR01 primer (SEQ ID NO: 8). The cDNA was then amplified by PCR (30 cycles: denaturation at 98°C for 10 seconds, annealing at 55°C for 15 seconds, extension at 68°C for 60 seconds) using the Rluc_WT_EcoF01 primer (SEQ ID NO: 9) and the Rluc_WT_BamR01 primer (SEQ ID NO: 8) with PrimeStar GXL DNA Polymerase (TaKaRa). The resulting cDNA was then cloned into the pUC19 plasmid as insert DNA as follows. The insert DNA and pUC19 plasmid were digested with EcoRI (TaKaRa) and BamHI (TaKaRa) at 37°C for 1 hour, followed by phenol / chloroform extraction and ethanol precipitation to purify each DNA. The pUC19 plasmid and insert DNA were mixed at a molar ratio of 1:3 after restriction enzyme digestion, and then ligated with the DNA Ligation Kit.<Mighty Mix> Ligation reactions were performed using a PCR product (TaKaRa). The resulting ligation sample was transformed into DH5α and cultured overnight at 37°C on an LB agar plate. Plasmids were then extracted using a QIAprep Spin Miniprep Kit (QIAGEN). The resulting plasmid DNA was sequenced to obtain a sequence in which A122 of Rluc_WT was mutated to G (Rluc_K41R) and a plasmid DNA in which Rluc_K41R was cloned (pUC19-Rluc_K41R).

[0217] Using pUC19-Rluc_K41R as a template, first PCR (30 cycles; denaturation: 98°C, 10 seconds; annealing: 55°C, 15 seconds; extension: 68°C, 40 seconds) was performed using PrimeStar GXL DNA Polymerase (TaKaRa) with the Rluc_NheF01 primer (sequence number 10) and RL_W104X_RV primer (sequence number 11) for the 5' fragment and the Rluc_XhoR01 primer (sequence number 12) and RL_W104X_FW primer (sequence number 13) for the 3' fragment. Next, each PCR product was diluted 100-fold, and a second PCR (30 cycles: denaturation at 98°C for 10 seconds, annealing at 55°C for 15 seconds, extension at 68°C for 60 seconds) was performed using the Rluc_NheF01 and Rluc_XhoR01 primers with PrimeStar GXL DNA Polymerase (TaKaRa). The product was then purified by phenol / chloroform extraction and ethanol precipitation to obtain DNA (Rluc_K41R_W104X) (SEQ ID NO: 16) having a sequence in which G311 of Rluc_K41R was mutated to A.

[0218] The obtained DNA (Rluc_K41R_W104X) and psiCHECK TM The -2 Vector (Promega) was digested with NheI (TaKaRa) and XhoI (TaKaRa) at 37°C for 1 hour, and then the DNA was purified by phenol / chloroform extraction and ethanol precipitation. TM -2 Vector was mixed at a molar ratio of 3:1 and ligated with the DNA Ligation Kit<Mighty Mix> Ligation reactions were performed using a ligation kit (TaKaRa). The resulting ligation sample was transformed into DH5α and cultured overnight at 37°C on an LB agar plate. Selected colonies were cultured overnight at 37°C in LB liquid medium, and the plasmid was extracted using a QIAprep Spin Miniprep Kit (QIAGEN). The sequence of the resulting plasmid was then confirmed by nucleotide sequence analysis.

[0219] Using the sequence-confirmed plasmid as a template, template DNA for in vitro transcription was amplified by PCR (30 cycles: denaturation at 98°C for 10 seconds, annealing at 55°C for 15 seconds, extension at 68°C for 20 seconds) using the T7_Rluc_sRNA_F01 primer (SEQ ID NO: 14) and the Rluc_sRNA_R01 primer (SEQ ID NO: 15) and PrimeStar GXL DNA Polymerase (TaKaRa). The amplified DNA was then purified by phenol / chloroform extraction and ethanol precipitation. In vitro transcription was performed using the AmpliScribe T7 Kit (Epicentre Biotechnologies) to obtain Rluc_sRNA (SEQ ID NO: 17). The synthesized Rluc_sRNA was excised and purified using a 5% polyacrylamide gel containing 8 M urea and used for subsequent experiments.

[0220] The sequences of Rluc_WT_RNA, Rluc_K41R_W104X, and Rluc_sRNA, as well as the sequences of the primers used above, are shown below. Note that the underlined residues in the table indicate the adenosine residues in the editing target.

[0221] [Table 6]

[0222] [Table 7]

[0223] [Table 8]

[0224] (B) Annealing reaction 0.3 μM Rluc_sRNA and 0.9 μM of the compounds of the Examples and Reference Examples (hereinafter sometimes referred to as gRNA) were heated at 80°C for 3 minutes in an annealing buffer (10 mM Tri-HCl (pH 7.6), 150 mM NaCl) and then cooled to 25°C over 15 minutes.

