Stable target editing guide RNA having phosphate moiety modification introduced thereinto

Abstract

Provided is an oligonucleotide capable of inducing the editing activity of ADAR in a cell and having excellent stability. The oligonucleotide comprises a first oligonucleotide and a second oligonucleotide linked to the 5'-side of the first oligonucleotide, and is capable of inducing the site-specific editing of target RNA. The first oligonucleotide includes: a target-corresponding nucleotide residue; an oligonucleotide that is located on the 5'-side of the target-corresponding nucleotide residue, is complementary to the target RNA, and is composed of 3 to 6 residues; and an oligonucleotide that is located on the 3'-side of the target-corresponding nucleotide residue, is complementary to the target RNA, and is composed of 10 to 24 residues. At least one of a phosphorus-containing linking group that links a first nucleoside residue to a second nucleoside residue and a phosphorus-containing linking group that links a fourth nucleoside residue to a fifth nucleoside residue is an alkylphosphonic acid residue, an alkyl phosphate residue or a substituted phosphoramide residue, wherein the nucleoside residues are numbered from the target-corresponding nucleotide in 3' direction. The second oligonucleotide has a nucleotide residue in which a nucleotide residue corresponding to the target RNA is deleted at the 3'-end or does not form a complementary pair with the target RNA. The second oligonucleotide is composed of 2 to 10 residues, wherein a nucleotide residue other than a nucleotide residue located at the 3'-end is complementary to the target RNA.

Description

Stable target-editing guide RNA with phosphate modification

[0001] The present invention relates to a stable target-editing guide RNA into which a phosphate moiety modification has been introduced.

[0002] With the development of genome editing technology, methods for controlling life phenomena by modifying genetic information, the blueprint of living organisms, in other words, DNA information within cells, are beginning to be used in the fields of medicine and drug discovery as a disease treatment approach. 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 transient genetic information molecule. Therefore, modifying RNA information can have a temporary, non-permanent effect on genetic information in the target organism.

[0003] As an RNA modification technique, for example, WO 2016 / 097212 describes an oligonucleotide construct for site-specific editing of nucleotides in a target RNA sequence, which comprises a targeting portion comprising an antisense sequence complementary to a portion of the target RNA, and a recruitment portion capable of binding to and recruiting an RNA editing entity present in cells and capable of editing nucleotides. Also, for example, WO 2017 / 010556 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. Also, WO 2022 / 124345 describes a stable target-editing guide RNA into which a chemically modified nucleic acid has been introduced.

[0004] One aspect of the present invention aims to provide an oligonucleotide that can induce ADAR editing activity in cells and has excellent stability in vivo.

[0005] Specific means for solving the above problems are as follows, and the present invention includes the following aspects.

[0006] (1-1) A nucleic acid sequence comprising: a first oligonucleotide that identifies a target RNA; and a second oligonucleotide linked to the 5' side of the first oligonucleotide, wherein the first oligonucleotide comprises: a target-corresponding nucleoside residue corresponding to an adenosine residue in the target RNA; an oligonucleotide of 3 to 6 residues that is linked to the 5' side of the target-corresponding nucleoside residue and has a base sequence complementary to the target RNA; and an oligonucleotide of 10 to 24 residues that is linked to the 3' side of the target-corresponding nucleoside residue and has a base sequence complementary to the target RNA; In the oligonucleotide linked to the 3' side of the target-corresponding nucleoside residue, at least one phosphorus-containing linking group selected from the group consisting of a phosphorus-containing linking group linking the first nucleoside residue and the second nucleoside residue and a phosphorus-containing linking group linking the fourth nucleoside residue and the fifth nucleoside residue, counting in the 3' direction from the target-corresponding nucleoside residue, is at least one type selected from the group consisting of an alkylphosphonic acid residue, an alkyl phosphate residue, and a substituted phosphoramide residue; the second oligonucleotide lacks a nucleoside residue corresponding to a nucleoside residue of the target RNA at its 3' end or has a nucleoside residue that does not form a complementary pair with a nucleoside residue of the target RNA; the second oligonucleotide has 2 to 10 residues, and at least nucleoside residues other than those at the 3' end form a double-stranded structure complementary to the target RNA; and at least one of the first oligonucleotide and the second oligonucleotide contains a phosphorothioate bond. An oligonucleotide that induces site-specific editing of the target RNA, or a pharmaceutically acceptable salt thereof.

[0007] (1-2) The oligonucleotide according to (1-1), or a pharmaceutically acceptable salt thereof, wherein the nucleoside residue linked to the 3'-side of the target-associated nucleoside residue is a 2'-deoxynucleoside residue.

[0008] (1-3) The oligonucleotide according to (1-1) or (1-2), or a pharmaceutically acceptable salt thereof, wherein in the oligonucleotide linked to the 3' side of the target-corresponding nucleoside residue, the third nucleoside residue counting in the 3' direction from the target-corresponding nucleoside residue is a 2'-deoxy-2'-fluoronucleoside residue.

[0009] (1-4) The oligonucleotide according to any one of (1-1) to (1-3), wherein the second oligonucleotide has 4 to 8 residues, or a pharmaceutically acceptable salt thereof.

[0010] (1-5) The oligonucleotide according to any one of (1-1) to (1-4), wherein the 3'-end of the second oligonucleotide lacks a nucleoside residue corresponding to a nucleoside residue in the target RNA, or a pharmaceutically acceptable salt thereof.

[0011] (1-6) The oligonucleotide according to any one of (1-1) to (1-5), or a pharmaceutically acceptable salt thereof, wherein the site-specific editing is caused by an enzymatic reaction catalyzed by adenosine deaminase 1.

[0012] (1-7) The oligonucleotide according to any one of (1-1) to (1-6), wherein the first oligonucleotide contains a phosphorothioate bond, or a pharmaceutically acceptable salt thereof.

[0013] (1-8) The oligonucleotide according to any one of (1-1) to (1-7), or a pharmaceutically acceptable salt thereof, wherein at least one phosphorus-containing linking group selected from the group consisting of a phosphorus-containing linking group linking the second nucleoside residue and the third nucleoside residue, a phosphorus-containing linking group linking the eighth nucleoside residue and the ninth nucleoside residue, a phosphorus-containing linking group linking the tenth nucleoside residue and the eleventh nucleoside residue, a phosphorus-containing linking group linking the eleventh nucleoside residue and the twelfth nucleoside residue, and a phosphorus-containing linking group linking the sixth nucleoside residue and the fifth nucleoside residue, counting in the 5' direction from the target-corresponding nucleoside residue, is a phosphate residue.

[0014] (1-9) The oligonucleotide according to any one of (1-1) to (1-8), wherein the first oligonucleotide is a nucleotide in which, counting in the 3' direction from the target-corresponding nucleoside residue, the second nucleoside residue and the third nucleoside residue are linked by a phosphodiester bond, or a pharmaceutically acceptable salt thereof.

[0015] (1-10) The oligonucleotide according to any one of (1-1) to (1-9), wherein the first oligonucleotide comprises at least one modified nucleoside residue selected from the group consisting of a 2'-O-alkylribonucleoside residue, a 2'-deoxy-2'-fluororibonucleoside residue, a bridged nucleoside residue, and a 2'-deoxyribonucleoside residue, or a pharmaceutically acceptable salt thereof.

[0016] (1-11) The oligonucleotide according to any one of (1-1) to (1-10), which comprises a partial structure in which the target-corresponding nucleoside residue is linked to an adjacent nucleoside residue via a phosphorothioate bond, or a pharmaceutically acceptable salt thereof.

[0017] (1-12) The oligonucleotide according to any one of (1-1) to (1-11), wherein the nucleoside residue linked to the 5' side of the target-corresponding nucleotide residue is a 2'-O-alkylribonucleoside residue, or a pharmaceutically acceptable salt thereof.

[0018] (1-13) The oligonucleotide according to any one of (1-1) to (1-12), wherein the second oligonucleotide comprises at least one modified nucleoside residue selected from the group consisting of a 2'-O-alkylribonucleoside residue, a 2'-deoxy-2'-fluororibonucleoside residue, and a bridged nucleoside residue, or a pharmaceutically acceptable salt thereof.

[0019] (1-14) The oligonucleotide according to any one of (1-1) to (1-13), wherein the oligonucleotide linked to the 3' side of the target-associated nucleoside residue has a base sequence in which 2'-deoxy-2'-fluoronucleoside residues and 2'-O-alkylribonucleoside residues are alternately linked, or a pharmaceutically acceptable salt thereof.

[0020] (1-15) The oligonucleotide according to any one of (1-1) to (1-14), wherein the oligonucleotide linked to the 3' side of the target-associated nucleoside residue has a base sequence in which bridged nucleoside residues and 2'-O-alkylribonucleoside residues are alternately linked, or a pharmaceutically acceptable salt thereof.

[0021] (1-16) The oligonucleotide according to any one of (1-1) to (1-15), wherein in the first oligonucleotide, the oligonucleotide linked to the 5' side of the target-corresponding nucleoside residue has a base sequence in which a 2'-O-alkylribonucleoside residue is linked, or a pharmaceutically acceptable salt thereof.

[0022] (1-17) The oligonucleotide according to any one of (1-1) to (1-16), wherein the second oligonucleotide has a base sequence in which 2'-O-alkylribonucleoside residues and bridged nucleoside residues are alternately linked, or a pharmaceutically acceptable salt thereof.

[0023] (1-18) The oligonucleotide according to any one of (1-1) to (1-17), or a pharmaceutically acceptable salt thereof, wherein the target-corresponding nucleoside residue is an N-alkylpyrimidine nucleoside residue or a 2'-deoxycytidine residue.

[0024] (1-19) The oligonucleotide according to any one of (1-1) to (1-18), wherein the second oligonucleotide is an oligonucleotide in which nucleoside residues are linked via phosphorothioate bonds, or a pharmaceutically acceptable salt thereof.

[0025] (1-20) The oligonucleotide according to any one of (1-1) to (1-19), wherein the first oligonucleotide has a bridged nucleoside residue at the 10th or subsequent nucleoside residue counting in the 3' direction from the target-corresponding nucleoside residue, or a pharmaceutically acceptable salt thereof.

[0026] (1-21) The first oligonucleotide is the oligonucleotide according to (1-20), or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide consisting of nucleoside residues from the 10th nucleoside residue onwards counting in the 3' direction from the target-corresponding nucleoside residue has a base sequence in which 2'-O-alkylribonucleoside residues and bridged nucleoside residues are alternately linked.

[0027] (1-22) The oligonucleotide according to any one of (1-1) to (1-21), wherein the second oligonucleotide has a bridged nucleoside residue in the second or subsequent nucleoside residue counting in the 5' direction from the 3'-terminal nucleoside residue, or a pharmaceutically acceptable salt thereof.

[0028] (1-23) The second oligonucleotide is the oligonucleotide according to (1-22), or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide consisting of the second and subsequent nucleoside residues counting in the 5' direction from the 3'-terminal nucleoside residue has a base sequence in which 2'-O-alkylribonucleoside residues and bridged nucleoside residues are alternately linked.

[0029] (1-24) The oligonucleotide according to any one of (1-1) to (1-7), which comprises a modified oligonucleotide having a chemical modification pattern represented by any one of the following formulas (1) to (20), or a pharmaceutically acceptable salt thereof:

[0030] (1) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (2) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (3) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (4) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (5) 5’- HO-LsMsMsMsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (6) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (7) 5’- HO-MsLsMsLsMsFsXFpMsMsdCsDqMpFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (8) 5’- HO-MsLsMsLsMsFsXFsMpMsdCsDqMsFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (9) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMsLpMsLsM-OH -3’ (10) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMpFsMsFsMsLpMsLsM-OH -3’ (11) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMsFsMpFsMsLpMsLsM-OH -3’ (12) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMpLpMsLsM-OH -3’ (13) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (14) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMsFsMsMsMsLsM-OH -3’(15) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMpFsMsMsMsLsM-OH -3' (16) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMpMsMsLsM-OH -3' (17) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMpFsMsMsMsLsM-OH -3' (18) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMsFsMpMsMsLsM-OH -3' (19) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMpFsMpMsMsLsM-OH -3' (20) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMpFsMpMsMsLsM-OH -3'

[0031] In each formula, D represents a 2'-deoxynucleoside residue, F represents a 2'-deoxy-2'-fluororibonucleoside residue, L represents a bridged nucleoside residue, M represents a 2'-O-methylribonucleoside residue, and dC represents a 2'-deoxycytidine residue, which is a target-corresponding nucleoside residue. X indicates that one nucleoside residue relative to the target RNA is deleted. p represents a phosphate residue, s represents a thiophosphate residue, and q represents an alkylphosphonate residue, an alkyl phosphate residue, or a substituted phosphoramide residue. HO- represents a hydroxyl group at the 5' end, and -OH represents a hydroxyl group at the 3' end.

[0032] (1-25) The oligonucleotide according to any one of (1-1) to (1-7), which comprises a modified oligonucleotide having a chemical modification pattern represented by any one of the following formulas (1a) to (20a), or a pharmaceutically acceptable salt thereof:

[0033] (1a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsLsMsLsM-OH -3’ (2a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsMsMsLsM-OH -3’ (3a) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsLsMsLsM-OH -3’ (4a) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsMsMsLsM-OH -3’ (5a) 5’- HO-LsMsMsMsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsMsMsLsM-OH -3’ (6a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMsLsMsLsM-OH -3’ (7a) 5’- HO-MsLsMsLsMsFsXFpMsMsdCsDrMpFsMrFsMsFsMsFsMsLsMsLsM-OH -3’ (8a) 5’- HO-MsLsMsLsMsFsXFsMpMsdCsDrMsFsMrFsMsFsMsFsMsLsMsLsM-OH -3’ (9a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMsLpMsLsM-OH -3’ (10a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMpFsMsFsMsLpMsLsM-OH -3’ (11a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMsFsMpFsMsLpMsLsM-OH -3’ (12a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMpLpMsLsM-OH -3’ (13a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMsMsMsLsM-OH -3’ (14a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMsFsMsMsMsLsM-OH -3’(15a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMpFsMsMsMsLsM-OH -3' (16a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMpMsMsLsM-OH -3' (17a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMpFsMsMsMsLsM-OH -3' (18a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMsFsMpMsMsLsM-OH -3' (19a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMpFsMpMsMsLsM-OH -3' (20a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMpFsMpMsMsLsM-OH -3'

[0034] In each formula, D represents a 2'-deoxynucleoside residue, F represents a 2'-deoxy-2'-fluororibonucleoside residue, L represents a bridged nucleoside residue, M represents a 2'-O-methylribonucleoside residue, and dC represents a 2'-deoxycytidine residue, which is a target-corresponding nucleoside residue. X indicates that one nucleoside residue relative to the target RNA is deleted. p represents a phosphate residue, s represents a thiophosphate residue, pm represents a methylphosphonate residue, and r represents an ethyl phosphate residue or a methoxyethyl phosphate residue. HO- represents a hydroxyl group at the 5' end, and -OH represents a hydroxyl group at the 3' end.

[0035] (1-26) The oligonucleotide according to any one of (1-1) to (1-7), which comprises a modified oligonucleotide having a chemical modification pattern represented by any one of the following formulas (1) to (22), or a pharmaceutically acceptable salt thereof:

[0036] (1) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (2) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (3) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (4) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (5) 5’- HO-LsMsMsMsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (6) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (7) 5’- HO-MsLsMsLsMsFsXFpMsMsdCsDqMpFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (8) 5’- HO-MsLsMsLsMsFsXFsMpMsdCsDqMsFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (9) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMsLpMsLsM-OH -3’ (10) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMpFsMsFsMsLpMsLsM-OH -3’ (11) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMsFsMpFsMsLpMsLsM-OH -3’ (12) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMpLpMsLsM-OH -3’ (13) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (14) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMsFsMsMsMsLsM-OH -3’(15) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMpFsMsMsMsLsM-OH -3' (16) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMpMsMsLsM-OH -3' (17) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMpFsMsMsMsLsM-OH -3' (18) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMsFsMpMsMsLsM-OH -3' (19) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMpFsMpMsMsLsM-OH -3' (20) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMpFsMpMsMsLsM-OH -3' (21) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMsFsMsFsMsFsMsLsMsLsM-OH -3' (22) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMsFsMsFsMsFsMsMsMsLsM-OH -3'

[0037] In each formula, D represents a 2'-deoxynucleoside residue, F represents a 2'-deoxy-2'-fluororibonucleoside residue, L represents a bridged nucleoside residue, M represents a 2'-O-methylribonucleoside residue, and dC represents a 2'-deoxycytidine residue, which is a target-corresponding nucleoside residue. X indicates that one nucleoside residue relative to the target RNA is deleted. p represents a phosphate residue, s represents a thiophosphate residue, and q represents an alkylphosphonate residue, an alkyl phosphate residue, or a substituted phosphoramide residue. HO- represents a hydroxyl group at the 5' end, and -OH represents a hydroxyl group at the 3' end.

[0038] (1-27) The oligonucleotide according to any one of (1-1) to (1-7), which comprises a modified oligonucleotide having a chemical modification pattern represented by any one of the following formulas (1a) to (22a), or a pharmaceutically acceptable salt thereof:

[0039] (1a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsLsMsLsM-OH -3’ (2a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsMsMsLsM-OH -3’ (3a) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsLsMsLsM-OH -3’ (4a) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsMsMsLsM-OH -3’ (5a) 5’- HO-LsMsMsMsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsMsMsLsM-OH -3’ (6a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMsLsMsLsM-OH -3’ (7a) 5’- HO-MsLsMsLsMsFsXFpMsMsdCsDrMpFsMrFsMsFsMsFsMsLsMsLsM-OH -3’ (8a) 5’- HO-MsLsMsLsMsFsXFsMpMsdCsDrMsFsMrFsMsFsMsFsMsLsMsLsM-OH -3’ (9a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMsLpMsLsM-OH -3’ (10a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMpFsMsFsMsLpMsLsM-OH -3’ (11a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMsFsMpFsMsLpMsLsM-OH -3’ (12a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMpLpMsLsM-OH -3’ (13a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMsMsMsLsM-OH -3’ (14a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMsFsMsMsMsLsM-OH -3’(15a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMpFsMsMsMsLsM-OH -3' (16a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMpMsMsLsM-OH -3' (17a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMpFsMsMsMsLsM-OH -3' (18a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMsFsMpMsMsLsM-OH -3' (19a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMpFsMpMsMsLsM-OH -3' (20a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMpFsMpMsMsLsM-OH -3' (21a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMsFsMsFsMsFsMsLsMsLsM-OH -3' (22a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMsFsMsFsMsFsMsMsMsLsM-OH -3'

[0040] In each formula, D represents a 2'-deoxynucleoside residue, F represents a 2'-deoxy-2'-fluororibonucleoside residue, L represents a bridged nucleoside residue, M represents a 2'-O-methylribonucleoside residue, and dC represents a 2'-deoxycytidine residue, which is a target-corresponding nucleoside residue. X indicates that one nucleoside residue relative to the target RNA is deleted. p represents a phosphate residue, s represents a thiophosphate residue, pm represents a methylphosphonate residue, and r represents an ethyl phosphate residue or a methoxyethyl phosphate residue. HO- represents a hydroxyl group at the 5' end, and -OH represents a hydroxyl group at the 3' end.

[0041] (2-1) An oligonucleotide or a pharmaceutically acceptable salt thereof that induces site-specific editing to convert an adenosine residue to be edited contained in a target RNA to an inosine residue, the oligonucleotide comprising a structure represented by the following formula (A), wherein the target-corresponding nucleoside residue corresponding to the adenosine residue to be edited is N: ６ It is expressed as N ６ and an oligonucleotide consisting of nucleoside residues excluding X, which has a base sequence complementary to a target RNA, and the base corresponding to X in the target RNA sequence corresponds to the base sequence as if it were present even when X is deleted, or a pharmaceutically acceptable salt thereof. １ -(N １ -L １ )-(N ２ -L ２ )-X-(N ３ -L ３ ) ｐ -(N ４ -L ４ )-(N ５ -L ５ )-(N ６ -L ６ )-(N ７ -L ７ )-(N ８ -L ８ )-(N ９ -L ９ )-(N １０ -L １０ ) -O ２ -O ３ -3'

[0042] In formula (A), O １ represents an oligonucleotide of 1 to 8 residues that forms a complementary pair with the corresponding oligonucleotide portion of the target RNA; １ From N ５ and N ７ From N １０ each represents a nucleoside residue that forms a complementary pair with a corresponding nucleoside residue of the target RNA, and N ６ represents a 2'-deoxycytidine residue, a 2'-deoxyguanosine residue, a 2'-deoxyadenosine residue, or a derivative thereof; O ２ and O３ each independently represents an oligonucleotide of 3 to 17 residues that forms a complementary pair with a corresponding oligonucleotide portion of a target RNA; ２ and O ３ The total number of residues of is 6 to 20, X represents a deletion of one nucleoside residue with respect to the target RNA, or represents a nucleoside residue that is not complementary to the corresponding nucleoside residue of the target RNA and a phosphorus-containing linking group, L １ , L ２ , L ３ , L ４ , L ５ , L ６ , L ８ and L ９ each independently represents a phosphorus-containing linking group selected from the group consisting of a phosphate residue and a thiophosphate residue; １ , L ２ , L ３ , L ４ , L ５ , L ６ , L ８ and L ９ at least one of L represents a thiophosphate residue; ７ and L １０ are each independently selected from the group consisting of a phosphate residue, a thiophosphate residue, an alkylphosphonate residue, an alkyl phosphate residue, and a substituted phosphoramide residue; L ７ and L １０ At least one of the groups represents a phosphorus-containing linking group selected from the group consisting of alkylphosphonic acid residues, alkyl phosphate residues, and substituted phosphoramide residues; and p represents an integer of 1 to 4.

[0043] (2-2) L １ , L ２ , L ３ , L ４ , L ５ , L ６ , L ８ and L ９ all of which represent phosphorothioate residues, or 1 to 5 of which represent phosphoric acid residues and the remaining represent phosphorothioate residues, or a pharmaceutically acceptable salt thereof.

[0044] (2-3) L７ and L １０ are each independently a phosphorus-containing linking group selected from the group consisting of alkylphosphonic acid residues, alkyl phosphate residues, and substituted phosphoramide residues, or a pharmaceutically acceptable salt thereof.

[0045] (2-4) L ８ is a phosphate residue, or a pharmaceutically acceptable salt thereof.

[0046] (2-5) O １ The oligonucleotide according to any one of (2-1) to (2-4), which comprises an oligonucleotide in which 2'-O-alkylribonucleoside residues and bridged nucleoside residues are alternately linked, or a pharmaceutically acceptable salt thereof.

[0047] (2-6) O ２ The oligonucleotide according to any one of (2-1) to (2-5), which comprises an oligonucleotide in which 2'-deoxy-2'-fluoronucleoside residues and 2'-O-alkylribonucleoside residues are linked alternately, or a pharmaceutically acceptable salt thereof.

[0048] (2-7) O ３ The oligonucleotide according to any one of (2-1) to (2-6), which comprises an oligonucleotide in which 2'-O-alkylribonucleoside residues and bridged nucleoside residues are alternately linked, or a pharmaceutically acceptable salt thereof.

[0049] (2-8) The oligonucleotide according to any one of (2-1) to (2-7), which comprises a modified oligonucleotide having a chemical modification pattern represented by any one of the following formulas (1) to (20), or a pharmaceutically acceptable salt thereof:

[0050] (1) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (2) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (3) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (4) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (5) 5’- HO-LsMsMsMsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (6) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (7) 5’- HO-MsLsMsLsMsFsXFpMsMsdCsDqMpFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (8) 5’- HO-MsLsMsLsMsFsXFsMpMsdCsDqMsFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (9) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMsLpMsLsM-OH -3’ (10) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMpFsMsFsMsLpMsLsM-OH -3’ (11) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMsFsMpFsMsLpMsLsM-OH -3’ (12) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMpLpMsLsM-OH -3’ (13) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (14) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMsFsMsMsMsLsM-OH -3’(15) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMpFsMsMsMsLsM-OH -3' (16) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMpMsMsLsM-OH -3' (17) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMpFsMsMsMsLsM-OH -3' (18) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMsFsMpMsMsLsM-OH -3' (19) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMpFsMpMsMsLsM-OH -3' (20) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMpFsMpMsMsLsM-OH -3'

[0051] In each formula, D represents a 2'-deoxynucleoside residue, F represents a 2'-deoxy-2'-fluororibonucleoside residue, L represents a bridged nucleoside residue, M represents a 2'-O-methylribonucleoside residue, and dC represents a 2'-deoxycytidine residue, which is a target-corresponding nucleoside residue. X indicates that one nucleoside residue relative to the target RNA is deleted. p represents a phosphate residue, s represents a thiophosphate residue, and q represents an alkylphosphonate residue, an alkyl phosphate residue, or a substituted phosphoramide residue. HO- represents a hydroxyl group at the 5' end, and -OH represents a hydroxyl group at the 3' end.