[0225] (C) In vitro editing reaction Assuming that Rluc_sRNA completely complexed with gRNA through the annealing reaction, the following Rluc_sRNA-gRNA complex concentrations were calculated: The editing reaction was performed by adding 12.5 nM or 6.25 nM recombinant hADAR2 to a 5 nM Rluc_sRNA-gRNA complex in editing reaction buffer (20 mM HEPES-KOH (pH 7.5), 2 mM MgCl2, 100 mM NaCl, 0.5 mM DTT, 0.01% Triton X-100, 5% glycerol) and incubating at 37°C for 1 hour.

[0226] (D) Editing and Analysis Method After the editing reaction, Rluc_sRNA was purified by phenol / chloroform extraction and ethanol precipitation. cDNA was synthesized using Primescript Reverse Transcriptase II (TaKaRa) with the Rluc_sRNA_R01 primer (SEQ ID NO: 18). The cDNA was then amplified by PCR (denaturation: 98°C, 10 s, annealing: 55°C, 15 s, extension: 68°C, 20 s) with the Rluc_sRNA_F01 primer (SEQ ID NO: 18) and the Rluc_sRNA_R01 primer (SEQ ID NO: 15) and PrimeStar GXL DNA Polymerase (TaKaRa). The resulting cDNA was sequenced using the Rluc_sRNA_F01 primer (SEQ ID NO: 18) and the Big Dye Terminator v3.1 Cycle Sequence Kit, and analyzed on an Applied Biosystem 3500 Genetic Analyzer (Thermo Fisher Scientific). The editing rate (%) was calculated from the peak height ratio (G / (G+A)) of the target site (A311) from the chromatogram obtained by sequencing.

[0227] [Table 9]

[0228] (E) Edit analysis results The editing rates (%) when using the compound of Reference Example 1 and the compounds of the Examples are shown in Figures 1A and 1B. The compound of Reference Example 1 showed an editing rate of over 90%. Even when modified nucleic acids were introduced, as in the compounds of the Examples (del03_02 to del03_07), they showed editing induction activity similar to that of the compound of Reference Example 1. In addition, a compound (del03_08) in which a phosphorothioate bond was introduced into the C paired with the target editing site, and a compound (del03_09) in which the loop in the ADAR induction region was replaced with a linker consisting of hexaethylenic glycol, also showed editing induction activity at the same level as the compound of Reference Example 1.

[0229] As shown in Figure 2, even when modified nucleic acids were introduced as in the compounds of the examples (del03_10 to del03_21), editing-inducing activity similar to that of the compound of Reference Example 1 was exhibited. Furthermore, the compound of Reference Example 2 (del03_22) has a Z-DNA stem-loop sequence and was created by adapting the gRNA design method described in WO2016 / 097212 to the Rluc_sRNA sequence. It can be seen that del03_22 has weaker editing-inducing activity than the compound of the Example (del03_21), which has the same Z-DNA stem sequence. On the other hand, even when modified nucleic acids were introduced, as in the Example compounds (del03_Am1 to del03_Am7), they showed editing-inducing activity comparable to that of the compound of Reference Example 1.

[0230] As shown in Figures 3A and 3B, even when modified nucleic acids were introduced, such as in the compounds of the examples (del03_23 to del03_29), the compounds showed editing-inducing activity similar to that of the compound of Reference Example 1.

[0231] As shown in Figures 4A and 4B, even when modified nucleic acids were introduced, such as in the compounds of the examples (del03_26, del03_30 to del03_32), the compounds showed editing-inducing activity similar to that of the compound of Reference Example 1.

[0232] As shown in Figures 5A and 5B, even when modified nucleic acids were introduced, such as in the compounds of the examples (del03_26, del03_34 to del03_41), editing-inducing activity similar to that of the compound of Reference Example 1 was exhibited.

[0233] (Test Example 2) Evaluation of editing induction ability in cultured cells (A) Cell culture HeLa cells were plated in a 24-well plate at 5.0 × 10 5 The cells were subcultured at 100 cells / well and cultured for 48 hours. 50 ng of psiCHECK2 was transfected using Lipofectamine 3000 (Thermo). TM The cells were transfected with _Rluc_K41R_W104X, 350 ng of pcDNA3.1(-)Hygro_ADAR2, and 10 nM of gRNA, a compound of the Examples or Reference Examples. As a control, 100 ng of a gRNA-expressing plasmid (described in Reference Example 2 of Japanese Patent Application No. 2017-234341) was transfected and used.