[0052] (2-9) The oligonucleotide according to any one of (2-1) to (2-7), which comprises a modified oligonucleotide having a chemical modification pattern represented by any one of the following formulas (1a) to (20a), or a pharmaceutically acceptable salt thereof:

[0053] (1a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsLsMsLsM-OH -3’ (2a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsMsMsLsM-OH -3’ (3a) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsLsMsLsM-OH -3’ (4a) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsMsMsLsM-OH -3’ (5a) 5’- HO-LsMsMsMsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsMsMsLsM-OH -3’ (6a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMsLsMsLsM-OH -3’ (7a) 5’- HO-MsLsMsLsMsFsXFpMsMsdCsDrMpFsMrFsMsFsMsFsMsLsMsLsM-OH -3’ (8a) 5’- HO-MsLsMsLsMsFsXFsMpMsdCsDrMsFsMrFsMsFsMsFsMsLsMsLsM-OH -3’ (9a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMsLpMsLsM-OH -3’ (10a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMpFsMsFsMsLpMsLsM-OH -3’ (11a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMsFsMpFsMsLpMsLsM-OH -3’ (12a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMpLpMsLsM-OH -3’ (13a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMsMsMsLsM-OH -3’ (14a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMsFsMsMsMsLsM-OH -3’(15a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMpFsMsMsMsLsM-OH -3' (16a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMpMsMsLsM-OH -3' (17a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMpFsMsMsMsLsM-OH -3' (18a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMsFsMpMsMsLsM-OH -3' (19a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMpFsMpMsMsLsM-OH -3' (20a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMpFsMpMsMsLsM-OH -3'

[0054] In each formula, D represents a 2'-deoxynucleoside residue, F represents a 2'-deoxy-2'-fluororibonucleoside residue, L represents a bridged nucleoside residue, M represents a 2'-O-methylribonucleoside residue, and dC represents a 2'-deoxycytidine residue, which is a target-corresponding nucleoside residue. X indicates that one nucleoside residue relative to the target RNA is deleted. p represents a phosphate residue, s represents a thiophosphate residue, pm represents a methylphosphonate residue, and r represents an ethyl phosphate residue or a methoxyethyl phosphate residue. HO- represents a hydroxyl group at the 5' end, and -OH represents a hydroxyl group at the 3' end.

[0055] (2-10) The oligonucleotide according to any one of (2-1) to (2-7), which comprises a modified oligonucleotide having a chemical modification pattern represented by any one of the following formulas (1) to (22), or a pharmaceutically acceptable salt thereof:

[0056] (1) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (2) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (3) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (4) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (5) 5’- HO-LsMsMsMsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (6) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (7) 5’- HO-MsLsMsLsMsFsXFpMsMsdCsDqMpFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (8) 5’- HO-MsLsMsLsMsFsXFsMpMsdCsDqMsFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (9) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMsLpMsLsM-OH -3’ (10) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMpFsMsFsMsLpMsLsM-OH -3’ (11) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMsFsMpFsMsLpMsLsM-OH -3’ (12) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMpLpMsLsM-OH -3’ (13) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (14) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMsFsMsMsMsLsM-OH -3’(15) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMpFsMsMsMsLsM-OH -3' (16) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMpMsMsLsM-OH -3' (17) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMpFsMsMsMsLsM-OH -3' (18) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMsFsMpMsMsLsM-OH -3' (19) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMpFsMpMsMsLsM-OH -3' (20) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMpFsMpMsMsLsM-OH -3' (21) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMsFsMsFsMsFsMsLsMsLsM-OH -3' (22) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMsFsMsFsMsFsMsMsMsLsM-OH -3'

[0057] In each formula, D represents a 2'-deoxynucleoside residue, F represents a 2'-deoxy-2'-fluororibonucleoside residue, L represents a bridged nucleoside residue, M represents a 2'-O-methylribonucleoside residue, and dC represents a 2'-deoxycytidine residue, which is a target-corresponding nucleoside residue. X indicates that one nucleoside residue relative to the target RNA is deleted. p represents a phosphate residue, s represents a thiophosphate residue, and q represents an alkylphosphonate residue, an alkyl phosphate residue, or a substituted phosphoramide residue. HO- represents a hydroxyl group at the 5' end, and -OH represents a hydroxyl group at the 3' end.

[0058] (2-11) The oligonucleotide according to any one of (2-1) to (2-7), which comprises a modified oligonucleotide having a chemical modification pattern represented by any one of the following formulas (1a) to (22a), or a pharmaceutically acceptable salt thereof:

[0059] (1a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsLsMsLsM-OH -3’ (2a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsMsMsLsM-OH -3’ (3a) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsLsMsLsM-OH -3’ (4a) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsMsMsLsM-OH -3’ (5a) 5’- HO-LsMsMsMsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsMsMsLsM-OH -3’ (6a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMsLsMsLsM-OH -3’ (7a) 5’- HO-MsLsMsLsMsFsXFpMsMsdCsDrMpFsMrFsMsFsMsFsMsLsMsLsM-OH -3’ (8a) 5’- HO-MsLsMsLsMsFsXFsMpMsdCsDrMsFsMrFsMsFsMsFsMsLsMsLsM-OH -3’ (9a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMsLpMsLsM-OH -3’ (10a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMpFsMsFsMsLpMsLsM-OH -3’ (11a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMsFsMpFsMsLpMsLsM-OH -3’ (12a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMpLpMsLsM-OH -3’ (13a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMsMsMsLsM-OH -3’ (14a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMsFsMsMsMsLsM-OH -3’(15a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMpFsMsMsMsLsM-OH -3' (16a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMpMsMsLsM-OH -3' (17a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMpFsMsMsMsLsM-OH -3' (18a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMsFsMpMsMsLsM-OH -3' (19a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMpFsMpMsMsLsM-OH -3' (20a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMpFsMpMsMsLsM-OH -3' (21a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMsFsMsFsMsFsMsLsMsLsM-OH -3' (22a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMsFsMsFsMsFsMsMsMsLsM-OH -3'

[0060] In each formula, D represents a 2'-deoxynucleoside residue, F represents a 2'-deoxy-2'-fluororibonucleoside residue, L represents a bridged nucleoside residue, M represents a 2'-O-methylribonucleoside residue, and dC represents a 2'-deoxycytidine residue, which is a target-corresponding nucleoside residue. X indicates that one nucleoside residue relative to the target RNA is deleted. p represents a phosphate residue, s represents a thiophosphate residue, pm represents a methylphosphonate residue, and r represents an ethyl phosphate residue or a methoxyethyl phosphate residue. HO- represents a hydroxyl group at the 5' end, and -OH represents a hydroxyl group at the 3' end.

[0061] According to one aspect of the present invention, an oligonucleotide can be provided that can induce ADAR editing activity in cells and has excellent stability in vivo.

[0062] 1 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in a cell-free system. 2 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in a cell-free system. 3 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in a cell-free system. 4 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in a cell-free system. 5 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in a cell-free system. 6 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in a cell-free system. 7 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in a cell-free system. 8 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in a cell-free system. 1 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in cells. FIG. 2 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in cells. FIG. 3 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in a cell-free system. FIG. 4 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in cells. FIG. 5 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in a cell-free system. FIG. 6 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in cells. FIG. 7 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in a cell-free system.1 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in a cell-free system. 2 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in a cell-free system. 3 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in a cell-free system. 4 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in a cell-free system. 5 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in a cell-free system. 6 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in a cell-free system. 7 is a graph showing the rate of editing of target RNA by oligonucleotides with hACTB in a cell-free system. 8 is a graph showing the rate of editing of target RNA by oligonucleotides with A1AT in a cell-free system. 9 is a graph showing the rate of editing of target RNA by oligonucleotides with A1AT in a cell. 10 is a graph showing the rate of editing of target RNA by oligonucleotides with mGAPDH in a cell-free system. 11 is a graph showing the rate of editing of target RNA by oligonucleotides with mGAPDH in a cell. 1 is a graph showing the percentage of target RNA editing by oligonucleotides relative to mGAPDH in cells.

[0063] In this specification, the term "process" refers not only to an independent process, but also to processes that cannot be clearly distinguished from other processes, as long as the intended purpose of the process is achieved. Furthermore, the content of each component in a composition refers to the total amount of the multiple substances present in the composition, unless otherwise specified, when multiple substances corresponding to each component are present in the composition. Furthermore, the upper and lower limits of the numerical ranges described in this specification can be arbitrarily selected and combined from the numerical values ​​exemplified as numerical ranges.

[0064] Furthermore, the term "oligonucleotide" as used herein may be interpreted as an oligonucleotide molecule or an oligonucleotide residue, depending on the context. Furthermore, in an oligonucleotide, the nucleoside residues constituting the oligonucleotide may be linked by natural phosphate residues, or by phosphorus-containing linking groups such as thiophosphate residues, alkylphosphonic acid residues, alkyl phosphate residues, and substituted phosphoramide residues (including N-sulfonyl-substituted phosphoramide residues and N-alkyl-substituted phosphoramide residues). In this specification, linking groups linking nucleoside residues, such as ordinary phosphate residues, thiophosphate residues, alkylphosphonic acid residues, alkyl phosphate residues, and substituted phosphoramide residues, are collectively referred to as phosphorus-containing linking groups.

[0065] Hereinafter, embodiments of the present invention will be described in detail, but the embodiments shown below are merely examples of oligonucleotides or pharmaceutically acceptable salts thereof for embodying the technical concept of the present invention, and the present invention is not limited to the oligonucleotides or pharmaceutically acceptable salts thereof shown below.

[0066]

[0023] Target-Editing Oligonucleotides

[0024] An oligonucleotide that induces site-specific editing of a target RNA (hereinafter also referred to as a target-editing oligonucleotide or a target-editing guide RNA) comprises a first oligonucleotide that specifies the target RNA and a second oligonucleotide linked to the 5' side of the first oligonucleotide. The first oligonucleotide may consist of a target-corresponding nucleoside residue corresponding to an adenosine residue in the target RNA, an oligonucleotide of 3 to 6 residues that is linked to the 5' side of the target-corresponding nucleoside residue and has a base sequence complementary to the target RNA, and an oligonucleotide of 10 to 24 residues that is linked to the 3' side of the target-corresponding nucleoside residue and has a base sequence complementary to the target RNA. In the first oligonucleotide, in the oligonucleotide linked to the 3' side of the target-corresponding nucleoside residue, at least one phosphorus-containing linking group selected from the group consisting of a phosphorus-containing linking group linking the first nucleoside residue to the second nucleoside residue and a phosphorus-containing linking group linking the fourth nucleoside residue to the fifth nucleoside residue, counting in the 3' direction from the target-corresponding nucleoside residue, may be at least one selected from the group consisting of alkylphosphonic acid residues, alkyl phosphate residues, and substituted phosphoramide residues. The second oligonucleotide may lack a nucleoside residue corresponding to a nucleoside residue in the target RNA at its 3' end, or may have a nucleoside residue that does not form a complementary pair with a nucleoside residue in the target RNA. The second oligonucleotide may have 2 to 10 residues, and at least nucleoside residues other than those at the 3' end may form a double-stranded structure complementary to the target RNA. At least one of the first oligonucleotide and the second oligonucleotide may contain a phosphorothioate bond.

[0067] The second oligonucleotide constituting the target-editing oligonucleotide has a base sequence that does not form a complementary pair with one nucleoside residue of the target RNA, and nucleoside residues other than that nucleoside residue form a double-stranded structure complementary to the target RNA. In other words, the second oligonucleotide has a base sequence that forms an incomplete double-stranded structure with the target RNA. A target-editing guide RNA having a short second oligonucleotide having such a characteristic base sequence on the 5' side of a first oligonucleotide that specifies the target RNA can preferentially induce the editing activity of ADAR in cells. This can be thought of, for example, as being because the presence of a nucleoside residue that does not form a complementary pair is advantageous for recruiting ADAR (e.g., ADAR1). Furthermore, in an oligonucleotide linked to the 3' side of a target-corresponding nucleoside residue, at least one phosphorus-containing linking group selected from the group consisting of a phosphorus-containing linking group linking the first nucleoside residue to the second nucleoside residue and a phosphorus-containing linking group linking the fourth nucleoside residue to the fifth nucleoside residue, counting in the 3' direction from the target-corresponding nucleoside residue, is at least one type selected from the group consisting of alkylphosphonic acid residues, alkyl phosphate residues, and substituted phosphoramide residues, thereby providing excellent stability in vivo and exhibiting excellent site-specific editing activity against target RNA.

[0068] The target-editing oligonucleotide of this embodiment is shorter than conventional target-editing oligonucleotides, and therefore production costs are simply reduced. Furthermore, chemical synthesis is facilitated, making it easier to apply modified nucleotides, such as existing artificial nucleic acids used in nucleic acid drug development, to target-editing oligonucleotides. This makes it possible to provide target-editing oligonucleotides with even higher functionality by improving resistance to intracellular degradation and cellular introduction efficiency. Furthermore, because the target-editing oligonucleotide is composed of the minimum number of residues required for editing induction, it is possible to suppress off-target editing at positions other than the adenosine residue, which is the editing target, while maintaining specificity for the target RNA.

[0069] The target-editing oligonucleotide may, for example, preferentially recruit ADAR1, one of the ADARs that catalyze target editing, to the target RNA, thereby efficiently inducing site-specific editing of the target RNA. ADAR is an enzyme that converts adenosine residues in double-stranded RNA to inosine residues through hydrolytic deamination, and is 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 or the like occur despite the absence of DNA mutations in the genome, thereby controlling the function of the target gene.

[0070] When introduced into mammalian cells, the target-editing oligonucleotide may preferentially recruit ADAR1 present in the cells to the target RNA and induce site-specific editing of the target RNA. Here, preferentially for ADAR1 may mean, for example, that in vitro, the ratio of editing-inducing activity for ADAR1 to editing-inducing activity for ADAR2 exceeds 1, and preferably is 1.5 or greater, 2 or greater, or 5 or greater. The editing-inducing activity for ADAR1 and ADAR2 in cells refers to the case where, when an ADAR1 expression plasmid and an ADAR2 expression plasmid constructed using the same expression vector are transfected and expressed in cells under the same conditions, ADAR1 has a higher editing induction efficiency at the same target editing site.

[0071] The first oligonucleotide contained in the target-editing oligonucleotide identifies the target RNA. That is, the first oligonucleotide may contain a base sequence complementary to the base sequence of the target RNA and form a complementary strand with 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 typically a protein-encoding mRNA precursor (including pre-mRNA) or 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 sequence of the target RNA may be mutated so as to encode a phenotype different from the native form. Furthermore, the residue linked to the 5' side of the adenosine residue that is the editing target in the target RNA is not particularly limited, but may preferably be a cytidine residue or a uridine residue.

[0072] The target RNA is preferably an RNA encoding a protein, such as a serotonin receptor, a glutamate receptor, a voltage-dependent potassium channel, or a phosphorylated protein involved in signal transduction, such as STAT3, NFkBIA, or MAPK14.

[0073] The target-editing oligonucleotide can be applied to, for example, the treatment of a genetic disease. The genetic disease may be, for example, a disease that can be treated by converting an adenosine residue in a target RNA to an inosine residue. The genetic disease may also be, for example, a genetic disease caused by a mutation from a guanosine residue to an adenosine residue in a gene. Examples of genetic diseases 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, epidermolysis bullosa, Fabry disease, factor V Leiden-related disorders, familial adenomatosis, 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 the like. -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-associated diseases such as 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, various forms of cancer (e.g., BRCA1 and 2-associated breast cancer, ovarian cancer, etc.).

[0074] Specific examples of genetic diseases include type I citrullinemia (ASS1; known mutations include Gly390Arg c.1168G>A (rs121908641)), type II citrullinemia (SLC25A13; known mutations include IVS11+1G>A (rs80338722)), hemophilia (Factor V Leiden; known mutations include Arg506Gln c.1601G>A (rs6025)), primary hyperoxaluria (AGXT; known mutations include G170R c.508G>A (rs121908529)), and distal myopathy (GNE; R266Q c.797G>A (rs121908622) and other known diseases), cystic fibrosis (CFTR; W1282X c.3846G>A (rs77010898) and other known diseases), homocystinuria (CBS; Asp444Asn c.1330G>A (rs28934891) and other known diseases), Hurler syndrome: mucopolysaccharidosis (IDUA (SLC26A1; Gly51Asp c.152G>A (rs794726877) and other known diseases), Alexander disease (GFAP; R239H, c.716G>A (rs59565950) and other known diseases), retinitis pigmentosa (EYS; W2940X, Examples of known diseases include phenylketonuria (PAH; R261Q c.782G>A (rs5030849) and the like), hemochromatosis (HFE; C282Y c.845G>A (rs1800562) and the like), and Gilbert's syndrome (UGT1A1; G71R c.211.G>A (rs4148323) and the like). The names of the target genes and examples of their gene mutations are shown in parentheses.

[0075] The c.1168G>A mutation (rs121908641) in the ASS1 gene, which causes type I citrullinemia, is a G to A mutation at nucleotide 1524 of Homo sapiens argininosuccinate synthase 1 (ASS1), transcript variant 1, mRNA (GenBank Accession No: NM_000050.4). In one embodiment, an oligonucleotide targets this adenosine residue. For example, in one embodiment, the nucleotide sequence of the oligonucleotide, excluding the target-corresponding nucleoside residue, may be completely complementary to a region of 20 to 40 consecutive bases contained in positions 1500 to 1540 of the ASS1 mRNA, preferably a region of 20 to 29 consecutive bases contained in positions 1504 to 1534, and more preferably a region of 20 to 25 consecutive bases contained in positions 1510 to 1534.

[0076] The c.1601G>A mutation (rs6025) in the Factor V gene, which causes hemophilia (thrombosis), is a G to A mutation at nucleotide 1696 of Homo sapiens coagulation factor V (F5) mRNA (GenBank Accession No: NM_000130.5). In one embodiment, an oligonucleotide targets this adenosine residue. For example, in one embodiment, the nucleotide sequence of the oligonucleotide, excluding the target-corresponding nucleoside residue, may be completely complementary to a region of 20 to 40 consecutive bases contained in positions 1672 to 1712 of the F5 mRNA, preferably a region of 20 to 29 consecutive bases contained in positions 1676 to 1706, and more preferably a region of 20 to 25 consecutive bases contained in positions 1682 to 1706.

[0077] Abnormalities in post-transcriptional modification of the GRIA2 gene can cause amyotrophic lateral sclerosis (ALS). Abnormalities in A-to-I RNA editing at adenosine 2135 of Homo sapiens glutamate ionotropic receptor AMPA type subunit 2 (GRIA2), transcript variant 1, mRNA (GenBank Accession No: NM_000826.4) can correspond to such abnormalities in post-transcriptional modification. In one embodiment, an oligonucleotide targets such adenosine residue. For example, in one embodiment, the nucleotide sequence of the oligonucleotide, excluding the target-corresponding nucleoside residue, may be completely complementary to a region of 20 to 40 consecutive bases included in positions 2111 to 2151 of the GRIA2 mRNA, preferably a region of 20 to 29 consecutive bases included in positions 2115 to 2145, and more preferably a region of 20 to 25 consecutive bases included in positions 2121 to 2145.

[0078] The c.1561G>A mutation (rs137852959) in the PANK2 gene, which causes pantothenate-related neurodegenerative disease, is a G to A mutation at nucleotide 1728 of Homo sapiens pantothenate kinase 2 (PANK2) transcript variant 1 mRNA (GenBank Accession No: NM_153638.3). In one embodiment, an oligonucleotide targets this adenosine residue. For example, in one embodiment, the nucleotide sequence of the oligonucleotide, excluding the target-corresponding nucleoside residue, may be completely complementary to a region of 20 to 40 consecutive bases included in positions 1704 to 1744 of the PANK2 mRNA, preferably a region of 20 to 29 consecutive bases included in positions 1708 to 1738, and more preferably a region of 20 to 25 consecutive bases included in positions 1714 to 1738.

[0079] The c.1330G>A mutation (rs28934891) in the CBS gene, which causes homocystinuria, is a G to A mutation at nucleotide 1575 of Homo sapiens cystationine beta-synthase (CBS), transcript variant 1, mRNA (GenBank Accession No: NM_000071.2). In one embodiment, an oligonucleotide targets this adenosine residue. For example, in one embodiment, the nucleotide sequence of the oligonucleotide, excluding the target-corresponding nucleoside residue, may be completely complementary to a region of 20 to 40 consecutive bases included in positions 1551 to 1591 of the CBS mRNA, preferably a region of 20 to 29 consecutive bases included in positions 1555 to 1585, and more preferably a region of 20 to 25 consecutive bases included in positions 1561 to 1585.

[0080] The c.413G>A mutation (rs74315342) in the NPHS2 gene, which causes monosegmental glomerulosclerosis, is a G to A mutation at nucleotide 497 of Homo sapiens NPHS2 stomatin family member, podocin (NPHS2), transcript variant 1, mRNA (GenBank Accession No: NM_014625.4). In one embodiment, an oligonucleotide targets this adenosine residue. For example, in one embodiment, the nucleotide sequence of the oligonucleotide, excluding the target-corresponding nucleoside residue, may be completely complementary to a region of 20 to 40 consecutive bases contained in the 473rd to 513th positions of the NPHS2 mRNA, preferably a region of 20 to 29 consecutive bases contained in the 477th to 507th positions, and more preferably a region of 20 to 25 consecutive bases contained in the 483rd to 507th positions.

[0081] The c.1096G>A mutation (rs28929474) in the SERPINA1 gene, which causes α1-antitrypsin deficiency, is a G to A mutation at nucleotide 1143 of Homo sapiens serpin family A member 1 (SERPINA1), transcript variant 1, mRNA (GenBank Accession No: NM_000295.5). In one embodiment, an oligonucleotide targets this adenosine residue. For example, in one embodiment, the nucleotide sequence of the oligonucleotide, excluding the target-corresponding nucleoside residue, may be completely complementary to a region of 20 to 40 consecutive bases included in positions 1119 to 1159 of the SERPINA1 mRNA, preferably a region of 20 to 29 consecutive bases included in positions 1123 to 1153, and more preferably a region of 20 to 25 consecutive bases included in positions 1129 to 1153.

[0082] The c.782G>A mutation (rs5030849) in the PAH gene, which causes phenylketonuria, is a G to A mutation at nucleotide 896 of Homo sapiens phenylalanine hydroxylase (PAH) transcript variant 1 mRNA (GenBank Accession No: NM_000277.3). In one embodiment, an oligonucleotide targets this adenosine residue. For example, in one embodiment, the nucleotide sequence of the oligonucleotide, excluding the target-corresponding nucleoside residue, may be completely complementary to a region of 20 to 40 consecutive bases included in positions 872 to 912 of the PAH mRNA, preferably a region of 20 to 29 consecutive bases included in positions 876 to 906, and more preferably a region of 20 to 25 consecutive bases included in positions 882 to 906.

[0083] The c.940+1G>A mutation (rs765249238) in the SLCO2A1 gene, which causes pachydermoperiostosis, is a G to A mutation at nucleotide 81,034 of the pre-mRNA of Homo sapiens solute carrier organic anion transporter family member 2A1 (SLCO2A1) mRNA (GenBank Accession No: NM_005630.3) (the editing target is located on the (-) strand of chromosome 3 at nucleotide 133,948,892). In one embodiment, an oligonucleotide targets this adenosine residue. For example, in one embodiment, the nucleotide sequence of the oligonucleotide, excluding the target-corresponding nucleoside residue, may be completely complementary to a region of 20 to 40 consecutive bases included in positions 81010 to 81050 of the SLCO2A1 pre-mRNA, preferably a region of 20 to 29 consecutive bases included in positions 81014 to 81044, and more preferably a region of 20 to 25 consecutive bases included in positions 81020 to 81044.

[0084] The c.716G>A mutation (rs59565950) in the GFAP gene that causes Alexander disease is a G to A mutation at nucleotide 2168 of Homo sapiens glial fibrillary acidic protein (GFAP) transcript variant 1 mRNA (GenBank Accession No: NM_002055.5). In one embodiment, an oligonucleotide targets this adenosine residue. For example, in one embodiment, the nucleotide sequence of the oligonucleotide, excluding the target-corresponding nucleoside residue, may be completely complementary to a region of 20 to 40 consecutive bases from positions 2144 to 2184 of the GFAP mRNA, preferably a region of 20 to 29 consecutive bases from positions 2148 to 2178, and more preferably a region of 20 to 25 consecutive bases from positions 2154 to 2178.

[0085] The c.508G>A mutation (rs121908529) in the AGXT gene, which causes primary hyperoxaluria, is a G to A mutation at nucleotide 550 of Homo sapiens alanine-glyoxylate and serine-pyruvate aminotransferase (AGXT) mRNA (GenBank Accession No: NM_000030.3). In one embodiment, an oligonucleotide targets this adenosine residue. For example, in one embodiment, the nucleotide sequence of the oligonucleotide, excluding the target-corresponding nucleoside residue, may be completely complementary to a region of 20 to 40 consecutive bases contained in the 526th to 566th positions of the AGXT mRNA, preferably a region of 20 to 29 consecutive bases contained in the 530th to 560th positions, and more preferably a region of 20 to 25 consecutive bases contained in the 536th to 560th positions.

[0086] The c.211G>A mutation (rs4148323) in the UGT1A1 gene that causes Gilbert syndrome is a G to A mutation at nucleotide 229 of Homo sapiens UDP glucuronosyltransferase family 1 member A1 (UGT1A1) mRNA (GenBank Accession No: NM_000463.3). In one embodiment, an oligonucleotide targets this adenosine residue. For example, in one embodiment, the nucleotide sequence of the oligonucleotide, excluding the target-corresponding nucleoside residue, may be completely complementary to a region of 20 to 40 consecutive bases included in positions 205 to 245 of the UGT1A1 mRNA, preferably a region of 20 to 29 consecutive bases included in positions 209 to 239, and more preferably a region of 20 to 25 consecutive bases included in positions 215 to 239.

[0087] The c.7919G>A mutation (rs527236066) in the EYS gene, which causes retinitis pigmentosa, is a G to A mutation at nucleotide 8478 of Homo sapiens eyes shut homolog (EYS), transcript variant 1, mRNA (GenBank Accession No: NM_001142800.2). In one embodiment, an oligonucleotide targets this adenosine residue. For example, in one embodiment, the nucleotide sequence of the oligonucleotide, excluding the target-corresponding nucleoside residues, may be completely complementary to a region of 20 to 40 consecutive bases contained in positions 8454 to 8494 of the EYS mRNA, preferably a region of 20 to 29 consecutive bases contained in positions 8458 to 8488, and more preferably a region of 20 to 25 consecutive bases contained in positions 8464 to 8488.

[0088] The c.797G>A mutation (rs121908622) in the GNE gene, which causes distal myopathy, is a G to A mutation at nucleotide 924 of Homo sapiens glucosamine (UDP-N-acetyl)-2-epimerase / N-acetylmannosamine kinase (GNE), transcript variant 1, mRNA (GenBank Accession No: NM_001128227.3). In one embodiment, an oligonucleotide targets this adenosine residue. For example, in one embodiment, the nucleotide sequence of the oligonucleotide, excluding the target-corresponding nucleoside residue, may be completely complementary to a region of 20 to 40 consecutive bases contained in positions 900 to 940 of the GNE mRNA, preferably a region of 20 to 29 consecutive bases contained in positions 904 to 934, and more preferably a region of 20 to 25 consecutive bases contained in positions 910 to 934.

[0089] The c.845G>A mutation (rs1800562) in the HFE gene, which causes hemochromatosis, is a G to A mutation at nucleotide 1005 of Homo sapiens homeostatic iron regulator (HFE), transcript variant 1, mRNA (GenBank Accession No: NM_000410.3). In one embodiment, an oligonucleotide targets this adenosine residue. For example, in one embodiment, the nucleotide sequence of the oligonucleotide, excluding the target-corresponding nucleoside residue, may be completely complementary to a region of 20 to 40 consecutive bases contained in positions 981 to 1021 of the HFE mRNA, preferably a region of 20 to 29 consecutive bases contained in positions 985 to 1015, and more preferably a region of 20 to 25 consecutive bases contained in positions 991 to 1015.

[0090] The first oligonucleotide comprises a target-corresponding nucleoside residue corresponding to the adenosine residue that is the editing target in the target RNA; a 10- to 24-residue 3'-side oligonucleotide linked to the 3'-side of the target-corresponding nucleoside residue and having a base sequence complementary to the corresponding base sequence in the target RNA; and a 3- to 6-residue 5'-side oligonucleotide linked to the 5'-side of the target-corresponding nucleoside residue and having a base sequence complementary to the corresponding base sequence in the target RNA. The oligonucleotides linked to the 3'-side and 5'-side of the target-corresponding nucleoside residue form a double-stranded structure with the target RNA, forming a complementary strand as a whole (hereinafter also referred to as the first complementary strand), thereby identifying the target RNA and the editing target site in the target RNA. Here, as used herein, the term "complementary base sequence" includes base sequences that form Watson-Crick base pairs as well as base sequences that form thermodynamically stable non-Watson-Crick wobble base pairs, such as a G-U base pair. The base pairs forming the first complementary strand may all be Watson-Crick base pairs except for the target-corresponding nucleotide residue, or at least some may be wobble base pairs. When the first complementary strand contains wobble base pairs, the number of wobble base pairs may be 1 or 2. Hereinafter, for convenience, a region consisting of three residues, namely, the target-corresponding nucleoside residue, one residue on the 5' side thereof, and one residue on the 3' side thereof, will be referred to as a counter region, and the portion of the first oligonucleotide other than the counter region will be referred to as a non-counter region.

[0091] In one embodiment, the 10- to 24-residue 3'-side oligonucleotide linked to the 3'-side of the target-corresponding nucleoside residue in the first oligonucleotide may have a first nucleoside residue on the 3'-side of the target-corresponding nucleoside residue that is a nucleoside residue that does not form a complementary pair with the corresponding nucleoside residue in the target RNA, and the other nucleoside residues form complementary pairs with the corresponding nucleoside residues in the target RNA to form a double-stranded structure with the target RNA. Specifically, for example, when a guanosine residue is linked to the 5'-side of an adenosine residue that is the editing target of the target RNA, the first nucleoside residue on the 3'-side of the target-corresponding nucleoside residue may be a 2'-deoxyinosine residue.

[0092] The target-corresponding nucleoside residue is a nucleoside residue corresponding to the adenosine residue that is the editing target, such as a cytidine residue, guanosine residue, adenosine residue, or derivatives thereof. The target-corresponding nucleoside 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. In one aspect, the target-corresponding nucleoside residue may be an N-alkylpyrimidine nucleoside residue, preferably an N-alkylcytidine residue, an N-alkyl-2'-deoxycytidine residue, an N-alkyluridine residue, or an N-alkyl-2'-deoxyuridine residue. The alkyl group in the N-alkylpyrimidine nucleoside residue may be an alkyl group having 1 to 6 or 1 to 4 carbon atoms, and preferably a methyl group or an ethyl group. When the target-corresponding nucleoside residue is an N-alkylpyrimidine nucleoside residue, editing-inducing activity may be improved. Furthermore, the sugar moiety of the N-alkylpyrimidine nucleoside residue may be further modified, or the phosphorus-containing linking group (e.g., phosphate residue) linking the adjacent nucleoside residue may be further modified. Modifications of the sugar moiety and phosphate residue are described below.

[0093] In one embodiment, the target-corresponding nucleoside residue may be a 2'-deoxycytidine residue, which may be further modified at the phosphate residue linked to the 2'-deoxycytidine residue.

[0094] In the first oligonucleotide, the number of residues of the oligonucleotide linked to the 3' side of the target-corresponding nucleoside residue and having a base sequence complementary to the target RNA may be 10 to 24, preferably 11 or more, 12 or more, or 13 or more, and also preferably 22 or less, 20 or less, 18 or less, 16 or less, or 15 or less. In one aspect, the number of residues of the oligonucleotide having a base sequence complementary to the target RNA may be 12 to 20, 12 to 18, 12 to 16, 14 to 18, or 14 to 16. Furthermore, in one aspect, the number of residues of the oligonucleotide having a base sequence complementary to the target RNA may be 14. When the number of residues is within the above range, selectivity for ADAR1 tends to be further improved.

[0095] In the first oligonucleotide, the 3'-side oligonucleotide linked to the 3'-side of the target-corresponding nucleoside residue may have a complementary base sequence that does not contain mismatched base pairs with respect to the target RNA. In this case, it is preferable that the target RNA does not have a guanosine residue linked to the 5'-side of the adenosine residue that is the editing target. That is, when the base linked to the 5'-side of the adenosine residue that is the editing target of the target RNA is an adenosine residue, a cytidine residue, or a uridine residue, it is preferable that the oligonucleotide linked to the 3'-side of the target-corresponding nucleoside residue in the first oligonucleotide has a complementary base sequence that does not contain mismatched base pairs with respect to the target RNA.

[0096] In the first oligonucleotide, the 3'-side oligonucleotide linked to the 3'-side of the target-corresponding nucleoside residue may, in some cases, contain a base non-complementary to the base sequence of the target RNA. For example, in a target RNA in which a guanosine residue is linked to the 5'-side of an adenosine residue that is the editing target, the editing-inducing activity may be reduced. Even in this case, the editing-inducing activity can be improved by having the first oligonucleotide have a base sequence including a base non-complementary to the guanosine residue. The base non-complementary to the guanosine residue may be, for example, a guanosine residue. In other words, a target-editing oligonucleotide for a target RNA in which a guanosine residue is linked to the 5'-side of an adenosine residue that is the editing target may have a guanosine residue linked to the 3'-side of the target-corresponding nucleoside residue.

[0097] In the first oligonucleotide, the 3' oligonucleotide linked to the 3' side of the target-corresponding nucleoside residue may optionally form a non-Watson-Crick wobble base pair with the corresponding nucleoside residue of the target RNA, and the 3' oligonucleotide linked to the 3' side of the target-corresponding nucleoside residue may optionally contain a 2'-deoxynucleoside residue, which may improve editing target activity.

[0098] For example, when a cytidine residue is linked to the 5' side of an adenosine residue that is the editing target in the target RNA, the first oligonucleotide may have a nucleoside residue having a hypoxanthine residue as a base linked to the 3' side of the target-corresponding nucleoside residue. That is, the cytidine residue linked to the 5' side of the adenosine residue that is the editing target and the nucleoside residue having a hypoxanthine residue as a base in the first oligonucleotide may form a wobble base pair. The nucleoside residue having a hypoxanthine residue as a base may be an inosine residue or a 2'-deoxyinosine residue, preferably a 2'-deoxyinosine residue. Furthermore, the nucleoside residue having a hypoxanthine residue as a base may be further modified in the sugar moiety, or the phosphorus-containing linking group (e.g., a phosphate residue) that links to the adjacent nucleoside residue may be further modified. Modifications of the sugar moiety and phosphate residue will be described below.

[0099] For example, when a uridine residue is linked to the 5' side of an adenosine residue that is the editing target in the target RNA, the first oligonucleotide may have a nucleoside residue having an adenyl group as a base linked to the 3' side of the target-corresponding nucleoside residue. That is, the uridine residue linked to the 5' side of the adenosine residue that is the editing target may form a base pair with the nucleoside residue that has an adenyl group as a base in the first oligonucleotide. The nucleoside residue that has an adenyl group as a base may be an adenosine residue or a 2'-deoxyadenosine residue, preferably a 2'-deoxyadenosine residue. Furthermore, the nucleoside residue that has an adenyl group as a base may be further modified in the sugar moiety, or the phosphorus-containing linking group (e.g., a phosphate residue) that links to the adjacent nucleoside residue may be further modified. Modifications of the sugar moiety and phosphate residue will be described below.

[0100] For example, when an adenosine residue is linked to the 5' side of an adenosine residue that is the editing target in the target RNA, the first oligonucleotide may have a nucleoside residue having a pyrimidinyl group as a base linked to the 3' side of the target-corresponding nucleoside residue. That is, the adenosine residue linked to the 5' side of the adenosine residue that is the editing target may form a base pair with a nucleoside residue that has a pyrimidinyl group as a base in the first oligonucleotide. The nucleoside residue having a pyrimidinyl group as a base may be a thymidine residue or a 2'-deoxyuridine residue. Furthermore, the nucleoside residue having a pyrimidinyl group as a base may be further modified in the sugar moiety, or the phosphorus-containing linking group (e.g., a phosphate residue) that links to the adjacent nucleoside residue may be further modified. Modifications of the sugar moiety and phosphate residue will be described below.

[0101] For example, when a guanosine residue is linked to the 5' side of an adenosine residue that is the editing target in the target RNA, the first oligonucleotide may have a nucleoside residue having a hypoxanthine residue as a base linked to the 3' side of the target-corresponding nucleoside residue. The nucleoside residue having a hypoxanthine residue may be a 2'-deoxyinosine residue. Furthermore, the nucleoside residue having a hypoxanthine residue may be further modified in the sugar moiety, or the phosphorus-containing linking group (e.g., phosphate residue) linking to the adjacent nucleoside residue may be further modified. Modifications of the sugar moiety and phosphate residue will be described below.

[0102] In the first oligonucleotide, the number of residues of the 5'-side oligonucleotide linked to the 5'-side of the target-corresponding nucleoside residue and having a base sequence complementary to the target RNA may be 3 or more and 6 or less, preferably 3 or more and 5 or less, 3 or more and 4 or less, or 3. When the number of residues is within the above range, selectivity for ADAR1 tends to be further improved.

[0103] The second oligonucleotide forms a double-stranded structure (hereinafter also referred to as the second complementary strand) with the target RNA, containing one or more nucleoside residues that do not form complementary base pairs. The number of residues in the second oligonucleotide may be 2 to 10 or 4 to 8. The number of residues in the second oligonucleotide is, for example, 3 or more, preferably 4 or more or 5 or more, and for example, 10 or less, preferably 9 or less, 8 or less, or 7 or less. In one aspect, the number of residues in the second oligonucleotide may be 6. When the number of residues is within the above range, editing induction tends to be more enhanced. The number of nucleoside residues in the second complementary strand that do not form complementary base pairs is, for example, 3 or less, or 2 or less, preferably 1.

[0104] The nucleoside residues that do not form a complementary base pair in the second complementary strand may be nucleoside residues that are present without base pairing due to the absence of a nucleoside residue corresponding to the other nucleoside residue in either the target RNA or the second oligonucleotide. Alternatively, the nucleoside residues that do not form a complementary base pair in the second complementary strand may be nucleoside residues that are present in both the target RNA and the second oligonucleotide due to a mismatched base pair (non-complementary base pair) in which the base pairs consisting of corresponding nucleoside residues in the target RNA and the second oligonucleotide have non-complementary nucleic acid bases. In one embodiment, the nucleoside residues that do not form a complementary base pair may be nucleoside residues that are present in either the target RNA or the second oligonucleotide and do not form base pairs, preferably nucleoside residues that are present in the target RNA and do not form base pairs. This tends to further enhance editing induction. Here, a mismatched base pair means that the nucleic acid bases of two corresponding nucleoside residues in the target RNA and the second oligonucleotide do not form a stable base pair.

[0105] The position of the nucleoside residue that does not form a complementary base pair in the second complementary strand may be the first residue on the 3' side from the binding position between the first complementary strand and the second complementary strand on the target RNA, or the position of the nucleoside residue that does not form a complementary base pair in the second complementary strand may be the first residue on the 5' side from the binding position between the first complementary strand and the second complementary strand on the second oligonucleotide.

[0106] The nucleoside residue in the second complementary strand that does not form a complementary base pair may be derived from a deletion in the second oligonucleotide of a nucleoside residue that corresponds to the first nucleoside residue in the target RNA that is located 3' from the binding position between the first complementary strand and the second complementary strand. Alternatively, the nucleoside residue in the second complementary strand that does not form a complementary base pair may be derived from a mismatch base pair formed between the first nucleoside residue in the target RNA that is located 3' from the binding position between the first complementary strand and the second complementary strand and the nucleoside residue at the 3' end of the second oligonucleotide.

[0107] In the second oligonucleotide, when the nucleoside residue corresponding to the nucleoside residue of the target RNA is deleted, the second oligonucleotide may have a base sequence complementary to the target RNA except for the part where the nucleoside residue is deleted.Also, when the second oligonucleotide has a nucleoside residue that does not form a complementary pair with the nucleoside residue corresponding to the nucleoside residue of the target RNA, the second oligonucleotide may have a base sequence complementary to the target RNA except for the nucleoside residue that does not form a complementary pair.

[0108] In one embodiment of the target-editing oligonucleotide, the first oligonucleotide that forms a first complementary strand with the target RNA has a cytidine residue as the target-corresponding nucleoside residue and a base sequence complementary to the base sequence of the target RNA, and includes a 3'-side oligonucleotide having 12 to 18 or 14 to 16 residues linked to the 3'-side of the target-corresponding nucleotide residue, and a 5'-side oligonucleotide having a base sequence complementary to the base sequence of the target RNA and having 3 residues linked to the 5'-side of the target-corresponding nucleoside residue, and the second oligonucleotide lacks a nucleoside residue corresponding to the first residue 3'-side from the binding position of the first complementary strand and the second complementary strand in the target RNA, and includes an oligonucleotide having 4 to 8 residues, and has a complementary base sequence from the second residue onwards.

[0109] The nucleotides constituting the target-editing oligonucleotide may be selected from natural ribonucleotides (RNA), natural deoxyribonucleotides (DNA), RNA / DNA chimeras, and modified nucleotides, which are modifications of these. The nucleotides constituting the target-editing oligonucleotide may be linked to each other by phosphorus-containing linking groups such as phosphodiester bonds attached to hydroxyl groups in the sugar moieties of the nucleosides. The bond between nucleosides via phosphorus-containing linking groups may be formed using the 2'-, 3'-, or 5'-hydroxyl groups in the sugar moieties. The nucleosides constituting the target-editing oligonucleotide may be linked by phosphate residues to form natural 3'-5' phosphodiester bonds. Furthermore, the nucleosides constituting the target-editing oligonucleotide may be linked by phosphorus-containing linking groups other than phosphate residues to form non-natural 3'-5' phosphorus-containing diester bonds. Here, non-natural phosphorus-containing diester bonds mean that the phosphodiester bond moiety is modified. At least one of the nucleosides constituting the target-editing oligonucleoside may be a modified nucleoside. Examples of modified nucleosides include those in which the sugar moiety is modified, those in which the base is modified, and combinations thereof.

[0110] Modifications of the sugar moiety include, for example, 2'-O-alkylated ribonucleotides of D-ribofuranose (e.g., 2'-O-methylated, 2'-O-ethylated, 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; Examples of suitable nucleotides include 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, and the like; 3'-deoxy-3'-amino-2'-deoxy-D-ribofuranose; 3'-deoxy-3'-amino-2'-deoxy-2'-fluoro-D-ribofuranose; 2'-deoxy-2'-fluoro-D-ribofuranose; and D-deoxyribose.

[0111] Modifications of the phosphodiester bond moiety include phosphorothioate bonds (including optically active forms derived from the asymmetry of the phosphorus atom), alkyl phosphate bonds, alkyl phosphonate bonds, alkylthiophosphonate bonds, phosphorodithioate bonds, phosphoramidate bonds, and the like.

[0112] Here, the phosphorothioate bond is formed by forming an ester bond between the thiophosphate and the hydroxyl groups of the sugar moieties of two nucleosides, respectively. That is, the phosphorothioate bond can be formed by the thiophosphate residue.

[0113] The alkyl phosphate bond can be formed by alkyl phosphate residues forming ester bonds with the hydroxyl groups of the sugar moieties of two nucleosides, respectively.

[0114] The alkylphosphonate bond is formed by forming an ester bond between the alkylphosphonic acid and the hydroxyl groups of the sugar moieties of two nucleosides, respectively. That is, the alkylphosphonate bond can be formed by an alkylphosphonic acid residue.

[0115] The alkylthiophosphonate bond is formed by forming an ester bond between the alkylthiophosphonic acid and the hydroxyl groups of the sugar moieties of two nucleosides. That is, the alkylthiophosphonate bond can be formed by the alkylthiophosphonic acid residue.

[0116] A phosphorodithioate bond is formed when a dithiophosphate forms an ester bond with the hydroxyl groups of the sugar moieties of two nucleosides, i.e., a phosphorodithioate bond can be formed by a dithiophosphate residue.

[0117] A phosphoramidate bond is formed when an optionally substituted phosphoric acid amide forms an ester bond with each of the hydroxyl groups of the sugar moieties of two nucleosides. That is, a phosphoramidate bond can be formed by a phosphoramidate residue that may have a substituent. The substituent in the phosphoric acid amide is a substituent on the nitrogen atom, and examples thereof include an alkyl group, an alkylsulfonyl group, and an imino group that may have a substituent. Specific examples of the alkyl group and the alkyl moiety of the alkylsulfonyl group include alkyl groups having 1 to 6 carbon atoms, such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a t-butyl group, a cyclobutyl group, a pentyl group, and a hexyl group, as well as substituted alkyl groups such as a 2-methoxyethyl group. Specific examples of the imino group that may have a substituent include a 1,3-dimethylimidazolidin-2-ylidene group.

[0118] The alkyl group in the alkyl phosphate, alkylphosphonic acid, alkylthiophosphonic acid, and phosphoric acid amide may be linear, branched, cyclic, or a combination thereof. The number of carbon atoms in the alkyl group may be, for example, 1 to 6, and preferably 1 to 3 or 1. Specific examples of the alkyl group include a methyl group, an ethyl group, a 2-methoxyethyl group, a propyl group, a 3-methoxypropyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a t-butyl group, a cyclobutyl group, a pentyl group, and a hexyl group. The alkyl group may further have a substituent. Examples of the substituent in the alkyl group include a halogen atom such as a fluorine atom or a chlorine atom, and an alkoxy group having 1 to 3 carbon atoms.

[0119] Specific examples of alkyl phosphates include methyl phosphate, ethyl phosphate, propyl phosphate, isopropyl phosphate, butyl phosphate, 2-methoxyethyl phosphate, cyclopropyl phosphate, and cyclobutyl phosphate. Specific examples of alkyl phosphonic acids include methyl phosphonic acid, ethyl phosphonic acid, propyl phosphonic acid, isopropyl phosphonic acid, butyl phosphonic acid, and 3-methoxypropyl phosphonic acid. Specific examples of alkyl thiophosphonic acids include methyl thiophosphonic acid, ethyl thiophosphonic acid, propyl thiophosphonic acid, and isopropyl thiophosphonic acid. Specific examples of substituted phosphoric acid amides include N-methyl phosphoric acid amide, N-ethyl phosphoric acid amide, and N-methanesulfonyl phosphoric acid amide (mesyl phosphoric acid amide).

[0120] Modifications of the base moiety 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-fluoronation, 5-bromination, 5-iodination, N4-methylation, etc. of cytosine; 5-demethylation (uracil), 5-fluoronation, 5-bromination, 5-iodination, etc. of thymine; N6-methylation, 8-bromination, etc. of adenine; and N2-methylation, 8-bromination, etc. of guanine.

[0121] In the target-editing oligonucleotide, in the oligonucleotide linked to the 3' side of the target-corresponding nucleoside residue, counting in the 3' direction from the target-corresponding nucleoside residue, at least one phosphorus-containing linking group selected from the group consisting of a phosphorus-containing linking group linking the first nucleoside residue to the second nucleoside residue and a phosphorus-containing linking group linking the fourth nucleoside residue to the fifth nucleoside residue may be at least one type selected from the group consisting of alkylphosphonic acid residues, alkyl phosphate residues, and substituted phosphoramide residues, preferably at least one type selected from the group consisting of alkylphosphonic acid residues and alkyl phosphate residues, and more preferably at least one type selected from the group consisting of methylphosphonic acid residues, ethyl phosphate residues, and 2-methoxyethyl phosphate residues. Furthermore, counting in the 3' direction from the target-corresponding nucleoside residue, the phosphorus-containing linking group linking the first nucleoside residue and the second nucleoside residue and the phosphorus-containing linking group linking the fourth nucleoside residue and the fifth nucleoside residue may each independently be at least one selected from the group consisting of alkylphosphonic acid residues, alkyl phosphate residues, and substituted phosphoramide residues, preferably at least one selected from the group consisting of alkylphosphonic acid residues and alkyl phosphate residues, and more preferably at least one selected from the group consisting of methylphosphonic acid residues, ethyl phosphate residues, and 2-methoxyethyl phosphate residues. Here, the first nucleoside residue counting in the 3' direction from the target-corresponding nucleoside residue is the first nucleoside residue counting not including the target-corresponding nucleoside residue itself, i.e., the nucleoside residue adjacent to the 3' side of the target-corresponding oligonucleoside residue.

[0122] In this specification, counting in the 3' direction from the target-corresponding nucleoside residue, the position of the phosphorus-containing linking group linking the target-corresponding nucleoside residue and the first nucleoside residue is sometimes referred to as an internucleoside distance of 0, and the position of the phosphorus-containing linking group linking the fourth nucleoside residue and the fifth nucleoside residue is sometimes referred to as an internucleoside distance of +4. Furthermore, counting in the 5' direction from the target-corresponding nucleoside residue, the position of the phosphorus-containing linking group linking the first nucleoside residue and the target-corresponding nucleoside residue is sometimes referred to as an internucleoside distance of -1, and the position of the phosphorus-containing linking group linking the seventh nucleoside residue and the sixth nucleoside residue is sometimes referred to as an internucleoside distance of -7. Similar designations may be used for other internucleosides. When the second oligonucleotide has a nucleoside residue at the 3' end that does not form a complementary base pair with a nucleoside residue of the target RNA, the nucleoside residue is not taken into consideration when specifying the position of the phosphorus-containing linking group.

[0123] The target-editing oligonucleotide includes, counting in the 3' direction from the target-corresponding nucleoside residue, a phosphorus-containing linking group (+2) linking the second nucleoside residue to the third nucleoside residue, a phosphorus-containing linking group (+6) linking the sixth nucleoside residue to the seventh nucleoside residue, a phosphorus-containing linking group (+8) linking the eighth nucleoside residue to the ninth nucleoside residue, a phosphorus-containing linking group (+10) linking the tenth nucleoside residue to the eleventh nucleoside residue, and a phosphorus-containing linking group (+11) linking the eleventh nucleoside residue. and the 12th nucleoside residue (+11); and, counting in the 5' direction from the target-corresponding nucleoside residue, at least one phosphorus-containing linking group selected from the group consisting of a phosphorus-containing linking group (-2) linking the second nucleoside residue and the first nucleoside residue, a phosphorus-containing linking group (-3) linking the third nucleoside residue and the second nucleoside residue, and a phosphorus-containing linking group (-6) linking the sixth nucleoside residue and the fifth nucleoside residue may be a phosphate residue. In the first oligonucleotide, preferably, between nucleosides, at least one phosphorus-containing linking group selected from the group consisting of a +2 phosphorus-containing linking group, a +8 phosphorus-containing linking group, a +10 phosphorus-containing linking group, a +11 phosphorus-containing linking group, and a -6 phosphorus-containing linking group may be a phosphate residue. In the first oligonucleotide, more preferably, at least the +2 phosphorus-containing linking group may be a phosphate residue. That is, in the first oligonucleotide, the second nucleoside residue and the third nucleoside residue, counting in the 3' direction from at least the target-corresponding nucleoside residue, may be linked by a phosphodiester bond. The number of phosphate residues linking the nucleoside residues constituting the first oligonucleotide may be, for example, 0 to 8, preferably 4 or less. The number of phosphate residues may be 1 or more.