[0234] (B) Editing and analysis method Total RNA was extracted from cells cultured in 24-well plates using Sepasol RNA I Super G (Nacalai). After DNase treatment using Recombinant DNase I (TaKaRa), the RNA was purified by phenol / chloroform extraction and ethanol precipitation. cDNA was amplified by reverse transcription using PrimeScript II Reverse Transcriptase (TaKaRa), 0.5 μg of total RNA, and 0.25 μM Oligo(dT)17 (SEQ ID NO: 19). Primary PCR was performed using PrimeStar GXL DNA polymerase (TaKaRa), Rluc_F01 primer (SEQ ID NO: 20), and 3'-Adp primer (SEQ ID NO: 21) for 30 cycles (denaturation at 98°C for 10 seconds, annealing at 55°C for 15 seconds, and extension at 68°C for 60 seconds). The Rluc fragment was amplified by a second PCR using PrimeStar GXL DNA polymerase (TaKaRa) and the Rluc_F01 primer (SEQ ID NO: 20) and Rluc_R01 primer (SEQ ID NO: 22) for 30 cycles (denaturation at 98°C for 10 seconds, annealing at 55°C for 15 seconds, and extension at 68°C for 60 seconds). The sequencing reaction was performed using the Big Dye Terminator v3.1 Cycle Sequence Kit (Thermo Fisher Scientific) and 0.165 μM Rluc_sRNA_F01 primer (SEQ ID NO: 18). The editing percentage (%) was calculated from the peak height ratio (G / (G+A)) of the target site (A311) on the chromatogram obtained by sequencing.

[0235] [Table 10]

[0236] (C) Luciferase reporter assay method The Dual-Luciferase Reporter Assay System (Promega) was used. Cells were cultured in a 24-well plate and 100 μL of Passive Lysis Buffer (Promega) was used to obtain a cell extract. 20 μL of the obtained cell extract was added to 100 μL of LARII, and after 60 seconds, the extract was lysed using GloMax (R) The luminescence intensity of Firefly luciferase (Fluc) was measured using a 20 / 20 Luminometer (Promega). Immediately thereafter, 100 μL of Stop&Glo Reagent was added, and the luminescence intensity of Renilla luciferase (Rluc) was measured 60 seconds later. The luminescence intensity was normalized by Fluc.

[0237] (D) Results of editing analysis and luciferase reporter assay. The editing rate (%) when using the compounds of the example (del03_24 to del03_26) is shown in Figure 6A. When transfected with a plasmid, the editing rate was approximately 10 to 20%. When transfected with modified nucleic acids with an increased number of phosphorothioate bonds, such as the compounds of the example (del03_25 and del03_26), editing induction activity was observed, and the rate was higher than when transfected with a plasmid.

[0238] The results of the luciferase reporter assay using the example compounds (del03_24 to del03_26) are shown in Figure 6B. When the example compounds (del03_25 and del03_26) were used, high luciferase activity was observed, which was equal to or higher than that observed when the plasmid was transfected.

[0239] Figure 6C shows the editing rate (%) when using the compound of Reference Example 1 (del03_01) and the compound of the Example (del03_26). When transfected with a plasmid, an editing rate of approximately 20% was observed. When transfected with the compound of Reference Example 1 (del03_01), no editing induction activity was observed. However, when transfected with the compound of the Example (del03_26), high editing induction activity was observed. Figure 6D shows the results of a luciferase reporter assay when using the compound of Reference Example 1 (del03_01) and the compound of the Example (del03_26). When the compound of the Reference Example (del03_01) was used, no luciferase activity was observed, but when the compound of the Example (del03_26) was used, much higher luciferase activity was observed than when transfected with a plasmid.

[0240] Figure 7A shows the editing rate (%) when using the compounds of the examples (del03_26, del03_30 to del03_33). When transfected with the compounds of the examples (del03_30 to del03_32), editing induction activity was observed at the same level as when transfected with the compound of the above example (del03_26). Figure 7B also shows the results of a luciferase reporter assay when using the compounds of the examples (del03_26, del03_30 to del03_33). When transfected with the compounds of the examples (del03_30 to del03_32), high luciferase activity was observed at the same level as when transfected with the compound of the above example (del03_26).

[0241] Figure 8A shows the editing rate (%) when the compounds of the examples (del03_26, del03_34 to del03_41) were used. When the compounds of the examples (del03_34 to del03_41) were transfected, editing induction activity equivalent to or greater than that observed when the compound of the above-mentioned example (del03_26) was transfected was observed. Figure 8B also shows the results of a luciferase reporter assay when the compounds of the examples (del03_26, del03_30 to del03_33) were used. When the compounds of the examples (del03_34 to del03_41) were transfected, high luciferase activity comparable to that observed when the compound of the above-mentioned example (del03_26) was transfected was observed.