[0124] In the target-editing oligonucleotide, some nucleoside residues may be linked by phosphorothioate bonds. In the target-editing oligonucleotide, the phosphorus-containing linking groups linking the nucleoside residues, other than phosphate residues, alkylphosphonic acid residues, alkyl phosphate residues, and substituted phosphoramide residues, may be phosphorus thioate residues. Furthermore, in the target-editing oligonucleotide, all phosphorus-containing linking groups other than phosphorus-containing linking groups in which the internucleoside bond is +1, +2, +4, +8, +10, +11, -2, -3, or -6 may be phosphorus thioate residues, and all phosphorus-containing linking groups other than at least one phosphorus-containing linking group in which the internucleoside bond is +1 or +4 and at least one phosphorus-containing linking group in which the internucleoside bond is +8, +10, +11, or -6 may be phosphorus thioate residues.

[0125] The counter region of the first oligonucleotide may contain a phosphorothioate bond, and at least one of the phosphorus-containing linking group linking the target-corresponding nucleoside residue to the nucleoside residue on its 3' side and the phosphorus-containing linking group linking the target-corresponding nucleoside residue to the nucleoside residue on its 5' side may be a thiophosphate residue, or both may be thiophosphate residues. That is, the first oligonucleotide may have a partial structure in which the target-corresponding nucleoside residue and the nucleoside residue on its 3' side are linked via a phosphorothioate bond, or may have a partial structure in which the target-corresponding nucleoside residue and the nucleoside residue on its 5' side are linked via a phosphorothioate bond, or may have a partial structure in which the target-corresponding nucleoside residue is linked to the nucleoside residue on its 3' side and the nucleoside residue on its 5' side via phosphorothioate bonds, respectively.

[0126] The target-editing oligonucleotide may comprise at least one modified nucleotide residue selected from the group consisting of a 2'-O-alkylribonucleoside residue, a 2'-deoxy-2'-fluororibonucleoside residue, a bridged nucleoside residue, and a 2'-deoxyribonucleoside residue.

[0127] In the target-editing oligonucleotide, the oligonucleoside residue linked to the 3' side of the target-corresponding nucleoside residue may be a 2'-deoxynucleoside residue, and the oligonucleoside residue linked to the 5' side of the target-corresponding nucleoside residue may be a 2'-O-alkylribonucleoside residue or a 2'-deoxynucleoside residue, preferably a 2'-O-alkylnucleoside residue.

[0128] The first oligonucleotide may have a base sequence in which the 3'-side oligonucleotide linked to the 3'-side of the target-corresponding nucleoside residue is alternately linked with two types of modified nucleoside residues selected from the group consisting of 2'-deoxy-2'-fluoronucleoside residues, 2'-O-alkylribonucleoside residues, and bridged nucleoside residues. That is, the 3'-side oligonucleotide linked to the 3'-side of the target-corresponding nucleoside residue may have a base sequence in which 2'-deoxy-2'-fluoronucleoside residues and 2'-O-alkylribonucleoside residues are alternately linked, may have a base sequence in which bridged nucleoside residues and 2'-O-alkylribonucleoside residues are alternately linked, or may have a base sequence in which 2'-deoxy-2'-fluoronucleoside residues and bridged nucleoside residues are alternately linked. As used herein, "having a base sequence in which two types of modified nucleoside residues are alternately linked" means either that the target oligonucleotide (here, the 3'-side oligonucleotide of the first oligonucleotide) has a base sequence in which two types of modified nucleoside residues are partially alternately linked, or that the entire target oligonucleotide has a base sequence in which two types of modified nucleoside residues are alternately linked, and this term is used in the same sense in the following explanations of this specification.

[0129] In the 3'-side oligonucleotide of the first oligonucleotide, the third nucleoside residue, counting in the 3' direction from the target-corresponding nucleoside residue, may be a modified nucleoside residue selected from the group consisting of a 2'-deoxy-2'-fluoronucleoside residue, a 2'-O-alkylribonucleoside residue, and a bridged nucleoside residue, preferably a 2'-deoxy-2'-fluoronucleoside residue. Furthermore, in the 3'-side oligonucleotide, the third nucleoside residue, counting in the 3' direction from the target-corresponding nucleoside residue, may be a modified nucleoside residue selected from the group consisting of a 2'-deoxy-2'-fluoronucleoside residue, a 2'-O-alkylribonucleoside residue, and a bridged nucleoside residue, and the fourth nucleoside residue may be a modified nucleoside residue different from the third modified nucleoside residue.

[0130] The first oligonucleotide may have at least one type of bridged nucleoside residue at the 10th or later nucleoside residue, preferably the 11th or later nucleoside residue, counting in the 3' direction from the target-corresponding nucleoside residue. The bridged nucleoside residue may be bridged between the 2' and 4' positions of the D-ribofuranose. The bridged nucleoside residue may include at least one of a 2'-O,4'-C-methylene-bridged nucleoside (LNA) and a 2'-O,4'-C-ethylene-bridged nucleoside (ENA). Furthermore, the base moiety of the bridged nucleoside residue may be further modified, or the phosphorus-containing linking group (e.g., a phosphate residue) linking the adjacent nucleoside residue may be further modified.

[0131] The first oligonucleotide may have at least one type of bridged nucleoside residue in at least one nucleoside residue selected from the group consisting of the fifth, fourth, third, second, and first nucleoside residues, counting from the 3'-end in the 5' direction. Preferably, at least one type of bridged nucleoside residue may be selected from the group consisting of the fifth, third, and first nucleoside residues, counting from the 3'-end in the 5' direction. Here, "fifth, counting from the 3'-end in the 5' direction" means the fifth nucleoside residue, counting from the nucleoside residue adjacent to the 5' side of the 3'-end nucleoside residue as the first.

[0132] The first oligonucleotide may have a base sequence in which 2'-O-alkylribonucleoside residues and bridged nucleoside residues are alternately linked, in an oligonucleotide consisting of nucleoside residues from the 10th position onwards, preferably from the 11th position onwards, counting in the 3' direction from the target-corresponding nucleoside residue. The number of repetitions of linkages between 2'-O-alkylribonucleoside residues and bridged nucleoside residues may be, for example, 1 to 3, and preferably 2. The base sequence in which 2'-O-alkylribonucleoside residues and bridged nucleoside residues are alternately linked may be linked alternately in the 3' direction from the 2'-O-alkylribonucleoside residue, or may be linked alternately in the 3' direction from the bridged nucleoside residue. Furthermore, the base moiety of the 2'-O-alkylribonucleoside residue or bridged nucleoside residue may be further modified, or the phosphorus-containing linking group (e.g., a phosphate residue) linking the adjacent nucleoside residue may be further modified. Here, the number of repetitions of the linkage between a 2'-O-alkylribonucleoside residue and a bridged nucleoside residue is counted as one unit, with the pair of linked 2'-O-alkylribonucleoside residue and bridged nucleoside residue being counted as one unit. That is, three alternately linked residues have one repeat number, and four alternately linked residues have two repeat numbers.

[0133] The 5'-side oligonucleotide linked to the 5'-side of the target-corresponding nucleoside residue of the first oligonucleotide may have a base sequence in which 2'-O-alkylribonucleoside residues are consecutive. The base sequence in which 2'-O-alkylribonucleoside residues are consecutive may be adjacent to the target-corresponding nucleoside residue. The number of residues in the base sequence in which 2'-O-alkylribonucleoside residues are consecutive may be, for example, 2 or 3. Furthermore, the 5'-side oligonucleotide may have a base sequence in which a 2'-O-alkylribonucleoside residue is linked to a 2'-deoxy-2'-fluoronucleoside residue or a bridged nucleoside residue.

[0134] The 5'-side oligonucleotide in the first oligonucleotide may be configured such that some of the nucleoside residues are linked via phosphorothioate bonds, or such that all of the nucleoside residues are linked via phosphorothioate bonds.

[0135] In the second oligonucleotide, at least one residue, at least three residues, or all residues may be modified in at least one of the sugar moiety and the phosphodiester bond, or at least the sugar moiety, at least the phosphodiester bond, or both the sugar moiety and the phosphodiester bond. Modifications of the sugar moiety in the second oligonucleotide may be, for example, 2'-O-alkylation, 2'-deoxy-2'-fluoro modification, cross-linking between the 2' and 4' positions, 2'-deoxylation (DNA modification), etc. When the second oligonucleotide has multiple modified nucleoside residues, the multiple modified nucleotide residues may be arranged consecutively or spaced apart.

[0136] The second oligonucleotide may have a base sequence in which 2'-O-alkylribonucleoside residues are consecutive. The second oligonucleotide may have a base sequence in which two types of nucleoside residues selected from the group consisting of 2'-deoxy-2'-fluoronucleoside residues, 2'-O-alkylribonucleoside residues, and bridged nucleoside residues are alternately linked, or may have a base sequence in which 2'-O-alkylribonucleoside residues and 2'-deoxy-2'-fluoronucleoside residues are alternately linked, or may have a base sequence in which 2'-O-alkylribonucleoside residues and bridged nucleoside residues are alternately linked. Furthermore, from the viewpoint of ADAR1 selectivity, the second nucleoside residue in the second oligonucleotide, counting in the 5' direction from the nucleoside residue at the 3' end of the second oligonucleotide, may be one selected from the group consisting of a 2'-deoxy-2'-fluoronucleoside residue, a 2'-O-alkylribonucleoside residue, and a bridged nucleoside residue, and may be a 2'-O-alkylribonucleoside residue. Here, the nucleoside residue at the 3' end of the second oligonucleotide means the nucleoside residue located farthest from the 5' end among the nucleoside residues that form a complementary pair with a nucleoside residue of the target RNA. Therefore, the second nucleoside residue counting in the 5' direction from the nucleoside residue at the 3' end of the second oligonucleotide refers to the nucleoside residue that forms a complementary pair with a nucleoside residue in the target RNA, excluding the nucleoside residue at the 3' end itself, but counting in the 5' direction, with the nucleoside residue adjacent to the 3' end oligonucleoside residue as the first nucleoside residue.

[0137] The second oligonucleotide may have at least one type of bridged nucleoside residue at the second or later nucleoside residue, preferably the third or later nucleoside residue, counting in the 5' direction from the 3'-terminal nucleoside residue. The bridged nucleoside residue may be bridged between the 2' and 4' positions of the D-ribofuranose. The bridged nucleoside residue may include at least one of a 2'-O,4'-C-methylene-bridged nucleoside (LNA) and a 2'-O,4'-C-ethylene-bridged nucleoside (ENA). Furthermore, the bridged nucleoside residue may be further modified at least in its base moiety and phosphate ester linkage.

[0138] The second oligonucleotide may have a base sequence in which 2'-O-alkylribonucleoside residues and bridged nucleoside residues are alternately linked in an oligonucleotide consisting of the second or subsequent nucleoside residues, preferably the third or subsequent nucleoside residues, counting in the 5' direction from the 3'-terminal nucleoside residue. The number of repetitions of linkages between 2'-O-alkylribonucleoside residues and bridged nucleoside residues may be, for example, 1 to 3, preferably 2. The base sequence in which 2'-O-alkylribonucleoside residues and bridged nucleoside residues are alternately linked may be linked in the 5' direction from the 2'-O-alkylribonucleoside residue, or may be linked in the 5' direction from the bridged nucleoside residue. Furthermore, at least one of the base moiety and the phosphate ester linkage moiety of the 2'-O-alkylribonucleoside residue or the bridged nucleoside residue may be further modified.

[0139] The second oligonucleotide may be configured such that some nucleoside residues are linked via phosphorothioate bonds, or such that all nucleoside residues are linked via phosphorothioate bonds. Furthermore, the phosphorus-containing linking group linking some nucleoside residues in the second oligonucleotide may be at least one selected from the group consisting of alkylphosphonic acid residues, alkyl phosphate residues, and substituted phosphoramide residues. The second oligonucleotide may include at least one phosphorus-containing linking group selected from the group consisting of the phosphorus-containing linking group linking the first nucleoside residue to the second nucleoside residue, the phosphorus-containing linking group linking the third nucleoside residue to the fourth nucleoside residue, and the phosphorus-containing linking group linking the fifth nucleoside residue to the sixth nucleoside residue, counting from the 3'-terminal nucleoside residue in the 5' direction, and the phosphorus-containing linking group selected from the group consisting of the phosphorus-containing linking group linking the fifth nucleoside residue to the sixth nucleoside residue may be at least one selected from the group consisting of alkylphosphonic acid residues, alkyl phosphate residues, and substituted phosphoramide residues.

[0140] In one aspect, the target-editing oligonucleotide may include a modified oligonucleotide having a chemical modification pattern represented by any of the following formulas (1) to (22), or may consist of a modified oligonucleotide having a chemical modification pattern represented by any of the following formulas (1) to (22).

[0141] (1) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (2) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (3) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (4) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (5) 5’- HO-LsMsMsMsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (6) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (7) 5’- HO-MsLsMsLsMsFsXFpMsMsdCsDqMpFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (8) 5’- HO-MsLsMsLsMsFsXFsMpMsdCsDqMsFsMqFsMsFsMsFsMsLsMsLsM-OH -3’ (9) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMsLpMsLsM-OH -3’ (10) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMpFsMsFsMsLpMsLsM-OH -3’ (11) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMsFsMpFsMsLpMsLsM-OH -3’ (12) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMpLpMsLsM-OH -3’ (13) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMsMsMsLsM-OH -3’ (14) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMsFsMsMsMsLsM-OH -3’(15) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMpFsMsMsMsLsM-OH -3' (16) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMpMsMsLsM-OH -3' (17) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMpFsMsMsMsLsM-OH -3' (18) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMsFsMpMsMsLsM-OH -3' (19) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMpFsMpMsMsLsM-OH -3' (20) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMpFsMpMsMsLsM-OH -3' (21) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMsFsMsFsMsFsMsLsMsLsM-OH -3' (22) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMsFsMsFsMsFsMsMsMsLsM-OH -3'

[0142] In each formula, D represents a 2'-deoxynucleoside residue, F represents a 2'-deoxy-2'-fluororibonucleoside residue, L represents a bridged nucleoside residue, M represents a 2'-O-methylribonucleoside residue, and dC represents a 2'-deoxycytidine residue, which is a target-corresponding nucleoside residue. X indicates that one nucleoside residue relative to the target RNA is deleted. p represents a phosphate residue, s represents a thiophosphate residue, and q represents an alkylphosphonate residue, an alkyl phosphate residue, or a substituted phosphoramide residue. HO- represents a hydroxyl group at the 5' end, and -OH represents a hydroxyl group at the 3' end.

[0143] In one aspect, the target-editing oligonucleotide may include a modified oligonucleotide having a chemical modification pattern represented by any of the following formulas (1a) to (22a), or may consist of a modified oligonucleotide having a chemical modification pattern represented by any of the following formulas (1a) to (22a).

[0144] (1a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsLsMsLsM-OH -3’ (2a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsMsMsLsM-OH -3’ (3a) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsLsMsLsM-OH -3’ (4a) 5’- HO-MsLsMsMsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsMsMsLsM-OH -3’ (5a) 5’- HO-LsMsMsMsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsMsMsLsM-OH -3’ (6a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMsLsMsLsM-OH -3’ (7a) 5’- HO-MsLsMsLsMsFsXFpMsMsdCsDrMpFsMrFsMsFsMsFsMsLsMsLsM-OH -3’ (8a) 5’- HO-MsLsMsLsMsFsXFsMpMsdCsDrMsFsMrFsMsFsMsFsMsLsMsLsM-OH -3’ (9a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMsLpMsLsM-OH -3’ (10a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMpFsMsFsMsLpMsLsM-OH -3’ (11a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMsFsMpFsMsLpMsLsM-OH -3’ (12a) 5’- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMpLpMsLsM-OH -3’ (13a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMsMsMsLsM-OH -3’ (14a) 5’- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMsFsMsMsMsLsM-OH -3’(15a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMpFsMsMsMsLsM-OH -3' (16a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMpMsMsLsM-OH -3' (17a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMpFsMsMsMsLsM-OH -3' (18a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMsFsMpMsMsLsM-OH -3' (19a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMpFsMpMsMsLsM-OH -3' (20a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMpFsMpMsMsLsM-OH -3' (21a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMsFsMsFsMsFsMsLsMsLsM-OH -3' (22a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMsFsMsFsMsFsMsMsMsLsM-OH -3'

[0145] In each formula, D represents a 2'-deoxynucleoside residue, F represents a 2'-deoxy-2'-fluororibonucleoside residue, L represents a bridged nucleoside residue, M represents a 2'-O-methylribonucleoside residue, and dC represents a 2'-deoxycytidine residue, which is a target-corresponding nucleoside residue. X indicates that one nucleoside residue relative to the target RNA is deleted. p represents a phosphate residue, s represents a thiophosphate residue, pm represents a methylphosphonate residue, and r represents an ethyl phosphate residue or a 2-methoxyethyl phosphate residue. HO- represents a hydroxyl group at the 5' end, and -OH represents a hydroxyl group at the 3' end.

[0146] In one aspect, the target-editing oligonucleotide is an oligonucleotide that induces site-specific editing to convert an adenosine residue to be edited contained in a target RNA into an inosine residue, or a pharmaceutically acceptable salt thereof, and the oligonucleotide may include a structure represented by the following formula (A) or may consist of a structure represented by the following formula (A).

[0147] (A) 5'-O １ -(N １ -L １ )-(N ２ -L ２ )-X-(N ３ -L ３ ) ｐ -(N ４ -L ４ )-(N ５ -L ５ )-(N ６ -L ６ )-(N ７ -L ７ )-(N ８ -L ８ )-(N ９ -L ９ )-(N １０ -L １０ ) -O ２ -O ３ -3'

[0148] In formula (A), the target nucleoside residue corresponding to the adenosine residue to be edited is N ６ It may be expressed as: N ６ An oligonucleotide consisting of nucleoside residues excluding X and X has a base sequence complementary to a target RNA, and the base corresponding to X in the target RNA sequence may be considered to be present even when X is deleted, so that the base sequence corresponds to that of the target RNA.

[0149] In formula (A), O １ may represent an oligonucleotide of 1 to 8 residues that forms a complementary pair with the corresponding oligonucleotide portion of the target RNA. １ From N ５ and N ７ From N １０ may each represent a nucleoside residue that forms a complementary pair with a corresponding nucleoside residue of a target RNA. ６may represent a 2'-deoxycytidine residue, a 2'-deoxyguanosine residue, a 2'-deoxyadenosine residue, or a derivative thereof. ２ and O ３ may each independently represent an oligonucleotide of 3 to 17 residues that forms a complementary pair with a corresponding oligonucleotide portion of the target RNA. ２ and O ３ The total number of residues of may be 6 to 20. X may represent a deletion of one nucleoside residue with respect to the target RNA, or may represent a nucleoside residue and a phosphorus-containing linking group that is not complementary to the corresponding nucleoside residue of the target RNA. １ , L ２ , L ３ , L ４ , L ５ , L ６ , L ８ and L ９ may each independently represent a phosphorus-containing linking group selected from the group consisting of a phosphate residue and a thiophosphate residue. １ , L ２ , L ３ , L ４ , L ５ , L ６ , L ８ and L ９ At least one of L may be a thiophosphate residue. ７ and L １０ may each independently be selected from the group consisting of a phosphate residue, a thiophosphate residue, an alkylphosphonate residue, an alkyl phosphate residue, and a substituted phosphoramide residue; L ７ and L １０ At least one of may represent a phosphorus-containing linking group selected from the group consisting of alkylphosphonic acid residues, alkyl phosphate residues, and substituted phosphoramide residues. p may represent an integer of 1 to 4. In formula (A), 5'- and -3' represent the 5'- and 3'-terminus of the oligonucleotide represented by formula (A), respectively.

[0150] The target-editing oligonucleotide has a structure represented by formula (A), which has excellent stability in vivo and can more effectively induce the editing activity of ADAR in cells.

[0151] In the structure represented by formula (A), from the 5' side to the 3' side (N ３ -L ３ ) ｐ -(N ４ -L ４ )-(N ５ -L ５ )-(N ６ -L ６ )-(N ７ -L ７ )-(N ８ -L ８ )-(N ９ -L ９ )-(N １０ -L １０ ) -O ２ -O ３ may correspond to a first oligonucleotide that specifies the target RNA, and may be represented from 5' to 3' by O １ -(N １ -L １ )-(N ２ -L ２ The oligonucleotide represented by -X may correspond to the second oligonucleotide linked to the 5' side of the first oligonucleotide.

[0152] In formula (A), L １ , L ２ , L ３ , L ４ , L ５ , L ６ , L ８ and L ９ All of L may represent thiophosphate residues, or 1 to 5, preferably 1 to 3, of L may represent phosphate residues and the rest may represent thiophosphate residues. １ , L ２ , L ３ , L ４ , L ５ , L ６ , L ８ and L ９ When any of L represents a phosphate residue, at least L ８ may represent a phosphate residue.

[0153] O １may preferably represent an oligonucleotide of 1 to 6 residues, 2 to 6 residues, or 4 residues. １ may comprise an oligonucleotide in which two types selected from the group consisting of 2'-deoxy-2'-fluoronucleoside residues, 2'-O-alkylribonucleoside residues, and bridged nucleoside residues are alternately linked, or may comprise an oligonucleotide in which 2'-O-alkylribonucleoside residues and bridged nucleoside residues are alternately linked.

[0154] O １ may be configured such that some of the nucleoside residues are linked via phosphorothioate bonds, or such that all of the nucleoside residues are linked via phosphorothioate bonds. １ The phosphorus-containing linking group linking some of the nucleoside residues may contain at least one selected from the group consisting of alkylphosphonic acid residues, alkyl phosphate residues, and substituted phosphoramide residues.

[0155] O ２ may represent an oligonucleotide of preferably 3 to 12 residues, 3 to 10 residues, 3 to 8 residues, or 3 to 6 residues. ２ may comprise an oligonucleotide in which two types of nucleoside residues selected from the group consisting of 2'-deoxy-2'-fluoronucleoside residues, 2'-O-alkylribonucleoside residues, and bridged nucleoside residues are alternately linked, or may comprise an oligonucleotide in which 2'-deoxy-2'-fluoronucleoside residues and 2'-O-alkylribonucleoside residues are alternately linked.

[0156] O ２ may be configured such that some of the nucleoside residues are linked via phosphorothioate bonds, or such that all of the nucleoside residues are linked via phosphorothioate bonds. ２ The phosphorus-containing linking group linking some of the nucleoside residues may contain at least one selected from the group consisting of alkylphosphonic acid residues, alkyl phosphate residues, and substituted phosphoramide residues.

[0157] O ３may represent an oligonucleotide of preferably 3 to 12 residues, 3 to 10 residues, 3 to 8 residues, or 3 to 6 residues. ３ may comprise an oligonucleotide in which two types selected from the group consisting of 2'-deoxy-2'-fluoronucleoside residues, 2'-O-alkylribonucleoside residues, and bridged nucleoside residues are alternately linked, or may comprise an oligonucleotide in which 2'-O-alkylribonucleoside residues and bridged nucleoside residues are alternately linked.

[0158] O ３ may be configured such that some of the nucleoside residues are linked via phosphorothioate bonds, or such that all of the nucleoside residues are linked via phosphorothioate bonds. ２ The phosphorus-containing linking group linking some of the nucleoside residues may contain at least one selected from the group consisting of alkylphosphonic acid residues, alkyl phosphate residues, and substituted phosphoramide residues.

[0159] The oligonucleotide containing the structure represented by the above formula (A) may also contain a modified oligonucleotide having a chemical modification pattern represented by any one of the above formulas (1) to (20).

[0160] The target-editing oligonucleotide can be synthesized using a commercially available synthesizer (for example, PerkinElmer's Model 392 using the phosphoramidite method) in accordance with the method described in known literature (for example, see Nucleic Acids Research, 12, 4539 (1984)). The phosphoramidite reagent used may be a commercially available reagent, or may be a reagent appropriately synthesized in accordance with the method described in known literature. After coupling with a phosphoramidite reagent, phosphorothioate bonds can be introduced by reacting with a reagent such as sulfur, tetraethylthiuram disulfide (TETD, Applied Biosystems), Beaucage reagent (Glen Research), xanthan hydride, phenylacetyl disulfide, or [(N,N-dimethylaminomethylidene)amino]-3H-1,2,4-dithiazoline-3-thione (DDTT) (see, for example, Tetrahedron Letters, 32, 3005 (1991), J. Am. Chem. Soc., 112, 1253 (1990), PCT / WO98 / 54198). Alternatively, instead of a phosphoramidite reagent, an alkylphosphonate bond, an alkylphosphonate bond, etc. can be introduced by using an alkylphosphonamidite reagent, an alkylphosphonamidite reagent, etc. Commercially available alkylphosphonamidite reagents and alkylphosphonamidite reagents may be used, or reagents appropriately synthesized according to methods described in known literature may be used.

[0161] A target-editing oligonucleotide (hereinafter also simply referred to as "oligonucleotide") may be used in the form of a pharmaceutically acceptable salt. A "pharmaceutically acceptable salt" is 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 salt, and the like. Examples of pharmaceutically acceptable salts of oligonucleotides include amine salts, such as organic salts like diethylammonium salts and tris(hydroxymethyl)aminomethane salts; hydrohalides like hydrofluoride, hydrochloride, hydrobromide, and hydroiodide; inorganic acid salts like nitrate, perchlorate, sulfate, and phosphate; lower alkanesulfonates like methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonates like benzenesulfonate and p-toluenesulfonate; organic acid salts like acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts like glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate. Pharmaceutically acceptable salt forms of oligonucleotides are preferably alkali metal salts of the oligonucleotides, more preferably sodium salts. These salts can be prepared by known methods.

[0162] Furthermore, the oligonucleotides and pharmaceutically acceptable salts thereof may also exist as solvates (for example, hydrates), and may be in the form of such solvates.

[0163] Oligonucleotides and pharmaceutically acceptable salts thereof may have optically active forms due to 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.).

[0164] The oligonucleotides and pharmaceutically acceptable salts thereof may have a delivery molecule such as GalNAc, cholesterol, or a fatty acid bound to them at the 5' or 3' end via a linker or a phosphodiester bond (including a phosphorothioate bond). Examples of such delivery molecules and linkers include those described in Bioconjugate Chem. 2019, 30, 366. Oligonucleotides bound to delivery molecules and pharmaceutically acceptable salts thereof can be produced by known production methods. The oligonucleotides and pharmaceutically acceptable salts thereof described herein also include forms bound to such delivery molecules.

[0165] When an oligonucleotide, a pharmaceutically acceptable salt or solvate thereof is used to treat a disease, it can be administered orally as it is or mixed with an appropriate pharmaceutically acceptable excipient, diluent or the like, in the form of tablets, capsules, granules, powders or syrups, or parenterally as injections, suppositories, patches or topical preparations.

[0166] These preparations contain excipients (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, bayle's 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 (e.g., stearic acid; metal stearates such as calcium stearate and magnesium stearate; talc; colloidal silica; waxes such as beeswax and Gay's 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, polyvinyl alcohol, polyvinylpyrrolidone, 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 crosslinked sodium carboxymethyl cellulose; chemically modified starches and celluloses such as carboxymethyl starch, carboxymethyl starch sodium, and crosslinked 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;The formulation is prepared by a known method using additives such as thimerosal, dehydroacetic acid, sorbic acid, etc.), flavoring agents (e.g., commonly used sweeteners, acidulants, flavoring agents, etc.), diluents, etc.

[0167] 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. Alternatively, the therapeutic agent may 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.

[0168] The carbohydrate is preferably at least one of monosaccharides and disaccharides. Examples of the carbohydrate and polyhydric alcohol include glucose, galactose, mannose, lactose, maltose, mannitol, and sorbitol. These may be used alone or in combination.

[0169] Preferred examples of surfactants include polyoxyethylene sorbitan mono- to tri-esters, alkylphenyl polyoxyethylene, sodium taurocholate, sodium cholate, and polyhydric alcohol esters. Of these, polyoxyethylene sorbitan mono- to tri-esters are particularly preferred, and particularly preferred esters here are oleate, laurate, stearate, and palmitate. These may be used alone or in combination.

[0170] Therapeutic agents containing oligonucleotides, pharmaceutically acceptable salts or solvates thereof may further preferably contain 0.03 M to 0.09 M of a pharmaceutically acceptable neutral salt, such as sodium chloride, potassium chloride and / or calcium chloride.

[0171] Therapeutic agents containing oligonucleotides, pharmaceutically acceptable salts, or solvates thereof may further preferably contain 0.002 M 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.

[0172] Furthermore, the above-mentioned therapeutic agent may be supplied in a solution state. However, when it is necessary to store the agent for a certain period of time, it is usually preferable to freeze-dry the agent in order to stabilize the oligonucleotide and prevent a decrease in the therapeutic effect. In this case, the agent is reconstituted with a solvent (such as distilled water for injection) at the time of use, i.e., in a liquid state for administration. Therefore, the therapeutic agent of the present invention also includes a freeze-dried agent that is reconstituted with a solvent so that each component has a predetermined concentration range. In order to promote the solubility of the freeze-dried agent, an amino acid such as albumin or glycine may be further contained.

[0173] 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 the number of administrations may be changed as appropriate depending on the type of disease, symptoms, age, administration method, etc.

[0174] Method for Treating Diseases The method for treating diseases comprises the step of administering the therapeutic agent to a subject. The method for treating diseases may comprise the step of administering a pharmacologically effective amount of the therapeutic agent to a subject. As used herein, "treatment" refers to any treatment performed for a disease, and includes, for example, treatment, amelioration, inhibition of progression (prevention of worsening), prevention, etc. (preferably, treatment or prevention) of a disease. The subject of treatment may be a warm-blooded animal including a human, a non-human warm-blooded animal, or a human.

[0175] 1. Method for site-specific editing of target RNA The method for site-specific editing of target RNA comprises contacting the target RNA with a target-editing oligonucleotide, which is an oligonucleotide that induces site-specific editing of the target RNA, in the presence of adenosine deaminase. The target-editing oligonucleotide forms a partial duplex with the target RNA and recruits adenosine deaminase, thereby site-specifically converting adenosine residues contained in the target RNA to inosine residues. The method for site-specific editing of target RNA may further comprise the step of preparing the target-editing oligonucleotide.

[0176] The method for site-specific editing of target RNA can be performed, for example, by introducing the above-mentioned target-editing oligonucleotide into a eukaryotic cell having the target RNA. The method for introducing the target-editing oligonucleotide into a eukaryotic cell can be appropriately selected and applied from various techniques used in nucleic acid medicines. The method for site-specific editing of target RNA may be performed in vitro or in vivo.

[0177] In other aspects, the present invention also includes the use of a target-editing oligonucleotide in the manufacture of a pharmaceutical composition used in the treatment of a disease (e.g., a genetic disease), the use of a target-editing oligonucleotide in the treatment of a disease (e.g., a genetic disease), and a target-editing oligonucleotide for use in the treatment of a disease (e.g., a genetic disease).

[0178] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0179] Reference Example 6 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine-3'-O-(ethyl-N,N-diisopropyl)phosphonamidite

[0180]

[0181] 5'-O-(4,4'-dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine (1.85 g, 2.81 mmol) was azeotropically dehydrated with anhydrous acetonitrile / anhydrous toluene (1 / 1), then dissolved in anhydrous dichloromethane (16 ml), and tetrazole N,N-diisopropylamine salt (0.48 g) was added. Under ice-cooling, a solution of p-ethyl-N,N,N',N'-tetraisopropylphosphonamidite (Tetrahedron Lett. 1989, 30, 2445-24482, 1.26 g, 5.62 mmol) in anhydrous dichloromethane (3 ml) was added, and the mixture was stirred under ice-cooling for 50 minutes and then overnight at room temperature. The solvent was concentrated under reduced pressure (to approximately 1 / 2 volume) and then diluted with toluene. The resulting solution was purified using silica gel chromatography (hexane (1% triethylamine added): ethyl acetate (1% triethylamine added) = 90:10 → 50 / 50 → 30 / 70) to obtain the target product (1.41 g, 1.73 mmol, 61%) as a colorless solid.

[0182] １ H-NMR (400MHz, CDCl ３ ): 0.9-1.20 (15H, m), 1.42-1.70 (2H, m), 2.55-2.72 (1H, m), 2.85 (1H, m), 3.29-3.56 (4H, m), 3.77 (6H, s), 4.28 (1H, m), 4.65 (1H, m), 6.49 (1H, m), 6.78 (4H, m), 7.17-7.32 (7H, m), 7.39 (2H, m), 7 .53 (2H, m), 7.61 (1H, m), 8.02 (2H, m), 8.19 (1H, m), 8.76 (1H, m), 9.03 (1H, m). FAB-MAS (mNBA): 817 (M+H) ＋

[0183] Reference Example 7 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine-3'-O-(n-propyl-N,N-diisopropyl)phosphonamidite

[0184]

[0185] P-propyl-N,N,N',N'-tetraisopropylphosphonamidite was synthesized as follows. Bis(diisopropylamino)chlorophosphine (5.18 g, 0.0194 mol) was suspended in anhydrous diethyl ether (340 ml). Under cooling in an ice / acetone bath, a 2 M solution of n-propylmagnesium chloride in diethyl ether (12 ml, 0.024 mol) was added dropwise, and the mixture was stirred under cooling in an ice / acetone bath for 1 hour, followed by stirring at room temperature for 1.5 hours. n-Hexane (350 ml) was added at room temperature, and insoluble matter was removed by filtration through Celite. The solvent was then concentrated under reduced pressure. The resulting residue was purified using NH silica gel chromatography (n-hexane 100%) to yield the target product (4.36 g, 0.0176 mol, 82%) as a colorless oil.

[0186] １ H-NMR (400MHz, CDCl ３ ): 1.00 (3H, t, J=7.3Hz), 1.13 (24H, dd, J1=40.3Hz, J2=6.7Hz), 1.33-1.47 (2H, m), 1.58-1.67 (2H, d, m), 3.33-3.58 (4H, m).

[0187] 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine (1.93 g, 2.93 mmol) was azeotropically dehydrated with anhydrous acetonitrile / anhydrous toluene (1 / 1), then dissolved in anhydrous dichloromethane (17 ml), and tetrazole N,N-diisopropylamine salt (0.5 g) was added. Under ice-cooling, a solution of P-propyl-N,N,N',N'-tetraisopropylphosphonamidite (1.45 g, 5.28 mmol) in anhydrous dichloromethane (3 ml) was added, and the mixture was stirred under ice-cooling for 50 minutes and then at room temperature overnight. The solvent was concentrated under reduced pressure (to approximately 1 / 2 volume) and then diluted with toluene. The resulting solution was purified using silica gel chromatography (hexane (1% triethylamine added): ethyl acetate (1% triethylamine added) = 90:10 → 50 / 50 → 25 / 75) to obtain the target product (1.89 g, 2.27 mmol, 76%) as a colorless solid.

[0188] １ H-NMR (400MHz, CDCl ３): 0.95-1.70 (19H, m), 2.51-2.72 (1H, m), 2.85 (1H, m), 3.29-3.57 (4H, m), 3.77 (6H, s), 4.28 (1H, m), 4.64 (1H, m), 6.49 (1H, m) , 6.78 (4H, m), 7.17-7.30 (7H, m), 7.39 (2H, m), 7.53 (2H, m), 7.60 (1H, m), 8.02 (2H, m), 8.18 (1H, m), 8.76 (1H, m), 9.03 (1H, m). FAB-MAS (mNBA): 831 (M+H) ＋

[0189] Reference Example 8 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine-3'-O-(isopropyl-N,N-diisopropyl)phosphonamidite

[0190]

[0191] P-Isopropyl-N,N,N',N'-tetraisopropylphosphonamidite was synthesized as follows. Bis(diisopropylamino)chlorophosphine (5.27 g, 0.0198 mol) was suspended in anhydrous diethyl ether (350 ml). Under cooling in an ice / acetone bath, a 1 M diethyl ether solution of isopropylmagnesium chloride (24 ml, 0.024 mol) was added dropwise, and the mixture was stirred under cooling in an ice / acetone bath for 1 hour, followed by stirring at room temperature for 1 hour. n-Hexane (350 ml) was added at room temperature, and insoluble matter was removed by filtration through Celite. The solvent was then concentrated under reduced pressure. The resulting residue was purified using NH silica gel chromatography (n-hexane 100%) to yield the target product (3.73 g, 0.0136 mol, 69%) as a colorless solid.

[0192] １ H-NMR (400MHz, CDCl ３ ): 1.03 (6H, dd, J1=17.1Hz, J2=6.7Hz), 1.17 (24H, dd, J1=40.3Hz, J2=6.7Hz), 2.19 (1H, m), 3.26-3.61 (4H, m).

[0193] A mixture of 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine (3 g, 4.56 mmol), 4,5-dicyanoimidazole (0.54 g), and anhydrous dichloromethane (25 ml) was stirred at room temperature for 10 minutes, and then a solution of P-isopropyl-N,N,N',N'-tetraisopropylphosphonamidite (1.91 g, 6.96 mmol) in anhydrous dichloromethane (5 ml) was added and stirred at room temperature overnight. The solvent was concentrated under reduced pressure (to about half its volume) and then diluted with toluene. The resulting solution was purified using NH silica gel chromatography (hexane (1% triethylamine added): ethyl acetate (1% triethylamine added) = 90:10 → 65 / 35 → 40 / 60) and then re-purified using silica gel chromatography (hexane (1% triethylamine added): ethyl acetate (1% triethylamine added) = 90:10 → 25 / 75) to obtain the target product as a colorless solid (306 mg, 0.37 mmol, 8%).

[0194] １ H-NMR (400MHz, CDCl ３ ): 0.86-1.20 (18H, m), 1.94 (1H, m), 2.58-2.77 (1H, m), 2.87 (1H, m), 3.29-3.58 (4H, m), 3.77 (6H, s), 4.28-4.38 (1H, m), 4.66 (1H, m), 6. 49 (1H, m), 6.78 (4H, m), 7.16-7.35 (7H, m), 7.39 (2H, m), 7.53 (2H, m), 7.60 (1H, m), 8.02 (2H, m), 8.18 (1H, m), 8.75 (1H, m), 9.01 (1H, m). FAB-MAS (mNBA): 831 (M+H) ＋

[0195] Reference Example 9 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine-3'-O-(n-butyl-N,N-diisopropyl)phosphonamidite

[0196]

[0197] 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine (1.500 g, 2.28 mmol) was suspended in anhydrous dichloromethane (12 ml), and tetrazole N,N-diisopropylamine salt (0.391 g) was added at room temperature, followed by stirring at the same temperature for 10 minutes. Next, a solution of separately prepared P-n-butyl-N,N,N',N'-tetraisopropylphosphonamidite (Tetraheron Lett. 1989, 30, 2445-2448; 0.987 g, 3.42 mmol) in anhydrous dichloromethane (3 ml) was added, followed by stirring at room temperature for 21 hours. The reaction solution was evaporated under reduced pressure, and the residue was purified by silica gel chromatography (hexane containing 1% triethylamine: ethyl acetate containing 1% triethylamine = 60:40 → 40 / 60) to obtain the target product (1.689 g, 2.00 mmol, 88%) as a white foamy solid.

[0198] １ H-NMR (500MHz, CDCl ３ ): 0.88-0.98 (4H, m), 1.03-1.95 (18H, m), 2.54-2.73 (1H, m), 2.80-2.89 (1H, m), 3.27-3 .58 (4H, m), 3.77 (6H, s), 4.24-4.33 (1H, m), 4.57-4.70 (1H, m), 6.46-6.53 (1H, m), 6.74- 6.81 (4H, m), 7.16-7.32 (6H, m), 7.35-7.42 (2H, m), 7.50-7.56 (2H, m), 7.58-7.63 (1H, m) , 7.99-8.05 (2H, m), 8.18 (1H, d, J = 9.3Hz), 8.75 (1H, d, J = 3.9Hz), 8.97 (1H, d, J = 5.9Hz). ESI-MAS: 843 (MH) ＋

[0199] Reference Example 10 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine-3'-O-(3-methoxypropyl-N,N-diisopropyl)phosphonamidite

[0200]

[0201] 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine (1.5 g, 2.3 mmol) and 4,5-dicyanoimidazole (340 mg, 2.6 mmol) were dissolved in anhydrous dichloromethane (10 ml) under a nitrogen stream and stirred for 10 minutes. A solution of separately prepared P-3-methoxypropyl-N,N,N',N'-tetraisopropylphosphonamidite (Nucleic Acids Research (2019), 47(11), 5465-5479; 870 mg, 2.9 mmol) in anhydrous dichloromethane (6 ml) was added dropwise at room temperature and stirred overnight. The reaction solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel chromatography (hexane containing 1% triethylamine: ethyl acetate containing 1% triethylamine = 2:3) to obtain the target product (907 mg, 1.13 mmol, 46%) as a white solid.

[0202] １ H-NMR (400MHz, CDCl ３ ): 1.06-1.19 (12H, m), 1.49-1.70 (4H, m), 2.53-2.61 and 2.65-2.72 (1H, eachm), 2.83-2.90 (1 H, m), 3.29-3.58 (10H, m), 3.77 (7H, s), 4.24-4.31 (1H, m), 4.60-4.69 (1H, m), 6.46-6.51 (1H, m), 6.76-6.80 (4H, m), 7.19-7.31 (6H, m), 7.37-7.41 (2H, m), 7.51-7.56 (2H, m), 7.58-7.64 (1 H, m), 8.00-8.04 (2H, m), 8.18 (1H, d, J = 7.3Hz), 8.75 (1H, d, J = 3.7Hz), 8.96 (1H, d, J = 4.3Hz). ESI-MAS: 778 (M-NiPr ２ +OH) ＋

[0203] Reference Example 11 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-O-methyl-4-N-benzoyladenosine-3'-O-(ethyl-N,N-diisopropyl)phosphonamidite

[0204]

[0205] 5'-O-(4,4'dimethoxytrityl)-2'-O-methyl-4-N-benzoyladenosine (1.2 g, 1.7 mmol) and 4,5-dicyanoimidazole (227 mg, 1.9 mmol) were dissolved in anhydrous dichloromethane (20 mL) under a nitrogen stream, and a separately prepared solution of P-ethyl-N,N,N',N'-tetraisopropylphosphonamidite (619 mg, 2.4 mmol) in anhydrous dichloromethane (5 mL) was added dropwise at room temperature, followed by stirring overnight. The reaction solvent was evaporated under reduced pressure, and the resulting residue was purified using silica gel chromatography (hexane containing 1% triethylamine: ethyl acetate containing 1% triethylamine = 1:1) to obtain the target product (1.0 g, 1.18 mmol, 70%) as a white solid.

[0206] １ H-NMR (400MHz, CDCl ３ ): 0.84-0.92 (4H, m), 0.97-1.09 (6H, m), 1.15 (2H, d, J = 6.7Hz), 1.56-1.67 (1H, m), 1.70-1.80 (1H, m), 3.32-3.4 0 (1H, m), 3.44-3.59 (6H, m), 3.78 (7H, s), 4.31-4.39 (1H, m), 4.46-4.54 (1H, m), 4.62-4.67 (1H, m), 6.18 and 6.21 (1H, eachd, J=6.1and3.7Hz), 6.78-6.82 (4H, m), 7.20-7.36 (8H, m), 7.42-7.47 (2H, m), 7.51-7.55 (2H, m), 7.59 -7.64 (1H, m), 8.00-8.04 (2H, m), 8.18 and 8.28 (1H, each), 8.73 and 8.76 (1H, each), 8.96 and 9.00 (1H, each). ESI-MAS: 764 (M-NiPr ２ +OH) ＋

[0207] Reference Example 12 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-O-methyl-4-N-benzoyladenosine-3'-O-(n-propyl-N,N-diisopropyl)phosphonamidite

[0208]

[0209] 5'-O-(4,4'dimethoxytrityl)-2'-O-methyl-4-N-benzoyladenosine (1.2 g, 1.7 mmol) and 4,5-dicyanoimidazole (227 mg, 1.9 mmol) were dissolved in anhydrous dichloromethane (20 mL) under a nitrogen stream and stirred for 10 minutes. A separately prepared solution of P-n-propyl-N,N,N',N'-tetraisopropylphosphonamidite (700 mg, 2.6 mmol) in anhydrous dichloromethane (5 mL) was added dropwise at room temperature, and the mixture was stirred overnight. The reaction solvent was evaporated under reduced pressure, and the resulting residue was purified using silica gel chromatography (hexane containing 1% triethylamine: ethyl acetate containing 1% triethylamine = 1:1) to obtain the target product (1.09 g, 1.27 mmol, 74%) as a white solid.

[0210] １ H-NMR (400MHz, CDCl ３ ): 0.88 and 0.94 (3H, eacht, J = 6.7 and 7.3Hz), 1.00-1.09 (12H, m), 1.15 (2H, d, J = 6.7Hz), 1.41-1.52 (2H, m), 1.58-1.66 (1H, m), 1.71-1.81 (1H, m), 3.33-3.40 (1H, m), 3.44-3.61 (6H, m), 3.78 (6H, s), 4.30-4.41 (1H, m), 4.45-4.54 (1H, m), 4.59-4.68 (1H, m), 6.17 (1H, d, J = 6.1Hz), 6.21 (1H, d, J = 3.7Hz), 6.78-6.82 (4H, m), 7.20-7.36 (6H, m), 7.41-7.47 (2H, m), 7.51-7.56 ( 2H, m), 7.59-7.64 (1H, m), 8.00-8.04 (2H, m), 8.18 and 8.26 (1H, each), 8.73 and 8.76 (1H, each), 8.96 and 9.00 (1H, each). ESI-MAS: 778 (M-NiPr ２ +OH) ＋

[0211] Reference Example 13 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-O-methyl-4-N-benzoyladenosine-3'-O-(isopropyl-N,N-diisopropyl)phosphonamidai

[0212]

[0213] 5'-O-(4,4'-dimethoxytrityl)-2'-O-methyl-4-N-benzoyladenosine (1.9 g, 2.8 mmol) and 4,5-dicyanoimidazole (364 mg, 3.1 mmol) were dissolved in anhydrous dichloromethane (10 mL) under a nitrogen stream and stirred for 10 minutes. A separately prepared solution of P-isopropyl-N,N,N',N'-tetraisopropylphosphonamidite (1.1 g, 4.2 mmol) in anhydrous dichloromethane (6 mL) was added dropwise at room temperature, and the mixture was stirred overnight. The reaction solvent was evaporated under reduced pressure, and the resulting residue was purified using silica gel chromatography (hexane containing 1% triethylamine: ethyl acetate containing 1% triethylamine = 2:1 to 1:1) to obtain the target product (310 mg, 0.36 mmol, 13%) as a white solid.

[0214] １ H-NMR (400MHz, CDCl ３ ): 0.86-1.23 (18H, m), 1.96-2.06 (1H, m), 3.34-3.56 (8H, m), 3.78 and 3.80 (6H, each s), 4.13-4.40 (1H, m), 4.43-4.56 (1H, m), 4.61-4.71 (1H, m), 6.01-6.22 (1H, m), 6.78 -6.82 (4H, m), 7.20-7.36 (6H, m), 7.39-7.56 (4H, m), 7.58-7.64 (1H, m), 7.96-8.05 (2 H, m), 8.16 and 8.27 (1H, each), 8.73 and 8.75 (1H, each), 8.99 and 9.05 (1H, each). ESI-MAS: 778 (M-NiPr ２ +OH) ＋

[0215] Reference Example 14 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-O-methyl-4-N-benzoyladenosine-3'-O-(n-butyl-N,N-diisopropyl)phosphonamidite

[0216]

[0217] 5'-O-(4,4'dimethoxytrityl)-2'-O-methyl-4-N-benzoyladenosine (1.5 g, 2.2 mmol) and 4,5-dicyanoimidazole (285 mg, 2.4 mmol) were dissolved in anhydrous dichloromethane (12 mL) under a nitrogen stream and stirred for 10 minutes. A separately prepared solution of p-n-butyl-N,N,N',N'-tetraisopropylphosphonamidite (825 mg, 2.9 mmol) in anhydrous dichloromethane (5 mL) was added dropwise at room temperature, and the mixture was stirred overnight. The reaction solvent was evaporated under reduced pressure, and the resulting residue was purified using silica gel chromatography (hexane containing 1% triethylamine: ethyl acetate containing 1% triethylamine = 1:1) to obtain the target product (1.3 g, 1.49 mmol, 68%) as a white solid.

[0218] １ H-NMR (400MHz, CDCl ３ ):0.88and0.92(3H,eacht,J=7.0and7.3Hz),1.03-1.16(12H,m),1.26-1.46(4H,m),1.56-1.79(2H,m),3.3 2-3.60 (7H, m), 3.78 (6H, s), 4.30-4.39 (1H, m), 4.46-4.54 (1H, m), 4.60-4.66 (1H, m), 6.18 and 6.21 (1H, eac hd, J=6.1and3.7Hz), 6.78-6.82 (4H, m), 7.20-7.36 (6H, m), 7.41-7.47 (2H, m), 7.49-7.56 (2H, m), 7.59-7.6 3 (1H, m), 8.01-8.04 (2H, m), 8.18 and 8.27 (1H, each), 8.73 and 8.75 (1H, each), 8.99 and 9.03 (1H, each). ESI-MAS: 792 (M-NiPr ２ +OH) ＋

[0219] Reference Example 15 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-O-methyl-4-N-benzoyladenosine-3'-O-(3-methoxypropyl-N,N-diisopropyl)phosphonamidite

[0220]

[0221] 5'-O-(4,4'dimethoxytrityl)-2'-O-methyl-4-N-benzoyladenosine (1.0 g, 1.5 mmol) and 4,5-dicyanoimidazole (190 mg, 1.7 mmol) were dissolved in anhydrous dichloromethane (5 mL) under a nitrogen stream and stirred for 10 minutes. A separately prepared solution of P-3-methoxypropyl-N,N,N',N'-tetraisopropylphosphonamidite (590 mg, 2.0 mmol) in anhydrous dichloromethane (4 mL) was added dropwise at room temperature, and the mixture was stirred overnight. The reaction solvent was evaporated under reduced pressure, and the resulting residue was purified using silica gel chromatography (hexane containing 1% triethylamine: ethyl acetate containing 1% triethylamine = 2:3) to obtain the target product (1.05 g, 1.18 mmol, 79%) as a white solid.