[0242] (Test Example 3) Evaluation of editing induction ability for RNA containing β-actin gene sequence (A) RNA synthesis An annealing reaction (heating at 80°C for 3 minutes, then cooling to 25°C over 15 minutes) was carried out using oligo DNAs ACTB_FW (SEQ ID NO: 23) and ACTB_RV (SEQ ID NO: 24) and NEBuffer 2 (NEB) to generate an insert. pUC19 was reacted with EcoRI (TaKaRa) and HindIII (TaKaRa) at 37°C for 1 hour, followed by phenol / chloroform extraction and ethanol precipitation to purify. The insert:plasmid was added at a molar ratio of 3:1, and the DNA Ligation Kit<Mighty Mix> Ligation reactions were performed using a PCR product (TaKaRa). The resulting ligation sample was transformed into DH5α and cultured overnight at 37°C on an LB agar plate. Selected colonies were cultured overnight at 37°C in LB liquid medium, and plasmids were extracted using a QIAprep Spin Miniprep Kit (QIAGEN). 100 ng of the resulting plasmid was subjected to sequencing using a Big Dye Terminator v3.1 Cycle Sequence Kit (Thermo Fisher Scientific) with 0.165 μM pUC19_seqFW primer (SEQ ID NO: 25) and 0.165 μM pUC19_seqRV primer (SEQ ID NO: 26). Sequence analysis was performed using an Applied Biosystems 3500 Genetic Analyzer (Thermo Fisher Scientific). Using the correctly constructed plasmid as a template, PCR was performed using T7_pUC19_FW (SEQ ID NO: 27) and M13_RV primers (SEQ ID NO: 28) with PrimeStar GXL DNA Polymerase (TaKaRa) (denaturation: 98°C for 10 seconds, annealing: 55°C for 15 seconds, extension: 68°C for 20 seconds) to prepare template DNA, which was then extracted with phenol / chloroform and purified by ethanol precipitation. In vitro transcription was performed using the AmpliScribe T7 Kit (Epicentre Biotechnologies), and the DNA was excised and purified using a 5% polyacrylamide gel containing 8 M urea.

[0243] [Table 11]

[0244] [Table 12]

[0245] (B) Evaluation of editing inducibility and its results The editing induction ability of the example compound (ACTB_26) was evaluated using the same method as in Test Example 1 (B)-(D). However, the primers used were as described below. The editing rate (%) when the example compound (ACTB_26) was used is shown in Figures 9A and 9B. As a result, the example compound (ACTB_26) showed an editing rate of approximately 50%.

[0246] [Table 13]

[0247] (Test Example 4) Evaluation of editing induction ability for RNA having GAPDH gene sequence (A) RNA synthesis The procedure was the same as that described in (A) RNA synthesis in Test Example 3, except that the oligo DNA used was as described below.

[0248] [Table 14]

[0249] (B) Evaluation of editing inducibility and its results The editing induction ability of the example compound (GAPDH_26) was evaluated using the same method as in "(B) Evaluation of editing induction ability and results" in Test Example 3. The editing rate (%) when the example compound (GAPDH_26) was used is shown in Figures 9A and 9B. As a result, the example compound (GAPDH_26) showed an editing rate of approximately 90%.

[0250] (Test Example 5) Evaluation of editing induction ability for RNA having GFAP gene sequence (A) Synthesis of target RNA The procedure was the same as that described in (A) RNA synthesis in Test Example 3, except that the oligo DNA used was as described below.

[0251] [Table 15]

[0252] (B) Synthesis of target-editing guide RNA (gRNA) Template DNA for in vitro transcription of GFAP_del03 (SEQ ID NO: 38) was prepared by annealing oligo DNAs of GFAP_del03_RV (SEQ ID NO: 34) and T7proGGG (SEQ ID NO: 29) in an annealing buffer (10 mM Tri-HCl (pH 7.6), 150 mM NaCl) at 80°C for 3 min and then cooled to 25°C over 15 min). Template DNA for 5'AS_GFAP_gRNA (SEQ ID NO: 37) was prepared by incubation of 5'AS_GFAP_FW (SEQ ID NO: 35), 5'AS_ADg_RV oligo DNA (SEQ ID NO: 36), and DNA Polymerase I, Large (Klenow) Fragment (NEB) at 25°C for 30 min. The resulting mixture was then purified by phenol / chloroform extraction and ethanol precipitation. In vitro transcription was performed using 1.0 μg of each template DNA and the AmpliScribe T7 Kit (Epicentre Biotechnologies), and the DNA was excised and purified using an 8% polyacrylamide gel containing 8 M urea.

[0253] [Table 16]

[0254] [Table 17]

[0255] (C) Evaluation of editing inducibility The editing-inducing ability of the example compound (GFAP_26) was evaluated using the same method as in Test Example 3, "(B) Evaluation of editing-inducing ability and results thereof."

[0256] (Test Example 6) Evaluation of the ability of hADAR1 p110 to induce editing of Rluc_sRNA (A) Annealing reaction 0.3 μM Rluc_sRNA and 0.9 μM of the compounds (gRNA) of the Examples and Reference Examples were heated at 80°C for 3 minutes in an annealing buffer (10 mM Tri-HCl (pH 7.6), 150 mM NaCl) and cooled to 25°C over 15 minutes.

[0257] (B) In vitro editing reaction Assuming that Rluc_sRNA completely complexed with gRNA during the annealing reaction, the following Rluc_sRNA-gRNA complex concentrations were calculated: The editing reaction was performed by adding recombinant hADAR1 p110 (synthesized and purified using a yeast expression system; see Macbeth, M.R. and Bass, B.L. Methods Enzymol. 424, 319-331 (2007), Fukuda, M. et al. Sci. Rep. srep41478 (2017)) to a final concentration of 250 nM to 5 nM Rluc_sRNA-gRNA complex in editing reaction buffer (20 mM HEPES-KOH (pH 7.5), 2 mM MgCl2, 100 mM NaCl, 0.5 mM DTT, 0.01% Triton X-100, 5% glycerol) and incubating at 37°C for 30 minutes.