[0222] １ H-NMR (400MHz, CDCl ３ ): 1.03-1.18 (12H, m), 1.25-1.32 (3H, m), 1.54-1.82 (3H, m), 3.28-3.61 (13H, m), 3.78 (6H, s), 4. 29-4.40 (1H, m), 4.49-4.54 (1H, m), 4.60-4.69 (1H, m), 6.16 and 6.19 (1H, eachd, J = 6.7 and 3.7Hz) , 6.77-6.83 (4H, m), 7.20-7.36 (6H, m), 7.40-7.47 (2H, m), 7.50-7.55 (2H, m), 7.59-7.64 (1H, m), 8.00-8.04 (2H, m), 8.17 and 8.25 (1H, each), 8.73 and 8.75 (1H, each), 8.95 and 8.99 (1H, each). ESI-MAS:808 (M-NiPr ２ +OH) ＋

[0223] Reference Example 16 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine-3'-O-(n-propyl-N,N-diisopropyl)phosphoramidite

[0224]

[0225] 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine (1.2 g, 1.8 mmol) and 4,5-dicyanoimidazole (240 mg, 2.0 mmol) were dissolved in anhydrous dichloromethane (10 ml) under a nitrogen stream and stirred for 15 minutes. A solution of separately prepared n-propyl-N,N,N',N'-tetraisopropylphosphoramidite (Nucleic Acids Research (2018), 46(8), 4013-4021; 630 mg, 2.2 mmol) in anhydrous dichloromethane (3 ml) was added dropwise at room temperature and stirred overnight. The reaction solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel chromatography (hexane containing 1% triethylamine: ethyl acetate containing 1% triethylamine = 1:1) to obtain the target product (920 mg, 1.09 mmol, 60%) as a white solid.

[0226] １ H-NMR (400MHz, CDCl ３ ):0.87and0.93(3H,eacht,J=7.3Hz),1.10-1.26(12H,m),1.50-1.66(2H,m),2.62-2.76(1H,m),2.8 5-2.95 (1H, m), 3.32-3.75 (6H, m), 3.77 (6H, s), 4.31-4.39 (1H, m), 4.73-4.81 and 5.07-5.12 (1H, each m), 6.52 (1H, t, J = 6.7Hz), 6.75-6.80 (4H, m), 7.18-7.30 (7H, m), 7.38-7.41 (2H, m), 7.50-7.55 (2H, m) , 7.58-7.63 (1H, m), 8.01-8.04 (2H, m), 8.20 (1H, d, J=7.9Hz), 8.74 (0H, s), 8.75 (1H, s), 9.02 (1H, s). ESI-MAS: 764 (M-NiPr ２ +OH) ＋

[0227] Reference Example 17 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine-3'-O-(n-butyl-N,N-diisopropyl)phosphoramidite

[0228]

[0229] 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine (1.3 g, 2.0 mmol) and 4,5-dicyanoimidazole (260 mg, 2.2 mmol) were dissolved in anhydrous dichloromethane (10 ml) under a nitrogen stream and stirred for 10 minutes. A solution of separately prepared n-butyl-N,N,N',N'-tetraisopropylphosphoramidite (Nucleic Acids Research (2018), 46(8), 4013-4021; 720 mg, 2.4 mmol) in anhydrous dichloromethane (4 ml) was added dropwise at room temperature and stirred overnight. The reaction solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel chromatography (hexane containing 1% triethylamine: ethyl acetate containing 1% triethylamine = 1:1) to obtain the target product (968 mg, 1.12 mmol, 56%) as a white solid.

[0230] １ H-NMR (400MHz, CDCl ３ ): 0.86-0.93 (3H, m), 1.09-1.28 (12H, m), 1.31-1.42 (2H, m), 1.47-1.61 (2H, m), 2.62-2.75 (1H, m), 2.8 6-2.96 (1H, m), 3.31-3.69 (5H, m), 3.73-3.81 (7H, m), 4.31-4.39 (1H, m), 4.72-4.82 and 5.06-5.12 (1H, e achm), 6.52 (1H, t, J = 6.7Hz), 6.76-6.80 (4H, m), 7.17-7.30 (7H, m), 7.38-7.41 (2H, m), 7.51-7.55 (2H, m), 7.58-7.63 (1H, m), 8.00-8.04 (2H, m), 8.20 (1H, d, J=6.1Hz), 8.74 (0H, s), 8.75 (1H, s), 8.98 (1H, s). ESI-MAS: 778 (M-NiPr ２ +OH) ＋

[0231] Reference Example 18 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine-3'-O-(2-methoxyethyl-N,N-diisopropyl)phosphoramidite

[0232]

[0233] 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine (1.5 g, 2.3 mmol) and 4,5-dicyanoimidazole (300 mg, 2.5 mmol) were dissolved in anhydrous dichloromethane (10 ml) under a nitrogen stream and stirred for 10 minutes. A solution of separately prepared 2-methoxyethyl-N,N,N',N'-tetraisopropylphosphoramidite (J. Am. Chem. Soc. (1990), 112(21), 7475-82; 916 mg, 3.0 mmol) in anhydrous dichloromethane (5 ml) was added dropwise at room temperature and stirred overnight. The reaction solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel chromatography (hexane containing 1% triethylamine: ethyl acetate containing 1% triethylamine = 1:1) to obtain the target product (1.2 g, 1.39 mmol, 62%) as a white solid.

[0234] １ H-NMR (400MHz, CDCl ３ ): 1.11-1.20 (11H, m), 2.65-2.77 (1H, m), 2.87-2.96 (1H, m), 3.30 and 3.33 (3H, eachs), 3.35-3.49 (3H, m), 3.53-3.82 (12H, m), 4.30-4.40 (1H, m), 4.73-4.82 (1H, m), 6.52 (1H, t , J=6.4Hz), 6.75-6.80 (4H, m), 7.18-7.30 (7H, m), 7.38-7.42 (2H, m), 7.50-7.54 (2H, m) , 7.59-7.62 (1H, m), 8.01-8.04 (2H, m), 8.20 (1H, d, J=5.5Hz), 8.75 (1H, s), 8.98 (1H, s). ESI-MAS: 780 (M-NiPr ２ +OH) ＋

[0235] Reference Example 19: Synthesis of 5'-O-(4,4'-dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine-3'-O-(cyclopropyl-N,N-diisopropyl)phosphoramidite

[0236]

[0237] Cyclopropyl-N,N,N',N'-tetraisopropylphosphoramidite was synthesized as follows. Cyclopropanol (1.0 g, 20.4 mmol) was mixed with THF (10 ml) under a nitrogen stream, and triethylamine (4.7 ml, 40.8 mmol) was added. Then, a separately prepared solution of P-chloro-N,N,N',N'-tetraisopropylphosphonamidite (3.7 g, 17 mmol) in THF (15 ml) was added dropwise under ice cooling. The mixture was then warmed to room temperature and stirred for 3 hours. The reaction solvent was evaporated under reduced pressure, and the resulting residue was purified using NH silica gel chromatography (eluted with hexane) to obtain the target product (2.8 g, 9.7 mmol, 57%) as a colorless oil.

[0238] １ H-NMR (400MHz, CDCl ３ ): 0.47-0.52 (2H, m), 0.64-0.68 (2H, m), 1.15 (12H, d, J = 6.7Hz), 1.17 (12H, d, J = 6.7Hz), 3.33-3.39 (1H, m), 3.46-3.57 (4H, m).

[0239] 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine (1.5 g, 2.3 mmol) and 4,5-dicyanoimidazole (300 mg, 2.5 mmol) were dissolved in anhydrous dichloromethane (10 mL) under a nitrogen stream and stirred for 10 minutes. A separately prepared solution of cyclopropyl-N,N,N',N'-tetraisopropylphosphoramidite (860 mg, 3.0 mmol) in anhydrous dichloromethane (5 mL) was added dropwise at room temperature, and the mixture was stirred overnight. The reaction solvent was evaporated under reduced pressure, and the resulting residue was purified using silica gel chromatography (hexane containing 1% triethylamine: ethyl acetate containing 1% triethylamine = 1:1) to obtain the target product (1.65 g, 1.95 mmol, 85%) as a white solid.

[0240] １ H-NMR (400MHz, CDCl ３): 0.44-0.48 and 0.52-0.57 (2H, eachm), 0.58-0.61 and 0.68-0.71 (2H, eachm), 1.11-1.26 (12H, m), 2.62-2. 74 (1H, m), 2.84-2.95 (1H, m), 3.26-3.45 (3H, m), 3.55-3.65 (2H, m), 3.74-3.80 (6H, m), 4.32-4.38 (1H, m), 4 .. 72-4.79 (1H, m), 6.51 (1H, t, J = 6.7Hz), 6.76-6.81 (4H, m), 7.18-7.31 (7H, m), 7.38-7.41 (2H, m), 7.50-7.56 (2H, m), 7.58-7.64 (1H, m), 8.00-8.04 (2H, m), 8.19 (1H, d, J = 9.2Hz), 8.75 (1H, d, J = 1.8Hz), 8.97 (1H, brs). ESI-MAS: 762 (M-NiPr ２ +OH) ＋

[0241] Reference Example 20 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine-3'-O-(cyclobutyl-N,N-diisopropyl)phosphoramidite

[0242]

[0243] 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-4-N-benzoyladenosine (1.5 g, 2.3 mmol) and 4,5-dicyanoimidazole (300 mg, 2.5 mmol) were dissolved in anhydrous dichloromethane (10 ml) under a nitrogen stream and stirred for 10 minutes. A solution of separately prepared cyclobutyl-N,N,N',N'-tetraisopropylphosphoramidite (Bioorg. Med. Chem. Lett. (2012), 22(18), 5924-5929; 860 mg, 3.0 mmol) in anhydrous dichloromethane (5 ml) was added dropwise at room temperature and stirred overnight. The reaction solvent was evaporated under reduced pressure, and the resulting residue was purified using NH silica gel chromatography (hexane containing 1% triethylamine: ethyl acetate containing 1% triethylamine = 1:1) to obtain the target product (1.63 g, 1.9 mmol, 82%) as a white solid.

[0244] １H-NMR (400MHz, CDCl ３ ): 1.09-1.20 (12H, m), 1.41-1.51 (1H, m), 1.59-1.69 (1H, m), 1.91-2.27 (4H, m), 2.62-2.77 (1H, m) , 2.86-2.96 (1H, m), 3.30-3.45 (2H, m), 3.52-3.65 (2H, m), 3.77 (6H, s), 4.14-4.40 (2H, m), 4.68-4. 78 (1H, m), 6.49-6.55 (1H, m), 6.75-6.81 (4H, m), 7.18-7.31 (7H, m), 7.38-7.43 (2H, m), 7.50-7.56 (2H, m), 7.59-7.63 (1H, m), 8.02 (2H, d, J = 7.3Hz), 8.20 (1H, d, J = 7.3Hz), 8.75 (1H, s), 8.99 (1H, s). ESI-MAS: 776 (M-NiPr ２ +OH) ＋

[0245] Reference Example 21 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-O-methoxy-4-N-benzoyladenosine-3'-O-(n-propyl-N,N-diisopropyl)phosphoramidite

[0246]

[0247] 5'-O-(4,4'dimethoxytrityl)-2'-O-methoxy-4-N-benzoyladenosine (1.3 g, 1.9 mmol) and 4,5-dicyanoimidazole (246 mg, 2.1 mmol) were dissolved in anhydrous dichloromethane (10 mL) under a nitrogen stream and stirred for 10 minutes. A separately prepared solution of n-propyl-N,N,N',N'-tetraisopropylphosphoramidite (660 mg, 2.1 mmol) in anhydrous dichloromethane (4 mL) was added dropwise at room temperature, and the mixture was stirred overnight. The reaction solvent was evaporated under reduced pressure, and the resulting residue was purified using silica gel chromatography (hexane containing 1% triethylamine: ethyl acetate containing 1% triethylamine = 1:1) to obtain the target product (983 mg, 1.16 mmol, 59%) as a white solid.

[0248] １ H-NMR (400MHz, CDCl ３): 0.80-0.98 (3H, m), 1.07-1.26 (12H, m), 1.43-1.49 (1H, m), 1.56-1.64 (1H, m), 3.34-3.69 (9H, m), 3.78 (6H, s), 4.39-4.47 (1H, m), 4.58-4.66 (2H, m), 6.16-6.21 (1H, m), 6.78-6.82 ( 4H, m), 7.20-7.28 (3H, m), 7.30-7.36 (4H, m), 7.42-7.46 (2H, m), 7.50-7.55 (2H, m), 7.59-7 .64 (1H, m), 8.00-8.05 (2H, m), 8.22 (1H, d, J = 12.2Hz), 8.73 (1H, t, J = 3.4Hz), 8.98 (1H, s). ESI-MAS: 794 (M-NiPr ２ +OH) ＋

[0249] Reference Example 22 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-O-methoxy-4-N-benzoyladenosine-3'-O-(n-butyl-N,N-diisopropyl)phosphoramidite

[0250]

[0251] 5'-O-(4,4'dimethoxytrityl)-2'-O-methoxy-4-N-benzoyladenosine (1.3 g, 1.9 mmol) and 4,5-dicyanoimidazole (246 mg, 2.1 mmol) were dissolved in anhydrous dichloromethane (10 mL) under a nitrogen stream and stirred for 10 minutes. A separately prepared solution of n-butyl-N,N,N',N'-tetraisopropylphosphoramidite (700 mg, 2.1 mmol) in anhydrous dichloromethane (4 mL) was added dropwise at room temperature, and the mixture was stirred overnight. The reaction solvent was evaporated under reduced pressure, and the resulting residue was purified using silica gel chromatography (hexane containing 1% triethylamine: ethyl acetate containing 1% triethylamine = 3:2) to obtain the target product (1.05 g, 1.18 mmol, 62%) as a white solid.

[0252] １ H-NMR (400MHz, CDCl ３):0.83and0.88(3H,eacht,J=each7.3Hz),1.05-1.26(12H,m),1.33-1.47(2H,m),1.54-1.60(2H,m),3.35- 3.46 (2H, m), 3.48 and 3.52 (3H, eachs), 3.54-3.71 (3H, m), 3.78 (7H, s), 4.38-4.48 (1H, m), 4.56-4.67 (2H, m) , 6.19 (1H, t, J = 4.9Hz), 6.77-6.82 (4H, m), 7.19-7.29 (3H, m), 7.30-7.36 (4H, m), 7.42-7.47 (2H, m), 7.51-7. 55 (2H, m), 7.59-7.63 (1H, m), 8.02 (2H, d, J = 7.3Hz), 8.22 (1H, d, J = 10.4Hz), 8.72-8.75 (1H, m), 8.99 (1H, s). ESI-MAS:808 (M-NiPr ２ +OH) ＋

[0253] Reference Example 23 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-O-methoxy-4-N-benzoyladenosine-3'-O-(2-methoxyethyl-N,N-diisopropyl)phosphoramidite

[0254]

[0255] 5'-O-(4,4'dimethoxytrityl)-2'-O-methoxy-4-N-benzoyladenosine (1.5 g, 2.2 mmol) and 4,5-dicyanoimidazole (286 mg, 2.4 mmol) were dissolved in anhydrous dichloromethane (10 ml) under a nitrogen stream and stirred for 10 minutes. A separately prepared solution of 2-methoxyethyl-N,N,N',N'-tetraisopropylphosphoramidite (809 mg, 2.6 mmol) in anhydrous dichloromethane (5 ml) was added dropwise at room temperature and stirred overnight. The reaction solvent was evaporated under reduced pressure, and the resulting residue was purified using silica gel chromatography (hexane containing 1% triethylamine: ethyl acetate containing 1% triethylamine = 1:1). The resulting crude product was dissolved in ethyl acetate, washed with water, and then dried over anhydrous sodium sulfate. After filtration, the filtrate solvent was evaporated under reduced pressure, and diethyl ether was added to the resulting residue to obtain the target product (1.2 g, 1.34 mmol, 61%) as a white solid.

[0256] １ H-NMR (400MHz, CDCl ３ ): 1.07-1.26 (12H, m), 3.21 (3H, s), 3.29 (3H, s), 3.34-3.68 (11H, m), 3.78 (6H, s), 3.82- 4.21 (1H, m), 4.37-4.48 (1H, m), 4.59-4.67 (2H, m), 6.18-6.20 (1H, m), 6.78-6.82 (4H, m) , 7.20-7.28 (3H, m), 7.31-7.35 (4H, m), 7.42-7.46 (2H, m), 7.51-7.55 (2H, m), 7.59-7.64 (1H, m), 8.02 (2H, t, J = 4.3Hz), 8.23 ​​(1H, d, J = 6.1Hz), 8.74 (1H, d, J = 1.2Hz), 8.99 (1H, s). ESI-MAS: 810 (M-NiPr ２ +OH) ＋

[0257] Reference Example 24 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-O-methoxy-4-N-benzoyladenosine-3'-O-(cyclopropyl-N,N-diisopropyl)phosphoramidite

[0258]

[0259] 5'-O-(4,4'dimethoxytrityl)-2'-O-methoxy-4-N-benzoyladenosine (1.5 g, 2.2 mmol) and 4,5-dicyanoimidazole (286 mg, 2.4 mmol) were dissolved in anhydrous dichloromethane (10 ml) under a nitrogen stream and stirred for 10 minutes. A separately prepared solution of cyclopropyl-N,N,N',N'-tetraisopropylphosphoramidite (761 mg, 2.6 mmol) in anhydrous dichloromethane (5 ml) was added dropwise at room temperature, and the mixture was stirred overnight. Water (10 ml) was added to the reaction solution, and the mixture was extracted with dichloromethane. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, the filtrate solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel chromatography (hexane containing 1% triethylamine: ethyl acetate containing 1% triethylamine = 1:1) to obtain the target product (1.32 g, 1.51 mmol, 69%) as a white solid.

[0260] １ H-NMR (400MHz, CDCl ３ ): 0.40-0.69 (4H, m), 1.07-1.23 (12H, m), 3.22-3.47 (2H, m), 3.47 (3H, s), 3.49 (3H, s), 3.50-3 .67 (3H, m), 3.78 (6H, s), 4.38-4.44 (1H, m), 4.57-4.66 (2H, m), 6.17 (1H, dd, J=11.6, 4.9Hz), 6. 78-6.84 (4H, m), 7.19-7.28 (3H, m), 7.30-7.36 (4H, m), 7.42-7.46 (2H, m), 7.51-7.55 (2H, m), 7. 59-7.64 (1H, m), 8.01-8.05 (2H, m), 8.21 (1H, d, J = 12.2Hz), 8.73 (1H, d, J = 1.8Hz), 9.00 (1H, s). ESI-MAS: 792 (M-NiPr ２ +OH) ＋

[0261] Reference Example 25 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-O-methoxy-4-N-benzoyladenosine-3'-O-(cyclobutyl-N,N-diisopropyl)phosphoramidite

[0262]

[0263] 5'-O-(4,4'dimethoxytrityl)-2'-O-methoxy-4-N-benzoyladenosine (1.5 g, 2.2 mmol) and 4,5-dicyanoimidazole (286 mg, 2.4 mmol) were dissolved in anhydrous dichloromethane (10 ml) under a nitrogen stream and stirred for 10 minutes. A separately prepared solution of cyclobutyl-N,N,N',N'-tetraisopropylphosphoramidite (800 mg, 2.6 mmol) in anhydrous dichloromethane (5 ml) was added dropwise at room temperature, and the mixture was stirred overnight. Water (10 ml) was added to the reaction solution, and the mixture was extracted with dichloromethane. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, the filtrate solvent was evaporated under reduced pressure, and the resulting residue was purified by silica gel chromatography (hexane containing 1% triethylamine: ethyl acetate containing 1% triethylamine = 1:1) to obtain the target product (1.36 g, 1.53 mmol, 69%) as a white solid.

[0264] １ H-NMR (400MHz, CDCl ３ ): 1.05-1.22 (12H, m), 1.38-1.48 (1H, m), 1.57-1.93 (2H, m), 1.99-2.25 (3H, m), 3.33- 3.42 (1H, m), 3.47-3.66 (6H, m), 3.78 (6H, s), 4.11-4.66 (4H, m), 6.18 (1H, t, J = 5.2Hz), 6.78-6.84 (4H, m), 7.19-7.27 (3H, m), 7.30-7.36 (4H, m), 7.42-7.47 (2H, m), 7.50-7.56 (2H, m), 7.58-7.64 (1H, m), 8.22 (1H, d, J = 12.2Hz), 8.74 (1H, d, J = 1.2Hz), 8.99 (1H, s). ESI-MAS:806(M-NiPr ２ +OH) ＋

[0265] Reference Example 26 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-2'-fluoro-4-N-benzoyladenosine-3'-O-(ethyl-N,N-diisopropyl)phosphoramidite

[0266]

[0267] 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-2'-fluoro-4-N-benzoyladenosine (1.24 g, 1.83 mmol) was dissolved in anhydrous dichloromethane (13 ml), and under ice-cooling, a solution of N,N-diisopropylethylamine (0.19 ml), 1H-tetrazole (154 mg), and ethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (J. Org. Chem. 1995, 60, 925-930; 0.92 g, 3.33 mmol) in anhydrous dichloromethane (2 ml) was added, and the mixture was stirred overnight while still ice-cooling (spontaneously warming to room temperature). The reaction mixture was purified directly using NH silica gel chromatography (hexane:ethyl acetate=70:30→15 / 85) and then re-purified using diol silica gel chromatography (hexane:ethyl acetate=90:10→35 / 65) to obtain the target product (1.25 g, 1.47 mmol, 80%) as a colorless solid.

[0268] １ H-NMR (400MHz, CDCl ３ ): 1.05-1.26 (15H, m), 3.49 (1H, m), 3.53-3.70 (5H, m), 3.77 (6H, s), 4.39 (1H, br), 4.79-5.02 (1H, m), 5.60-5.78 (1H, m), 6.28-6.36 ( 1H, m), 6.78 (4H, m), 7.16-7.32 (7H, m), 7.39 (2H, m), 7.53 (2H, m), 7.62 (1H, m), 8.02 (2H, m), 8.25 (1H, m), 8.77 (1H, m), 8.98 (1H, br). FAB-MAS (mNBA): 851 (M+H) ＋

[0269] Reference Example 27 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-2'-fluoro-2-N-isobutyrylguanosine-3'-O-(ethyl-N,N-diisopropyl)phosphoramidite

[0270]

[0271] 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-2'-fluoro-2-N-isobutyrylguanosine (1.17 g, 1.78 mmol) was dissolved in anhydrous dichloromethane (10 ml), and under ice-cooling, a solution of N,N-diisopropylethylamine (0.18 ml), 1H-tetrazole (150 mg), and ethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (0.89 g, 3.22 mmol) in anhydrous dichloromethane (2 ml) was added and stirred overnight while still ice-cooled (naturally warmed to room temperature). The reaction mixture was purified directly using diol silica gel chromatography (hexane:ethyl acetate = 80:20 → 20 / 80) to obtain the target product (1.19 g, 1.43 mmol, 80%) as a colorless solid.

[0272] １ H-NMR (400MHz, CDCl ３ ): 0.74-1.23 (21H, m), 1.62-1.76 (1H, m), 3.14 (1H, m), 3.44-3.68 (11H, m), 4.31 (1H, m), 4.73 (1H, m), 5.65-5.83 (1H , m), 6.00-6.11 (1H, m), 6.79 (4H, m), 7.20-7.26 (3H, m), 7.38 (4H, m), 7.49-7.59 (3H, m), 7.84 (1H, m), 11.90 (1H, br). FAB-MAS (mNBA): 833 (M+H) ＋

[0273] Reference Example 28 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-2'-fluoro-4-N-benzoylcytidine-3'-O-(ethyl-N,N-diisopropyl)phosphoramidite

[0274]

[0275] 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-2'-fluoro-4-N-benzoylcytidine (1.37 g, 2.1 mmol) was dissolved in anhydrous dichloromethane (12 ml), and a solution of N,N-diisopropylethylamine (0.21 ml), 1H-tetrazole (177 mg), and ethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (1.05 g, 3.8 mmol) in anhydrous dichloromethane (2 ml) was added under ice cooling. The mixture was stirred overnight while still ice-cooled (spontaneously warming to room temperature). The reaction mixture was purified directly using NH silica gel chromatography (hexane:ethyl acetate = 70:30 → 15:85) to give the target product (1.59 g, 1.92 mmol, 91%) as a colorless solid.

[0276] １ H-NMR (400MHz, CDCl ３ ): 1.00-1.24 (15H, m), 3.47-3.75 (6H, m), 3.83 (6H, s), 4.32 (1H, m), 4.42-4.70 (1H, m), 4.98-5.14 (1H, m), 6.13-6.18 (1H, m), 6.87 (4H, m), 7.10 (1H, br), 7.28-7.37 (7H, m), 7.40-7.47 (2H, m), 7.49-7.54 (2H, m), 7.61 (1H, m), 7.87 (2H, m), 8.61 (1H, br). FAB-MAS (mNBA): 827 (M+H) ＋

[0277] Reference Example 29 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-2'-fluorouridine-3'-O-(ethyl-N,N-diisopropyl)phosphoramidite

[0278]

[0279] 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-2'-fluorouridine (1.58 g, 2.88 mmol) was dissolved in anhydrous dichloromethane (14 ml), and a solution of N,N-diisopropylethylamine (0.29 ml), 1H-tetrazole (242 mg), and ethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (1.44 g, 5.21 mmol) in anhydrous dichloromethane (2 ml) was added under ice cooling and stirred overnight (naturally warmed to room temperature). The reaction mixture was purified directly using NH silica gel chromatography (hexane:ethyl acetate = 50:50 → 0:100 → ethyl acetate / methanol = 95:5) to give the target product (1.94 g, 2.68 mmol, 93%) as a colorless solid.

[0280] １ H-NMR (400MHz, CDCl ３ ): 1.04-1.29 (15H, m), 3.48-3.80 (12H, m), 4.24 (1H, m), 4.48-4.73 (1H, m), 4.90-5.05 (1H, m), 5.19 (1H, m), 6.08 (1H, m), 6.84 (4H, m), 7.24-7.33 (7H, m), 7.36-7.42 (2H, m), 8.03 (1H, m), 8.26 (1H, br). FAB-MAS (mNBA): 724 (M+H) ＋

[0281] Reference Example 30 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-deoxyinosine-3'-O-(ethyl-N,N-diisopropyl)phosphoramidite

[0282]

[0283] 5'-O-(4,4'-dimethoxytrityl)-2'-deoxyinosine (2.00 g, 3.61 mmol) was dissolved in anhydrous dichloromethane (20 ml) and dicyanoimidazole (426 mg, 3.61 mmol) was added. Ethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (1.49 g, 5.42 mmol) was added dropwise to the mixture, and the mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified using silica gel chromatography (eluted with 1% triethylamine in hexane:1% triethylamine in ethyl acetate = 7:3 to 0:10) to yield the target product (1.67 g, 2.29 mmol, 63%) as a colorless foamy solid.