[0258] (C) Editing and Analysis Method After the editing reaction, Rluc_sRNA was purified by phenol / chloroform extraction and ethanol precipitation. cDNA was synthesized using Primescript Reverse Transcriptase II (TaKaRa) with the Rluc_sRNA_R01 primer. The cDNA was then amplified by PCR (denaturation: 98°C, 10 s, annealing: 55°C, 15 s, extension: 68°C, 20 s) with the Rluc_sRNA_F01 primer (SEQ ID NO: 18) and the Rluc_sRNA_R01 primer (SEQ ID NO: 15) and PrimeStar GXL DNA Polymerase (TaKaRa). The resulting cDNA was sequenced using the Rluc_sRNA_F01 primer (SEQ ID NO: 18) and the Big Dye Terminator v3.1 Cycle Sequence Kit, and analyzed on an Applied Biosystems 3500 Genetic Analyzer. The editing rate (%) was calculated from the peak height ratio (G / (G+A)) of the target site (A311) from the chromatogram obtained by sequencing.

[0259] (D) Edit analysis results The editing rate (%) when using the example compounds (del03_21, del03_26, and del03_32) is shown in Figure 10. In the figure, ADg(-) indicates the case where no gRNA was added. The example compound (del03_21) exhibited hADAR1 p110-mediated editing induction activity at the same level as the compound of Reference Example 1 (del03_01) and the compound of Reference Example 2 (del03_22). Furthermore, even when modified nucleic acids were introduced, as in the example compounds (del03_26 and del03_32), editing induction activity by hADAR1 p110 was also exhibited.

[0260] Examples 51 to 66 The oligonucleotide compounds of Examples 51 to 57 were obtained by incorporating modified nucleotides as shown below based on the sequence of the oligonucleotide of Reference Example 1. The oligonucleotide compounds of Examples 58 to 63 have first oligonucleotides (ASR) of various lengths corresponding to hGAPDH and were obtained by incorporating modified nucleotides as shown below. The oligonucleotide compounds of Examples 64 to 66 have first oligonucleotides (ASR) of the sequences shown below and were obtained by incorporating modified nucleotides as shown below. These oligonucleotide compounds were synthesized by the phosphoramidite method in the same manner as in Examples 1 to 50.

[0261] [Table 18]

[0262] The "molecular weight" in the table indicates the actual value measured by negative ion ESI mass spectrometry. In the "sequence" section, uppercase letters indicate RNA, lowercase letters indicate DNA, N(M) indicates 2'-O-methylation of D-ribofuranose, N(F) indicates 2'-deoxy-2'-fluoroation of D-ribofuranose, N(L) indicates 2'-O,4'-C-methylenation of D-ribofuranose, and N(E) indicates 2'-O,4'-C-ethylenation of D-ribofuranose. "9" indicates a linker represented by -O(CH2CHO)3-, and "18" indicates a linker represented by -O(CH2CHO)6-. "^" indicates a linkage between nucleoside units via -P(=S)(OH)-. Unless otherwise specified, this indicates a linkage between nucleoside units or between a nucleoside unit and a linker via -P(=O)(OH)-. The 5' and 3' ends of the oligonucleotide are hydroxyl groups in which a hydrogen atom is bonded to an oxygen atom in the chemical formula.

[0263] (Test Example 7) Evaluation of editing induction ability for Rluc_sRNA (2) (A) Annealing reaction, in vitro editing reaction, and editing analysis method The experiment was carried out in the same manner as in Test Example 1 or Test Example 6. In the case of hADAR2, recombinant hADAR2 was added to 5 nM of the Rluc_sRNA-gRNA complex to a concentration of 12.5 nM, and the mixture was incubated at 37°C for 1 hour. In the case of hADAR1 p110, recombinant hADAR1 p110 was added to 5 nM of the Rluc_sRNA-gRNA complex to a concentration of 250 nM, and the mixture was incubated at 37°C for 30 minutes.

[0264] (B) Edit analysis results Figures 11A and 12A show the results for hADAR2, and Figures 11B and 12B show the results for hADAR1 p110. As shown in Figure 11A, even when modified nucleic acids were introduced, such as in the example compounds (del03_32, 39, 42, 43, 44, and 45), they exhibited ADAR2 editing induction activity similar to the compound of Reference Example 1. Furthermore, as shown in Figure 11B, even when modified nucleic acids were introduced, such as in the example compounds (del03_32, 39, 43, and 45), they exhibited ADAR1 p110 editing induction activity similar to the compound of Reference Example 1. On the other hand, when modified nucleic acids such as in the example compounds (del03_42 and 44) ​​were introduced, the editing induction activity by ADAR1 p110 was significantly reduced compared to the compound of Reference Example 1. From the above, it was revealed that the example compounds (del03_42 and 44) ​​act selectively on ADAR2.

[0265] As shown in Figure 12A, even when modified nucleic acids were introduced, such as in the example compounds (del03_26, and del03_46 to del03_48), they exhibited ADAR2 editing induction activity similar to the compound of Reference Example 1. Furthermore, as shown in Figure 12B, even when modified nucleic acids were introduced, such as in the example compounds (del03_26, del03_46, and del03_47), they exhibited ADAR1 p110 editing induction activity similar to the compound of Reference Example 1. On the other hand, when modified nucleic acids such as the example compound (del03_48) were introduced, the ADAR1 p110 editing induction activity was significantly reduced compared to the compound of Reference Example 1. From the above, it was revealed that the example compound (del03_48) having a linker between the first oligonucleotide and the second oligonucleotide selectively acts on ADAR2.

[0266] (Test Example 8) Evaluation of editing induction ability in cultured cells (2) (A) Cell culture HeLa cells were plated in a 24-well plate at 5.0 × 10 5 The cells were subcultured at 100 cells / well and cultured for 48 hours. 50 ng of psiCHECK2 was transfected using Lipofectamine 3000 (Thermo). TM _Rluc_K41R_W104X, 350 ng of an ADAR expression plasmid (pcDNA3.1(-)Hygro_ADAR2, pcDNA3.1(-)Hygro_ADAR1 p110, or pcDNA3.1(-)Hygro_ADAR1 p150), and 20 nM of gRNA, a compound of the Examples or Reference Examples, were transfected. As a control, cells transfected with pSuper_neo instead of gRNA, a compound of the Examples or Reference Examples, were used.

[0267] (B) Editing analysis method and luciferase reporter assay method The same procedure as in Test Example 2 was carried out.

[0268] (C) Results of editing analysis and luciferase reporter assay. The editing rates (%) when using the compounds of the examples (del03_26, and del03_34 to del03_40) are shown in Figure 13A. When transfected with a plasmid expressing ADAR1 p110 or ADAR1 p150, the editing rates were approximately 10% to 40%, which were significantly higher than when transfected with only the ADAR1 p110 or ADAR1 p150 plasmid.

[0269] The results of luciferase reporter assays using example compounds (del03_26, and del03_34 to del03_40) are shown in Figure 13B. When example compounds (del03_26, and del03_38 to del03_40) were used, significantly higher luciferase activity was observed than when ADAR2, ADAR1 p110, or ADAR1 p150 plasmids were transfected alone. Furthermore, when example compounds (del03_34 to del03_37) were used, significantly higher luciferase activity was observed than when ADAR2 plasmids were transfected alone. However, the luciferase activity was very weak when ADAR1 p110 or ADAR1 p150 plasmids were transfected. These results demonstrate that example compounds (del03_34 to del03_37) having a linker between the first and second oligonucleotides are specific to ADAR2.

[0270] The editing rates (%) when using the compounds of the examples (del03_26, and del03_32, 39, 43, and 45) are shown in Figure 14A. When transfected with a plasmid expressing ADAR2, an editing rate of about 60% was observed, which was clearly higher than when transfected with only the ADAR2 plasmid. When transfected with a plasmid expressing ADAR1 p110 or ADAR1 p150, an editing rate of about 10% to 40% was observed, which was clearly higher than when transfected with only the ADAR1 p110 or ADAR1 p150 plasmid.

[0271] The results of luciferase reporter assays using the example compounds (del03_26, and del03_32, 39, 43, and 45) are shown in Figure 14B. When the example compounds (del03_26, and del03_32, 39, 43, and 45) were used, significantly higher luciferase activity was observed than when ADAR2, ADAR1 p110, or ADAR1 p150 plasmid alone was transfected.

[0272] The editing rates (%) when using the example compounds (del03_26, and del03_42, 44, 46, 47, and 48) are shown in Figure 15A. When transfected with a plasmid expressing ADAR2, the editing rates were approximately 30% to 60%, which were clearly higher than when transfected with only the ADAR2 plasmid. When transfected with the example compounds (del03_26, 46, and 47) with a plasmid expressing ADAR1 p110 or ADAR1 p150, the editing rates were approximately 10% to 30%, which were clearly higher than when transfected with only the ADAR1 p110 or p150 plasmid. On the other hand, when the compounds of the examples (del03_42, 44, and 48) were used to transfect a plasmid expressing ADAR1 p110 or ADAR1 p150, the rate was similar to that when only the ADAR1 p110 or ADAR1 p150 plasmid was transfected, and almost no editing activity was observed.

[0273] The results of luciferase reporter assays using the example compounds (del03_26, and del03_42, 44, 46, 47, and 48) are shown in Figure 15B. When the example compounds (del03_26, and del03_46 and 47) were used, significantly higher luciferase activity was observed than when ADAR2, ADAR1 p110, or p150 plasmids were transfected alone. On the other hand, when the example compounds (del03_42, 44, and 48) were used to transfect a plasmid expressing ADAR1 p110 or ADAR1 p150, the rate was similar to that when ADAR1 p110 or ADAR1 p150 plasmids were transfected alone, and almost no luciferase activity was observed. From the above, it was revealed that the example compounds (del03_42, 44) and the example compound (del03_48) having a linker between the first oligonucleotide and the second oligonucleotide act selectively on ADAR2.