[0284] １ H-NMR (400MHz, CDCl ３ ) 1.05-1.31 (15H, m), 2.58-2.79 (2H, m), 3.31-3.39 (2H, m), 3.51-3.73 (4H, m), 3.77 (6H, s), 4.30-4.39 (1H, m), 4.66-4.75 (1 H, m), 6.42 (1H, t, J=7.0Hz), 6.76-6.82 (4H, m), 7.15-7.26 (3H, m), 7.27-7.33 (4H, m), 7.38-7.44 (2H, m), 7.96-8.00 (2H, m). ESI-MAS (hexafluoroisopropanol / Et ３ N):831(M+Et ３ N+H) ＋

[0285] Reference Example 31 Synthesis of 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-purine riboside-3'-O-(ethyl-N,N-diisopropyl) phosphoramidite

[0286]

[0287] 5'-O-(4,4'dimethoxytrityl)-2'-deoxy-purine riboside (Nucleic Acids Research (1986), 14(20), 8135-53; 2.00 g, 3.72 mmol) was dissolved in anhydrous dichloromethane (20 ml), and diisopropylammonium tetrazole (699 mg, 4.09 mmol) was added. Ethyl-N,N,N',N'-tetraisopropylphosphorodiamidite (1.54 g, 5.57 mmol) was added dropwise to the mixture, and the mixture was stirred at room temperature for 7 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified using silica gel chromatography (eluted with 1% triethylamine in hexane:1% triethylamine in ethyl acetate = 4:1 to 0:10) to obtain the target product (2.86 g, 3.72 mmol, 100%) as a colorless foamy solid.

[0288] １ H-NMR (400MHz, CDCl ３ ) 1.06-1.30 (15H, m), 2.61-2.96 (2H, m), 3.33-3.76 (6H, m), 3.78 (6H, s), 4.36 (1H, dd, J = 16.0, 4.0Hz), 4.72-4.82 (1H, m), 6.52-6.57 (1H, m), 6.75-6.80 (4H, m), 7.17-7.25 (3H, m), 7.27-7.32 (4H, m), 7.36-7.42 (2H, m), 8.27 (1H, d, J=8.0Hz), 8.91 (1H, s), 9.12 (1H, s). ESI-MAS (hexafluoroisopropanol / Et ３ N):714(M+H) ＋

[0289] Reference Example 1 Human β-actin (hACTB; GenBank accession No. NM_001101.5) was selected as the target RNA, and the base sequence shown in Table 1 (SEQ ID NO: 1) was selected as the base sequence of the target-editing oligonucleotide. In Table 1, SEQ ID NO: 2 indicates a region that contains an adenosine residue that is the editing target in the target RNA and corresponds to the target-editing oligonucleotide (SEQ ID NO: 1), and the underlined portion corresponds to the target-corresponding nucleoside residue. The target-editing oligonucleotide (SEQ ID NO: 1; lower row) whose base sequence is shown in Table 1 is thought to be able to form, for example, the following structure with the target RNA (SEQ ID NO: 2; upper row).

[0290] Furthermore, the base sequences shown in Table 1 (SEQ ID NOs: 126 and 127) were selected as the base sequences of target-editing oligonucleotides whose target RNA was the human A1AT gene (hA1AT) or the mouse GAPDH gene (mGAPDH).

[0291]

[0292]

[0293] Examples 1 to 83 and Comparative Examples 1 to 22 The target-editing oligonucleotides of Examples 1 to 83 and Comparative Examples 1 to 22 were obtained by introducing modified oligonucleosides and phosphorus-containing linking groups as shown in the modification patterns of Tables 2 to 5, based on the base sequences of the target-editing oligonucleotides in Table 1. The target-editing oligonucleotides of Examples 1 to 83 and Comparative Examples 1 to 22 were synthesized using the phosphoramidite method (Nucleic Acids Research, 12, 4539 (1984), Nature Communications 6, Article number: 6317 (2015)) using the raw materials shown below.

[0294] "2'-O-methylnucleosides" linked by phosphodiester or phosphorothioate bonds were synthesized using the phosphoramidite form described in Nucleic Acids Research 17, 3373 (1989). "DNA" linked by phosphodiester or phosphorothioate bonds was synthesized using the phosphoramidite form described in Nucleic Acids Research 11, 4539 (1984). "2'-deoxy-2'-fluoronucleosides" linked by phosphodiester or phosphorothioate bonds were synthesized using the phosphoramidite form described in J. Med. Chem. 36, 831 (1993). The "2'-O,4'-C-methylene nucleosides" linked by a phosphodiester bond or a phosphorothioate bond were synthesized using the phosphoramidite compounds described in International Publication WO99 / 14226. The "2'-O,4'-C-methylene nucleosides" linked by a phosphodiester bond or a phosphorothioate bond were synthesized using the phosphoramidite compounds described in Example 14 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-6-N-benzoyladenosine-3'-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite) and Example 27 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-2-N-isobutyrylguanosine-3'-O-(2 The synthesis was carried out using the compounds described in Example 22 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-4-N-benzoyl-5-methylcytidine-3'-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite), Example 23 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-4-N-benzoyl-5-methylcytidine-3'-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite), and Example 9 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-5-methyluridine-3'-O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite)."2'-deoxyinosine" having a phosphodiester bond or a phosphorothioate bond was synthesized using a phosphoramidite (deoxy Inosine CED phosphoramidite (ANP-6411, manufactured by ChemGenes)).

[0295] DNA linked with methylphosphonate (PM) or methylthiophosphonate (PSM) was synthesized using P-methylphosphonamidites (deoxyadenosine (N-bz) P-methylphosphonamidite (ANP-7551, manufactured by ChemGenes); deoxyinosine P-methylphosphonamidite (ANP-7558, manufactured by ChemGenes)). The "2'-O-methylnucleoside" linked with "methylphosphonate (PM)" or "methylthiophosphonate (PSM)" is a phosphonamidite (2'-O-Methyladenosine (N-bz) P-methylphosphonamidite (ANP-8551, manufactured by ChemGenes); The synthesis was carried out using 2'-O-MethylCytidine(N-acetyl)P-methylphosphonamidite (ANP-8556, manufactured by ChemGenes); 2'-O-MethylGuanosine(N-ibu)P-methylphosphonamidite (ANP-8553, manufactured by ChemGenes); and 2'-O-MethylUridine P-methylphosphonamidite (ANP-8554, manufactured by ChemGenes). "2'-deoxyadenosine" linked with "ethyl phosphate (POE)" was synthesized using a phosphoramidite (deoxyadenosine (N-bz) P-ethoxyphosphoramidite (ANP-6541, manufactured by ChemGenes)).The "2'-O-methylnucleoside" linked with "ethyl phosphate (POE)" can be prepared in the form of a phosphoramidite (2'-O-MethylAdenosine(N-bz)ethylphosphoramidite (ANP-7221, manufactured by ChemGenes); 2'-O-MethylCytidine(N-bz)ethylphosphoramidite (ANP-7222, manufactured by ChemGenes); 2'-O-MethylGuanosine(N-ibu)ethylphosphoramidite (ANP-7223, manufactured by ChemGenes); 2'-O-MethylUridine 2'-Deoxyadenosine linked with isopropyl phosphate (POIP) was synthesized using a phosphoramidite (deoxyadenosine (N-bz) P-isopropyl phosphate (ANP-6901, ChemGenes)).

[0296] 2'-Deoxyadenosine linked with ethyl phosphonate (PE) was synthesized using the phosphonamidite of Reference Example 6. 2'-Deoxyadenosine linked with propyl phosphonate (PP) was synthesized using the phosphonamidite of Reference Example 7. 2'-Deoxyadenosine linked with isopropyl phosphonate (PIP) was synthesized using the phosphonamidite of Reference Example 8. 2'-Deoxyadenosine linked with butyl phosphonate (PB) was synthesized using the phosphonamidite of Reference Example 9. 2'-Deoxyadenosine linked with methoxypropyl phosphonate (PMOP) was synthesized using the phosphonamidite of Reference Example 10. 2'-O-methyladenosine linked with ethyl phosphonate (PE) was synthesized using the phosphonamidite of Reference Example 11. 2'-O-methyladenosine linked with propyl phosphonate (PP) was synthesized using the phosphonamidite of Reference Example 12. 2'-O-methyladenosine linked with isopropyl phosphonate (PIP) was synthesized using the phosphonamidite of Reference Example 13. 2'-O-methyladenosine linked with butyl phosphonate (PB) was synthesized using the phosphonamidite of Reference Example 14. 2'-O-methyladenosine linked with methoxypropyl phosphonate (PMOP) was synthesized using the phosphonamidite of Reference Example 15.

[0297] 2'-Deoxyadenosine linked with propyl phosphate (POP) was synthesized using the phosphoramidite form of Reference Example 16. 2'-Deoxyadenosine linked with butyl phosphate (POB) was synthesized using the phosphoramidite form of Reference Example 17. 2'-Deoxyadenosine linked with methoxyethyl phosphate (POMOE) was synthesized using the phosphoramidite form of Reference Example 18. 2'-Deoxyadenosine linked with cyclopropyl phosphate (POcP) was synthesized using the phosphoramidite form of Reference Example 19. 2'-Deoxyadenosine linked with cyclobutyl phosphate (POcB) was synthesized using the phosphoramidite form of Reference Example 20. 2'-O-methyladenosine linked with propyl phosphate (POP) was synthesized using the phosphoramidite form of Reference Example 21. 2'-O-methyladenosine linked with butyl phosphate (POB) was synthesized using the phosphoramidite form of Reference Example 22. 2'-O-methyladenosine linked with methoxyethyl phosphate (POMOE) was synthesized using the phosphoramidite form of Reference Example 23. 2'-O-methyladenosine linked with cyclopropyl phosphate (POcP) was synthesized using the phosphoramidite form of Reference Example 24. 2'-O-methyladenosine linked with cyclobutyl phosphate (POcB) was synthesized using the phosphoramidite form of Reference Example 25.

[0298] "2'-deoxy-2'-fluoroadenosine" linked with "ethyl phosphate (POE)" was synthesized using the phosphoramidite form of Reference Example 26. "2'-deoxy-2'-fluoroguanosine" linked with "ethyl phosphate (POE)" was synthesized using the phosphoramidite form of Reference Example 27. "2'-deoxy-2'-fluorocytidine" linked with "ethyl phosphate (POE)" was synthesized using the phosphoramidite form of Reference Example 28. "2'-deoxy-2'-fluorouridine" linked with "ethyl phosphate (POE)" was synthesized using the phosphoramidite form of Reference Example 29.

[0299] 2'-Deoxyinosine linked with ethyl phosphate (POE) was synthesized using the phosphoramidite form of Reference Example 30. 2'-Deoxypurine riboside or nebularine linked with ethyl phosphate (POE) was synthesized using the phosphoramidite form of Reference Example 31.

[0300] The moiety containing "N-methanesulfonyl phosphoric acid amide (PMs)" was synthesized using the method described in Nucleic Acids Research, (2021) 49, 9026-9041. The moiety containing "1,3-dimethylimidazolidin-2-ylidene phosphate (PN)" was synthesized using the method described in Front. Pharmacol. 2019;10:1049.

[0301] Deprotection of the acyl group of an oligonucleotide containing an alkylphosphonate, alkylthiophosphonate, or alkyl phosphate triester was carried out using ethylenediamine (Tetrahedron Letter, 37, 8691 (1996)).

[0302] The modification patterns of the target-editing oligonucleotides of Examples 1 to 83 and Comparative Examples 1 to 22 are shown in Tables 2 to 5.

[0303]

[0304]

[0305]

[0306]

[0307] The "molecular weight" in the table indicates the actual measured value by negative ion ESI mass spectrometry. In the "sequence" in the table, 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. Here, N indicates a nucleic acid base. As for the bond between nucleoside units, "p" indicates a phosphate residue (-P(=O)(OH)-), and "^" indicates a thiophosphate residue (-P(=S)(OH)-). (PM) indicates a methylphosphonate residue, and (PSM) indicates a methylthiophosphonate residue. (PE) represents an ethyl phosphonate residue, (PP) represents a propyl phosphonate residue, (PIP) represents an isopropyl phosphonate residue, (PB) represents a butyl phosphonate residue, and (PMOP) represents a methoxypropyl phosphonate residue. (POE) represents an ethyl phosphate residue, (POP) represents a propyl phosphate residue, (POIP) represents an isopropyl phosphate residue, (POB) represents a butyl phosphate residue, (POMOE) represents a methoxyethyl phosphate residue, (POcP) represents a cyclopropyl phosphate residue, and (POcB) represents a cyclobutyl phosphate residue. (PMs) represents an N-mesyl phosphate amide residue, and (PN) represents a 1,3-dimethylimidazolidin-2-ylidene phosphate residue. Specific structures are shown below. The 5' and 3' ends of the oligonucleotide are hydroxyl groups, with hydrogen atoms bonded to the oxygen atoms in the diagram.

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317] The compounds of Reference Examples 1 to 5 and Reference Example 32 were synthesized in the same manner as the compounds of Examples 1 to 83. The compounds of Reference Examples 1 to 5 and Reference Example 32 are shown in Table 6.

[0318]

[0319] The explanations for "molecular weight" and "sequence" in Table 6 are the same as those in Tables 2 to 5.

[0320] Test Example 1 Evaluation of the editing induction ability of target-editing oligonucleotides for RNA having the hACTB gene sequence in a cell-free environment

[0321] (A) Synthesis of ACTB_RNA pUC19_ACTB (a plasmid having the β-actin gene obtained in Test Example 3 (A) described in the specification of WO 2020 / 246560) as a template, T7_pUC19_FW primer (SEQ ID NO: 3) and M13_RV primer (SEQ ID NO: 4), PrimeStar GXL DNA Polymerase (TaKaRa) was used for PCR (denaturation: 98 ° C, 10 seconds, annealing: 55 ° C, 15 seconds, extension: 68 ° C, 20 seconds), template DNA was prepared, phenol / chloroform extraction, and purified by ethanol precipitation. In vitro transcription (37 ° C, 3 hours) was performed using AmpliScribe T7 Kit (Epicentre Biotechnologies) to synthesize RNA. DNase (final concentration: 2 U) was then added, and the mixture was treated at 37°C for 30 minutes. RNA was purified by phenol / chloroform extraction and ethanol precipitation. The resulting RNA was excised using 8M Urea PAGE (8%), extracted by crushing and immersion, and purified using a 0.22 μm filter (Millipore) and gel filtration (BIO RAD).

[0322] (B) Annealing reaction ACTB_RNA (final concentration: 0.3 μM) and target-editing oligonucleotide (hereinafter also referred to as gRNA) (final concentration: 0.9 μM) 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 to obtain an RNA complex.

[0323] (C) In vitro editing reaction Assuming that ACTB_RNA completely formed a complex with the target editing oligonucleotide through the annealing reaction, the following complex concentration was calculated. The resulting RNA complex (final concentration: 0.3 μM) was diluted with dH ２ After dilution, recombinant hADAR1p110 (synthesized and purified using a yeast expression system; see Macbeth, M.R. and Bass, B.L. Methods Enzymol. 424, 319-331 (2007), and Fukuda, M. et al. Sci. Rep. srep41478 (2017)) was added to the RNA complex (final concentration: 5 nM) to a final concentration of 250 nM, and the mixture was incubated at 37°C for 30 minutes.

[0324] (D) Editing Analysis After the editing reaction, ACTB_RNA was purified by phenol / chloroform extraction and ethanol precipitation. The resulting precipitates of each RNA complex were dissolved in 5 μL of TE buffer, and reverse transcription was performed using Primescript Reverse Transcriptase II (TaKaRa) and M13_RV primer (SEQ ID NO: 4) at 65°C for 5 minutes, followed by rapid cooling, at 42°C for 15 minutes, and at 70°C for 15 minutes. The resulting cDNA was used as a template for PCR amplification (denaturation: 98°C, 10 seconds; annealing: 55°C, 15 seconds; extension: 68°C, 20 seconds) using the T7_pUC19_FW primer (SEQ ID NO: 3) and the M13_RV primer (SEQ ID NO: 4) and PrimeStar GXL DNA Polymerase (TaKaRa). The amplification of the cDNA was confirmed using the DNA-500 kit (Shimadzu) and MultiNA (Shimadzu). A sequencing reaction (denaturation: 96°C, 10 seconds, annealing: 50°C, 5 seconds, extension: 60°C, 30 seconds) was performed using the T7proGGG primer (SEQ ID NO: 5) and the SuperDye v3.1 Cycle Sequencing Kit (Edge BioSystems), followed by purification by ethanol precipitation. The resulting precipitate was dissolved in 15 μL of Hi-Di formamide (Thermo Fisher Scientific), and the base sequence was analyzed using an Applied Biosystem 3500 Genetic Analyzer (Thermo Fisher Scientific). The peak heights of A and G at the target editing site in the sequencing chromatogram obtained by the analysis were analyzed, and the editing rate (%) was calculated from the peak height ratio (G / (G+A)).

[0325]

[0326] (E) Results of editing analysis The results of the editing rate by ADAR1p110 are shown in Figure 1A. The RNA editing efficiency by ADAR1p110 is compared to Reference Example Compound 1 (AD1_hACTB.39), in which all internucleoside bonds in the target editing oligonucleotide (gRNA) are phosphorothioate bonds, Example Compound 2 (AD1_hACTB.39_PM02), in which the internucleoside +1 site is a methylphosphonate bond, Example Compound 3 (AD1_hACTB.39_PM03), in which the internucleoside +4 site is a methylphosphonate bond, showed a high editing rate. Furthermore, Example Compound 1 (AD1_hACTB.39_PM01), in which the internucleoside +1 and +4 sites are methylphosphonate bonds, showed an even higher editing rate. On the other hand, in the comparative example compounds 1 to 3 (AD1_hACTB.39_PM04 to 06) in which the internucleoside position +2, -9, or -7 was methylphosphonate bonded, a slight improvement or decrease in the editing rate was observed.

[0327] As shown in Figure 2A, in comparative example compounds 4 to 9 (AD1_hACTB.39_PM07 to 12) in which the internucleoside site is -5, -1, +6, +8, +10 or +12 and a methylphosphonate bond was used, the improvement in the editing rate was slight. Also, as shown in Figure 3A, in comparative example compound 10 (AD1_hACTB.39_PM13) in which the internucleoside site is -2 and a methylphosphonate bond was used, compared to reference example compound 1 (AD1_hACTB.39), a decrease in the editing rate was observed. These results indicate that in gRNA, particularly in which the internucleoside site is +1 and +4, a high editing rate is shown when a methylphosphonate bond is used at least one site selected from the group consisting of. Furthermore, as shown in Figure 3D, comparative example compound 22 (AD1_hACTB.39_PM08a), in which the internucleoside position -1 was substituted with a methylphosphonate bond, did not show any improvement in the editing rate.

[0328] The results of the editing rate by ADAR1p110 are shown in Figure 3B. The RNA editing efficiency by ADAR1p110 is compared to Reference Example Compound 1 (AD1_hACTB.39), in which all bonds between nucleosides in the gRNA are phosphorothioate bonds, Example Compound 9 (AD1_hACTB.39_PSM02), in which the site where the internucleoside is +1 is a methylthiophosphonate bond, Example Compound 10 (AD1_hACTB.39_PSM03), in which the site where the internucleoside is +4 is a methylthiophosphonate bond, showed a high editing rate. Furthermore, Example Compound 8 (AD1_hACTB.39_PSM01), in which the site where the internucleoside is +1 and +4 is a methylthiophosphonate bond, showed an even higher editing rate. These findings indicate that the use of methylthiophosphonate bonds in gRNA, especially at internucleoside positions +1 and / or +4, results in a high editing rate.

[0329] The results of the editing rate by ADAR1p110 are shown in Figure 4A. The RNA editing efficiency by ADAR1p110 is compared to Reference Example Compound 1 (AD1_hACTB.39), in which all bonds between nucleosides in the gRNA are phosphorothioate bonds, Example Compound 5 (AD1_hACTB.39_POE02) in which the site between nucleosides is a phosphate ethyl bond, Example Compound 7 (AD1_hACTB.39_POIP02) in which the site between nucleosides is a phosphate isopropyl bond, Example Compound 6 (AD1_hACTB.39_POE03) in which the site between nucleosides is a phosphate ethyl bond, showed a high editing rate. Furthermore, Example Compound 4 (AD1_hACTB.39_POE01), in which the internucleoside positions in gRNA were phosphate ethyl bonds, showed an even higher editing rate. These results indicate that the use of phosphate ethyl bonds at the internucleoside positions +1 and / or +4, or particularly phosphate isopropyl bonds at the internucleoside positions +1, in gRNA, shows a high editing rate.

[0330] The results of the editing rate by ADAR1p110 are shown in Figure 4B. The RNA editing efficiency by ADAR1p110 is compared to Reference Example Compound 1 (AD1_hACTB.39), in which all bonds between nucleosides in the gRNA are phosphorothioate bonds, Example Compound 11 (AD1_hACTB.39_PE02), in which the site where the internucleoside is +1 is an ethyl phosphonate bond, Example Compound 12 (AD1_hACTB.39_PP02), and Example Compound 13 (AD1_hACTB.39_PIP02), in which the internucleoside is +1, showed a high editing rate. These results indicate that in the gRNA, particularly the site where the internucleoside is +1, an ethyl phosphonate bond, a propyl phosphonate bond, or an isopropyl phosphonate bond is used to show a high editing rate.

[0331] The results of the editing rate by ADAR1p110 are shown in Figure 6A. The RNA editing efficiency by ADAR1p110 is compared to Reference Example Compound 1 (AD1_hACTB.39), in which all bonds between nucleosides in the gRNA are phosphorothioate bonds, Comparative Example Compound 12 (AD1_hACTB.39_PO02), in which the site where the internucleoside is +1 is a phosphodiester bond, Comparative Example Compound 13 (AD1_hACTB.39_PO03), in which the site where the internucleoside is +4 is a phosphodiester bond, showed a high editing rate. Furthermore, Comparative Example Compound 11 (AD1_hACTB.39_PO01), in which the site where the internucleoside is +1 and +4 is a phosphodiester bond, showed an even higher editing rate. These findings indicate that the use of phosphodiester bonds in gRNA, especially at internucleoside positions +1 and / or +4, results in a high editing rate.

[0332] The results of the editing rate by ADAR1p110 are shown in Figure 7A. The RNA editing efficiency by ADAR1p110 was higher in the comparative example compound 1 (AD1_hACTB.39), in which all internucleoside bonds in the gRNA were phosphorothioate bonds, than in the comparative example compound 14 (AD1_hACTB.39_POE14), in which the internucleoside position was -4 was replaced with an ethyl phosphate bond, and in the comparative example compound 15 (AD1_hACTB.39_POE14), in which the internucleoside position was -3 was replaced with an ethyl phosphate bond. POE15), comparative example compound 17 (AD1_hACTB.39_POE17) in which the site between the nucleosides is an ethyl phosphate bond, comparative example compound 18 (AD1_hACTB.39_POE18) in which the site between the nucleosides is an ethyl phosphate bond, comparative example compound 19 (AD1_hACTB.39_POE19) in which the site between the nucleosides is an ethyl phosphate bond, showed similar editing rates. On the other hand, in the gRNA, comparative example compound 16 (AD1_hACTB.39_POE16) in which the site between the nucleosides is an ethyl phosphate bond, showed a slight decrease in editing rate. Furthermore, in gRNA, Example compound 19 (AD1_hACTB.39_POE20) in which three sites where the internucleoside is -5, +1, and +4 are phosphate ethyl bonds, Example compound 20 (AD1_hACTB.39_POE21) in which three sites where the internucleoside is +1, +4, and +8 are phosphate ethyl bonds, and Example compound 21 (AD1_hACTB.39_POE22) in which three sites where the internucleoside is +1, +4, and +10 are phosphate ethyl bonds, showed a high editing rate. These indicate that in gRNA, using phosphate ethyl bonds at sites where the internucleoside is +1 and / or +4 shows a high editing rate. In addition, in particular, in addition to sites where the internucleoside is +1 and +4, those having phosphate ethyl bonds at either -5, +8, or +10 also show a high editing rate.

[0333] The results of the editing rate by ADAR1p110 are shown in Figure 8A. The RNA editing efficiency by ADAR1p110 was compared with Reference Example Compound 1 (AD1_hACTB.39), in which all bonds between nucleosides in the gRNA were phosphorothioate bonds. Example Compound 34 (AD1_hACTB.39_PB02), in which the site where the internucleoside bond was +1 was a butyl phosphonate bond, Example Compound 35 (AD1_hACTB.39_PMOP02), in which the site where the internucleoside bond was +1 was a methoxypropyl phosphonate bond, and Example Compound 54 (AD1_hACTB.39_PO P02), Example compound 55 (AD1_hACTB.39_POB02) in which the site between the nucleosides is +1 is a butyl phosphate bond, Example compound 56 (AD1_hACTB.39_POMOE02) in which the site between the nucleosides is +1 is a methoxyethyl phosphate bond, Example compound 57 (AD1_hACTB.39_POcP02) in which the site between the nucleosides is +1 is a cyclopropyl phosphate bond, and Example compound 58 (AD1_hACTB.39_POcB02) in which the site between the nucleosides is +1 is a cyclobutyl phosphate bond, showed a high editing rate. These indicate that in gRNA, particularly in the site between the nucleosides is +1, a butyl phosphonate bond, a methoxypropyl phosphonate bond, a propyl phosphate bond, a butyl phosphate bond, a methoxyethyl phosphate bond, a cyclopropyl phosphate bond, or a cyclobutyl phosphate bond shows a high editing rate.