[0274] (Test Example 9) Evaluation of editing induction ability for RNA having GAPDH gene sequence (2) (A) Evaluation of RNA synthesis and editing induction ability The same procedure as in Test Example 4 was carried out, except that hADAR1 p110 described in Test Example 6 was used in addition to hADAR2. Here, in the case of hADAR2, recombinant hADAR2 was added to 5 nM of Rluc_sRNA-gRNA complex to make it 12.5 nM, and the mixture was incubated at 37 ° C for 1 hour. In addition, in the case of hADAR1 p110, recombinant hADAR1 p110 was added to 5 nM of Rluc_sRNA-gRNA complex to make it 250 nM, and the mixture was incubated at 37 ° C for 30 minutes.

[0275] (B) Results of editing induction evaluation The editing induction ability of the example compounds (GAPDH_26, and GAPDH_49 to 51) was evaluated using the same method as in (B) Editing induction ability evaluation and results described in Test Example 4. The editing rate (%) when the example compounds (GAPDH_26, and GAPDH_49 to 51) were used is shown in Figures 16A and 16B. As a result, the example compounds (GAPDH_26, and GAPDH_49 to 51) showed a high editing rate when hADAR2 and hADAR1 p110 were used.

[0276] The editing induction ability of the example compounds (GAPDH_26, and GAPDH_39, 52, and 53) was evaluated using the same method as in (B) Editing induction ability evaluation and the results thereof described in Test Example 4. The editing rate (%) when the example compounds (GAPDH_26, and GAPDH_39, 52, and 53) were used is shown in Figures 17A and 17B. As a result, when hADAR2 and hADAR1 p110 were used, the example compounds (GAPDH_26, and GAPDH_39, 52, and 53) showed a higher editing rate than when gRNA was not added.

[0277] (Test Example 10) Evaluation of editing induction ability for RNA having GAPDH gene sequence in cultured cells (A) Cell culture HEK293 cells were plated in a 24-well plate at 5.0 × 10 4 The cells were passaged to 100 cells / well and cultured for 48 hours. Using Lipofectamine 3000 (Thermo), 500 ng of ADAR expression plasmid (pcDNA3.1(-)Hygro_ADAR2, pcDNA3.1(-)Hygro_ADAR1 p110, or pcDNA3.1(-)Hygro_ADAR1 p150) and 50 nM of gRNA, a compound of the Examples or Reference Examples, were transfected. Cultured cells not transfected with gRNA, a compound of the Examples or Reference Examples, were used as a control.

[0278] (B) Evaluation of editing inducibility and its results The editing induction ability of the example compounds (GAPDH_26, and GAPDH_49 to 51) was evaluated using the same method as in Test Example 4 (B) Evaluation of editing induction ability and the results thereof. The editing rate (%) when the example compounds (GAPDH_26, and GAPDH_49 to 51) were used is shown in Figure 16C. As a result, the example compounds (GAPDH_26, and GAPDH_49 to 51) showed high editing rates when hADAR2, hADAR1 p110, and hADAR1 p150 were used.

[0279] The method for evaluating the editing induction ability of the example compounds (GAPDH_26, and GAPDH_39, 52, and 53) was the same as that used in Test Example 4 (B) Evaluation of editing induction ability and the results thereof. The editing rate (%) when the example compounds (GAPDH_26, and GAPDH_39, 52, and 53) were used is shown in Figure 18. As a result, when hADAR2, hADAR1 p110, and hADAR1 p150 were used, the example compounds (GAPDH_26, and GAPDH_39, 52, and 53) showed a higher editing rate compared to when gRNA was not added.

Claims

1. a first oligonucleotide that identifies a target RNA; a second oligonucleotide linked to the 3' side of the first oligonucleotide; a third oligonucleotide capable of forming a complementary pair with the second oligonucleotide; a first linking portion linking the second oligonucleotide and the third oligonucleotide, the first oligonucleotide having a target-corresponding nucleotide residue corresponding to an adenosine residue in the target RNA; an oligonucleotide of 12 to 30 residues linked to the 5' side of the target-corresponding nucleotide residue and having a base sequence complementary to the target RNA; an oligonucleotide of 3 to 6 residues having a base sequence complementary to the target RNA, linked to the 3' side of the target-corresponding nucleotide residue, wherein all nucleotide residues are linked by phosphorothioate bonds; the second oligonucleotide has 5 to 8 residues; the third oligonucleotide has 5 to 8 residues; at least one residue selected from the counter region consisting of the target-corresponding nucleotide residue and one residue each on the 3' side and 5' side thereof is a nucleotide residue other than a naturally occurring ribonucleotide residue; the first linking portion consists solely of a polyalkyleneoxy group consisting of 1 to 8 alkyleneoxy units, the first oligonucleotide has a base sequence in which an oligonucleotide linked to the 5' side of a target-corresponding nucleotide residue is alternately linked with two types of modified oligonucleotide residues selected from the group consisting of 2'-deoxy-2'-fluoronucleotide residues, 2'-O-alkylribonucleotide residues, and bridged nucleotide residues; the second oligonucleotide and the third oligonucleotide have a base sequence in which 2'-O-alkylribonucleotide residues are linked, all nucleotide residues of the third oligonucleotide are linked via phosphorothioate bonds; An oligonucleotide that induces site-specific editing of the target RNA, or a pharmaceutically acceptable salt thereof.

2. The oligonucleotide according to claim 1, or a pharmaceutically acceptable salt thereof, comprising a second linking moiety comprising an alkyleneoxy unit between the first and second oligonucleotides.

3. 3. The oligonucleotide according to claim 1, or a pharmaceutically acceptable salt thereof, wherein at least one of the residues on the 3' side and the 5' side of the target-corresponding nucleotide residue is at least one modified nucleotide residue selected from the group consisting of 2'-O-alkylribonucleotide residues and 2'-deoxy-2'-fluororibonucleotide residues.

4. The oligonucleotide according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue has a base sequence in which 2'-deoxy-2'-fluoronucleotide residues and 2'-O-alkylribonucleotide residues are alternately linked.

5. The oligonucleotide according to claim 4, or a pharmaceutically acceptable salt thereof, wherein in the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue, the third nucleotide residue counting in the 5' direction from the target-corresponding nucleotide is a 2'-deoxy-2'-fluoronucleotide residue.

6. The oligonucleotide according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue has a base sequence in which bridged nucleotide residues and 2'-O-alkylribonucleotide residues are alternately linked.

7. 7. The oligonucleotide according to claim 6, or a pharmaceutically acceptable salt thereof, wherein in the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue, the third nucleotide residue counting in the 5' direction from the target-corresponding nucleotide is a bridged nucleotide residue.

8. The oligonucleotide according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue has a base sequence in which 2'-deoxy-2'-fluoronucleotide residues and bridged nucleotide residues are alternately linked.

9. The oligonucleotide according to claim 8, or a pharmaceutically acceptable salt thereof, wherein in the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue, the third nucleotide residue counting in the 5' direction from the target-corresponding nucleotide is a 2'-deoxy-2'-fluoronucleotide residue.

10. The oligonucleotide according to any one of claims 1 to 9, wherein in the first oligonucleotide, the oligonucleotide linked to the 3' side of the target-corresponding nucleotide residue has a base sequence in which a 2'-O-alkylribonucleotide residue is linked, or a pharmaceutically acceptable salt thereof.

11. The oligonucleotide according to any one of claims 1 to 9, wherein in the first oligonucleotide, the oligonucleotide linked to the 3' side of the target-corresponding nucleotide residue has a base sequence in which 2'-O-alkylribonucleotide residues and bridged nucleotide residues are alternately linked, or a pharmaceutically acceptable salt thereof.

12. 12. The oligonucleotide according to any one of claims 1 to 11, wherein in the first oligonucleotide, the oligonucleotide linked to the 3' side of the target-corresponding nucleotide residue consists of 4 to 6 residues, or a pharmaceutically acceptable salt thereof.

13. The oligonucleotide according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, wherein the second oligonucleotide comprises nucleotide residues linked via phosphorothioate bonds.

14. The oligonucleotide according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein the site-specific editing is caused by an enzymatic reaction catalyzed by adenosine deaminase.

15. A pharmaceutical comprising the oligonucleotide according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof.

16. 15. A therapeutic agent for a genetic disease, comprising the oligonucleotide according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof.

17. A pharmaceutical composition comprising, as an active ingredient, the oligonucleotide according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof.

18. The pharmaceutical composition according to claim 17, for the prevention or treatment of a genetic disease.

19. 19. The pharmaceutical composition of claim 18, wherein the genetic disease is a disease that can be treated by converting adenosine residues in the target RNA to inosine residues.

20. The pharmaceutical composition according to claim 18, wherein the genetic disease is caused by a mutation from a guanosine residue to an adenosine residue in a gene.

21. 15. Use of the oligonucleotide according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention or treatment of a disease.

22. 15. The oligonucleotide according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, for use in the prevention or treatment of a disease.

23. A method for preventing or treating a disease by administering a pharmacologically effective amount of the oligonucleotide described in any one of claims 1 to 14, or a pharmacologically acceptable salt thereof, to a warm-blooded animal (excluding humans).

24. 24. The method of claim 23, wherein the disease is a genetic disease.

25. 25. The method of claim 24, wherein the genetic disease is a disease that can be treated by converting adenosine residues in the target RNA to inosine residues.

26. The method according to claim 24, wherein the genetic disease is caused by a mutation from a guanosine residue to an adenosine residue in a gene.

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