[0334] The results of the editing rate by ADAR1p110 are shown in Figure 9A. The RNA editing efficiency by ADAR1p110 was higher in Example Compound 22 (AD1_hACTB.39_POE23), which has phosphate ethyl bonds at the +1 and +4 internucleoside positions and a phosphodiester bond at the +2 internucleoside position, compared to Reference Example Compound 1 (AD1_hACTB.39), in which all internucleoside bonds in the gRNA are phosphorothioate bonds. Example compound 23 (AD1_hACTB.39_POE24) has phosphate diester bonds at the positions where the internucleosides are -3 and +2; Example compound 24 (AD1_hACTB.39_POE25) has phosphate ethyl bonds at the positions where the internucleosides are +1 and +4 and has phosphate diester bonds at the positions where the internucleosides are -8, -6, +2, +11, and +13; Further, Example Compound 25 (AD1_hACTB.39_POE26) has phosphate diester bonds at the positions where the internucleosides are -7, -5, +2, +10, and +12; Example Compound 26 (AD1_hACTB.39_POE27) has phosphate ethyl bonds at the positions where the internucleosides are +1 and +4 and has phosphate diester bonds at the positions where the internucleosides are +2, +3, and +5; Furthermore, Example compound 27 (AD1_hACTB.39_POE28), which has phosphate diester bonds at the internucleoside positions of -3, -2, -1, 0, +2, +3, and +5, and Example compound 28 (AD1_hACTB.39_POE29), which has phosphate ethyl bonds at the internucleoside positions of +1 and +4 and has phosphate diester bonds at the internucleoside positions of -3 to -1, 0, +2, +3 and +5 to +9, showed high editing rates.

[0335] In addition, the RNA editing efficiency by ADAR1p110 is compared to Reference Example Compound 4 (AD1_hACTB.39_POE31), in which all bonds between nucleosides in the gRNA are phosphodiester bonds, and Comparative Example Compound 20 (AD1_hACTB.39_POE30), which has phosphate ethyl bonds at the sites where the internucleosides are +1 and +4, and other bonds are phosphodiester bonds, showed a slightly higher editing rate. These facts indicate that, in particular, using phosphate ethyl bonds at the sites where the internucleosides are +1 and +4 in the gRNA shows a high editing rate.

[0336] The results of the editing rate by ADAR1p110 are shown in Figure 10A. The RNA editing efficiency by ADAR1p110 was higher in Reference Example Compound 1 (AD1_hACTB.39), in which all internucleoside bonds in the gRNA were phosphorothioate bonds, than in Example Compound 36 (AD1_hACTB.39_POE32), which had phosphate ethyl bonds at the positions where the internucleosides were +1 and +4, and a phosphodiester bond at the position where the internucleosides were -1, and in Example Compound 36 (AD1_hACTB.39_POE32), which had phosphate ethyl bonds at the positions where the internucleosides were +1 and +4, and a phosphodiester bond at the position where the internucleosides were -2. Example compound 37 (AD1_hACTB.39_POE33) has phosphate ethyl bonds at the positions where the internucleosides are +1 and +4, and further has phosphate diester bonds at the positions where the internucleosides are -6, +2, and +11; and Example compound 39 (AD1_hACTB.39_POE35) has phosphate ethyl bonds at the positions where the internucleosides are +1 and +4, and further has phosphate diester bonds at the positions where the internucleosides are -6, +2, +6, and +11. Example compound 40 (AD1_hACTB.39_POE36) has ethyl phosphate bonds at the positions where the internucleosides are +1 and +4 and further has phosphodiester bonds at the positions where the internucleosides are -6, +2, +8 and +11; Example compound 41 (AD1_hACTB.39_POE37) has ethyl phosphate bonds at the positions where the internucleosides are +1 and +4 and further has phosphodiester bonds at the positions where the internucleosides are -6, +2, +10 and +11; Example compound 42 (AD1_hACTB.39_POE38) has a phosphate diester bond at the internucleoside positions of -6, -5, +2, and +11; Example compound 43 (AD1_hACTB.39_POE39) has a phosphate ethyl bond at the internucleoside positions of +1 and +4, and a phosphate diester bond at the internucleoside positions of -6, -5, +2, +10, and +11; Example compound 44 (AD1_hACTB.39_POE39) has a phosphate ethyl bond at the internucleoside positions of +1 and +4, and a phosphate diester bond at the internucleoside positions of -6, -3, +2, +30, +40;Example compound 44 (AD1_hACTB.39_POE40) has phosphate diester bonds at the positions where +6 and +11 are internucleoside, Example compound 45 (AD1_hACTB.39_POE41) has phosphate ethyl bonds at the positions where +1 and +4 are internucleoside, and further has phosphate diester bonds at the positions where -6, -3, +2, +8, and +11 are internucleoside, Example compound 46 (AD1_hACTB.39_POE46) has phosphate ethyl bonds at the positions where +1 and +4 are internucleoside, and further has phosphate diester bonds at the positions where -6, -3, +2, +10, and +11 are internucleoside D1_hACTB.39_POE42), Example compound 47 (AD1_hACTB.39_POE43), which has a phosphate ethyl bond at the internucleoside positions +1 and +4, and a phosphate diester bond at the internucleoside positions -6, -5, -3, +2, and +11, and Example compound 48 (AD1_hACTB.39_POE44), which has a phosphate ethyl bond at the internucleoside positions +1 and +4, and a phosphate diester bond at the internucleoside positions -6, -5, -3, +2, +10, and +11, showed a high editing rate. These results indicate that, particularly in gRNA, using a phosphate ethyl bond at the internucleoside positions +1 and +4 shows a high editing rate. ,

[0337] The results of the editing rate by ADAR1p110 are shown in Figure 11A. The RNA editing efficiency by ADAR1p110 was compared with that of Reference Example Compound 1 (AD1_hACTB.39), in which all internucleoside bonds in the gRNA were phosphorothioate bonds, with Example Compound 29 (AD1_hACTB.39_PE03), in which the internucleoside position +4 was replaced with an ethylphosphonate bond; Example Compound 30 (AD1_hACTB.39_PP03), in which the internucleoside position +4 was replaced with a propylphosphonate bond; Example Compound 31 (AD1_hACTB.39_PIP03), in which the internucleoside position +4 was replaced with an isopropylphosphonate bond; Example Compound 32 (AD1_hACTB.39_PB03), in which the internucleoside position +4 was replaced with a butylphosphonate bond; and Example Compound 33 (AD1_hACTB.39_PB03), in which the internucleoside position +4 was replaced with a methoxypropylphosphonate bond. Example compound 33 (AD1_hACTB.39_PMOP03) in which a nate bond was used, Example compound 49 (AD1_hACTB.39_POP03) in which a propyl phosphate bond was used at the +4 internucleoside position, Example compound 50 (AD1_hACTB.39_POB03) in which a butyl phosphate bond was used at the +4 internucleoside position, Example compound 51 (AD1_hACTB.39_POMOE03) in which a methoxyethyl phosphate bond was used at the +4 internucleoside position, Example compound 52 (AD1_hACTB.39_POcP03) in which a cyclopropyl phosphate bond was used at the +4 internucleoside position, and Example compound 53 (AD1_hACTB.39_POcB03) in which a cyclobutyl phosphate bond was used at the +4 internucleoside position showed high editing rates. These findings indicate that a high editing rate is achieved when an ethyl phosphonate bond, a propyl phosphonate bond, an isopropyl phosphonate bond, a butyl phosphonate bond, a methoxypropyl phosphonate bond, a propyl phosphate bond, a butyl phosphate bond, a methoxyethyl phosphate bond, a cyclopropyl phosphate bond, or a cyclobutyl phosphate bond is used at a +4 internucleoside site in a gRNA.

[0338] The results of the editing rate by ADAR1p110 are shown in Figure 11C. The RNA editing efficiency by ADAR1p110 was compared with Reference Example Compound 32 (AD1_hACTB.86), in which all bonds between nucleosides in the gRNA were phosphorothioate bonds, Example Compound 73 (AD1_hACTB.86_POE45), in which the positions where the internucleosides were +1 and +4 were phosphate ethyl bonds, Example Compound 74 (AD1_hACTB.86_POE46), in which the positions where the internucleosides were +1 and +4 were phosphate diester bonds, and Example Compound 75 (AD1_hACTB.86_POE46), in which the positions where the internucleosides were +2 were phosphate diester bonds. Example compound 75 (AD1_hACTB.86_POE47) has phosphate ethyl bonds at the positions where the internucleosides are +1 and +4 and a phosphodiester bond at the positions where the internucleosides are +2 and +6; Example compound 76 (AD1_hACTB.86_POE48) has phosphate ethyl bonds at the positions where the internucleosides are +1 and +4 and a phosphodiester bond at the positions where the internucleosides are +2 and +8; Example compound 77 (AD1_hACTB.86_POE49) has phosphate ethyl bonds at the positions where the internucleosides are +1 and +4 and a phosphodiester bond at the positions where the internucleosides are +2 and +10; Example compound 77 (AD1_hACTB.86_POE49) has phosphate diester bonds at the positions where the internucleosides are +1 and +4, and also has phosphate diester bonds at the positions where the internucleosides are +2, +6, and +8; Example compound 78 (AD1_hACTB.86_POE50) has phosphate ethyl bonds at the positions where the internucleosides are +1 and +4, and also has phosphate diester bonds at the positions where the internucleosides are +2, +6, and +10; and Example compound 79 (AD1_hACTB.86_PO E51), Example compound 80 (AD1_hACTB.86_POE52) having ethyl phosphate bonds at the positions where the internucleosides are +1 and +4 and further having phosphodiester bonds at the positions where the internucleosides are +2, +8, and +10, Example compound 81 (AD1_hACTB.86_POE53) having ethyl phosphate bonds at the positions where the internucleosides are +1 and +4 and further having phosphodiester bonds at the positions where the internucleosides are +2, +6, +8, and +10,Example compound 82 (AD1_hACTB.86_POMOE46) has an ethyl phosphate bond at the site where the nucleoside is +4, and a phosphate diester bond at the site where the nucleoside is +2, and the +1 nucleoside is nebularine, and has an ethyl phosphate bond at the site where the nucleoside is +1 and +4, and a phosphate diester bond at the site where the nucleoside is +2 Example compound 83 (AD1_hACTB.86p_POE46), showed a high editing rate. These facts indicate that, particularly in gRNA, a phosphate ethyl bond at the site where the nucleoside is +1 and +4, or a methoxyethyl phosphate bond at the site where the nucleoside is +1, and a phosphate ethyl bond at the site where the nucleoside is +4 shows a high editing rate. Furthermore, in addition to the above, it has been shown that a high editing rate is achieved when a phosphodiester bond is used at at least one internucleoside position selected from the group consisting of +2, +6, +8, and +10.

[0339] Test Example 2: Evaluation of the ability of target-editing oligonucleotides to induce editing of RNA having the hACTB gene sequence in cultured cells

[0340] (A) Cell culture. When interferon α was added: HEK293 cells (JCRB Cell Bank) were cultured in a 24-well plate at 5.0 × 10 ４ When evaluating under conditions where ADAR1p150 was overexpressed, human interferon α (PBL Assay Science) was added at 300 U / well. After 48 hours, the medium was replaced and the cells were incubated with Lipofectamine. ＴＭ The target-editing oligonucleotide was transfected using RNAiMAX Transfection Reagent (Thermo Fisher Scientific) to a final concentration of 50 nM. After 48 hours, the cells were harvested.

[0341] Without the addition of interferon α: HEK293 cells (JCRB Cell Bank) were cultured in a 24-well plate at 5.0 × 10 ４After 48 hours, the cells were cultured using Lipofectamine. ＴＭ The target-editing oligonucleotide was transfected using RNAiMAX Transfection Reagent (Thermo Fisher Scientific) (final concentration: 3 nM, 10 nM, 50 nM, etc.). After 48 hours, the cells were harvested.

[0342] (B) Editing analysis Total RNA was extracted from cells cultured in a 24-well plate using RNeasy Plus Mini Kit (QIAGEN). （Ｒ） Reverse transcription was performed using qPCR RT Master Mix with gDNA Remover (TOYOBO) and 0.5 μg of total RNA to amplify the cDNA. The ACTB fragment was amplified by PCR (denaturation: 98°C, 10 seconds; annealing: 55°C, 15 seconds; extension: 68°C, 30 seconds) using PrimeStar GXL DNA polymerase (TaKaRa) and endoACTB_F02 primer (SEQ ID NO: 6) and endoACTB_R02 primer (SEQ ID NO: 7). Amplification was confirmed using the DNA-1000 kit (SHIMADZU) and MultiNA (SHIMADZU). A sequencing reaction (denaturation: 96°C, 10 seconds; annealing: 50°C, 5 seconds; extension: 60°C, 30 seconds) was performed using the endoACTB_seqF04 primer (SEQ ID NO: 8) and the SupreDye v3.1 Cycle Sequencing Kit (Edge BioSystems), followed by purification by ethanol precipitation.

[0343]

[0344] (C) Results of editing analysis The results of the RNA editing rate using a target editing oligonucleotide (gRNA) are shown in Figure 1B. When intracellular editing induction was evaluated when gRNA, an example compound or a comparative example compound, was transfected to a concentration of 50 nM, a higher editing rate was shown compared to gRNA (-). In addition, compared to Reference Example Compound 1 (AD1_hACTB.39), in which all bonds between nucleosides in the RNA are phosphorothioate bonds, Example Compound 2 (AD1_hACTB.39_PM02), in which the site where the internucleoside is +1 is a methylphosphonate bond, and Example Compound 3 (AD1_hACTB.39_PM03), in which the site where the internucleoside is +4 is a methylphosphonate bond, showed an improvement in editing rate. Furthermore, Example Compound 1 (AD1_hACTB.39_PM01), in which the sites where the internucleosides are +1 and +4 are methylphosphonate bonds in the gRNA, showed a further improvement in editing rate. On the other hand, in Comparative Example Compounds 1 to 3 (AD1_hACTB.39_PM04 to 06), in which the internucleoside site was a methylphosphonate bond at +2, -9, or -7, compared to Reference Example Compound 1 (AD1_hACTB.39), a slight improvement or decrease in the editing rate was observed. As shown in Figure 2B, in Comparative Example Compounds 4 to 9 (AD1_hACTB.39_PM07 to 12), in which the internucleoside site was a methylphosphonate bond at either -5, -1, +6, +8, +10, or +12, a slight improvement in the editing rate was observed. As shown in Figure 3C, Comparative Example Compound 10 (AD1_hACTB.39_PM13), in which the internucleoside site was a methylphosphonate bond at -2, compared to Reference Example Compound 1 (AD1_hACTB.39), had a similar editing rate. In addition, as shown in Figure 3E, comparative compound 21 (AD1_hACTB.39_PM08a), in which the internucleoside position was a methylphosphonate bond, did not show an improvement in the editing rate. These results indicate that, in particular, using a methylphosphonate bond at the internucleoside position +1 and / or +4 in the gRNA shows a high editing rate.

[0345] The results of the RNA editing rate using the example compounds are shown in Figure 3C. Compared to Reference Example Compound 1 (AD1_hACTB.39), in which all internucleoside bonds in the gRNA are phosphorothioate bonds, Example Compound 9 (AD1_hACTB.39_PSM02), in which the internucleoside +1 site is a methylthiophosphonate bond, and Example Compound 10 (AD1_hACTB.39_PSM03), in which the internucleoside +4 site is a methylthiophosphonate bond, showed an improvement in the editing rate. Furthermore, Example Compound 8 (AD1_hACTB.39_PSM01), in which the internucleoside +1 and +4 sites are a methylthiophosphonate bond, showed a further improvement in the editing rate. These facts indicate that, in particular, using a methylthiophosphonate bond at the internucleoside +1 and / or +4 site in the gRNA shows a high editing rate.

[0346] The results of the RNA editing rate using the example compounds are shown in Figure 4C. Compared to Reference Example Compound 1 (AD1_hACTB.39), in which all internucleoside bonds in the gRNA are phosphorothioate bonds, Example Compound 5 (AD1_hACTB.39_POE02), in which the internucleoside +1 site is an ethyl phosphate bond, and Example Compound 6 (AD1_hACTB.39_POE03), in which the internucleoside +4 site is an ethyl phosphate bond, showed a high editing rate. Furthermore, Example Compound 4 (AD1_hACTB.39_POE01), in which the internucleoside +1 and +4 sites are ethyl phosphate bonds, showed an even higher editing rate. These results indicate that, in particular, using an ethyl phosphate bond at the internucleoside +1 and / or +4 site in the gRNA shows a high editing rate.

[0347] The results of the RNA editing rate using the example compounds are shown in Figure 4D. All bonds between nucleosides in the gRNA are phosphorothioate bonds Reference Example Compound 1 (AD1_hACTB.39) compared to Example Compound 7 (AD1_hACTB.39_POIP02) in which the site between nucleosides is a phosphate isopropyl bond, Example Compound 11 (AD1_hACTB.39_PE02) in which the site between nucleosides is an ethyl phosphonate bond, Example Compound 12 (AD1_hACTB.39_PP02) in which the propyl phosphonate bond, and Example Compound 13 (AD1_hACTB.39_PIP02) in which the isopropyl phosphonate bond, showed a high editing rate. These findings indicate that a high editing rate is achieved when a phosphate isopropyl bond, an ethyl phosphonate bond, a propyl phosphonate bond, or an isopropyl phosphonate bond is used at a +1 internucleoside site in a gRNA.

[0348] The results of the RNA editing rate using the example compounds and comparative example compounds are shown in Figure 5. All bonds between nucleosides in the gRNA are phosphorothioate bonds. Compared to Reference Example Compound 2 (AD1_hACTB.39e), Example Compound 60 (AD1_hACTB.39e_PMs01) in which the site between nucleosides is a mesyl phosphoramidate bond (AD1_hACTB.39e_PMs01), Example Compound 61 (AD1_hACTB.39_PMs02) in which the site between nucleosides is a mesyl phosphoramidate bond (AD1_hACTB.39_PMs02) showed a high editing rate. Furthermore, in gRNA, the internucleoside positions +1 and +4 were 1,3-dimethylimidazolidin-2-ylidene phosphate bonds Example compound 62 (AD1_hACTB.39e_PN01), and the internucleoside positions +1 and +4 were mesyl phosphoramidate bonds Example compound 59 (AD1_hACTB.39e_PMs04), which showed even higher editing rates. On the other hand, comparative example compound 21 (AD1_hACTB.39e_PMs3), in which the internucleoside positions +2 were mesyl phosphoramidate bonds, showed a decrease in editing rate. These facts indicate that, in particular, in gRNA, the internucleoside positions +1 and / or +4 were mesyl phosphoramidate bonds, or 1,3-dimethylimidazolidin-2-ylidene phosphate bonds, when used, show high editing rates.

[0349] The results of the RNA editing rate using example compounds with different LNA number and position in gRNA are shown in Figure 5. Example compound 1 (AD1_hACTB.39_PM01), in which the internucleoside positions are methylphosphonate bonds in gRNA, has four LNAs, but the number and / or positions of Example compound 63 (AD1_hACTB.86_PM01), Example compound 64 (AD1_hACTB.87_PM01), Example compound 65 (AD1_hACTB.88_PM01), and Example compound 66 (AD1_hACTB.89_PM01) showed similar or higher editing rates. These results indicate that, in particular, gRNAs with methylphosphonate bonds at the internucleoside positions of +1 and / or +4 in gRNA exhibit high editing rates.

[0350] The results of the RNA editing rate using the comparative compound are shown in Figure 6B. Compared to Reference Example Compound 1 (AD1_hACTB.39), in which all bonds between nucleosides in the gRNA are phosphorothioate bonds, Comparative Example Compound 12 (AD1_hACTB.39_PO02), in which the site where the internucleoside is +1 is a phosphodiester bond, and Comparative Example Compound 13 (AD1_hACTB.39_PO03), in which the site where the internucleoside is +4 is a phosphodiester bond, showed a high editing rate. Furthermore, Comparative Example Compound 11 (AD1_hACTB.39_PO01), in which the site where the internucleoside is +1 and +4 is a phosphodiester bond, showed an even higher editing rate. These results indicate that using a phosphodiester bond in the gRNA, especially in the site where the internucleoside is +1 and / or +4, shows a high editing rate.

[0351] The results of the RNA editing rates using the example compounds and the comparative compounds are shown in Figure 7B. All bonds between nucleosides in the gRNA are phosphorothioate bonds Reference Example Compound 1 (AD1_hACTB.39) compared to, the nucleoside -4 site is a phosphate ethyl bond Comparative Example Compound 14 (AD1_hACTB.39_POE14), the nucleoside -3 site is a phosphate ethyl bond Comparative Example Compound 15 (AD1_hACTB.39_POE15), the nucleoside +5 site is a phosphate ethyl bond Comparative Example Compound 17 (AD1_hACTB.39_POE17), the nucleoside +7 site is a phosphate ethyl bond Comparative Example Compound 18 (AD1_hACTB.39_POE18), the nucleoside +9 site is a phosphate ethyl bond Comparative Example Compound 19 (AD1_hACTB.39_POE19) showed similar editing rates. On the other hand, in gRNA, the internucleoside +3 site was an ethyl phosphate bond Comparative Example Compound 15 (AD1_hACTB.39_POE16), a slight decrease in the editing rate was observed. Also, in gRNA, the internucleoside -5, +1 and +4 three sites were ethyl phosphate bonds Example Compound 19 (AD1_hACTB.39_POE20), the internucleoside +1, +4 and +8 three sites were ethyl phosphate bonds Example Compound 20 (AD1_hACTB.39_POE21), and the internucleoside +1, +4 and +10 three sites were ethyl phosphate bonds Example Compound 21 (AD1_hACTB.39_POE22), showed a high editing rate. These results indicate that, in particular, using an ethyl phosphate bond at a site where the internucleoside bond is +1 and / or +4 in gRNA shows a high editing rate. In addition, in addition to the sites where the internucleoside bond is +1 and +4, those having an ethyl phosphate bond at either -5, +8, or +10 also show a high editing rate.

[0352] The results of the RNA editing rate using the example compounds are shown in Figure 8B. Compared to Reference Example Compound 1 (AD1_hACTB.39), in which all bonds between nucleosides in the gRNA are phosphorothioate bonds, Example Compound 34 (AD1_hACTB.39_PB02), in which the site where the internucleoside bond is +1 is a butyl phosphonate bond, Example Compound 35 (AD1_hACTB.39_PMOP02), in which the site where the internucleoside bond is +1 is a methoxypropyl phosphonate bond, Example Compound 54 (AD1_hACTB.39_POP02), in which the site where the internucleoside bond is +1 is a P-propoxy bond, Example compound 55 (AD1_hACTB.39_POB02) in which the site between the nucleosides is +1 is a butyl phosphate bond, Example compound 56 (AD1_hACTB.39_POMOE02) in which the site between the nucleosides is +1 is a methoxyethyl phosphate bond, Example compound 57 (AD1_hACTB.39_POcP02) in which the site between the nucleosides is +1 is a cyclopropyl phosphate bond, and Example compound 58 (AD1_hACTB.39_POcB02) in which the site between the nucleosides is +1 is a cyclobutyl phosphate bond, showed a high editing rate. These indicate that, in particular, in the gRNA, the site between the nucleosides is +1 is a butyl phosphonate bond, a methoxypropyl phosphonate bond, a propyl phosphate bond, a butyl phosphate bond, a phosphorus methoxyethyl bond, a cyclopropyl phosphate bond, or a cyclobutyl phosphate bond shows a high editing rate.

[0353] The results of the RNA editing rate using the example compounds are shown in Figure 9B. Compared to Reference Example Compound 1 (AD1_hACTB.39), in which all internucleoside bonds in the gRNA are phosphorothioate bonds, Example Compound 22 (AD1_hACTB.39_POE23), which has phosphate ethyl bonds at the positions where the internucleosides are +1 and +4 and a phosphodiester bond at the position where the internucleosides are +2, and Example Compound 23 (AD1_hACTB.39_POE23), which has phosphate ethyl bonds at the positions where the internucleosides are +1 and +4 and a phosphodiester bond at the position where the internucleosides are -3 and Example compound 23 (AD1_hACTB.39_POE24) has phosphate diester bonds at the positions where the internucleosides are +1 and +4, and further has phosphate diester bonds at the positions where the internucleosides are -8, -6, +2, +11, and +13; Example compound 24 (AD1_hACTB.39_POE25) has phosphate ethyl bonds at the positions where the internucleosides are +1 and +4, and further has phosphate diester bonds at the positions where the internucleosides are -8, -6, +2, +11, and +13; Example compound 25 (AD1_hACTB.39_POE26) has phosphate diester bonds at the positions of -7, -5, +2, +10, and +12; Example compound 26 (AD1_hACTB.39_POE27) has phosphate ethyl bonds at the positions of +1 and +4 internucleosides and further has phosphate diester bonds at the positions of +2, +3, and +5 internucleosides; Example compound 27 (AD1_hACTB.39_POE28), which has phosphate diester bonds at the internucleoside positions of -3, -2, -1, 0, +2, +3, and +5, and example compound 28 (AD1_hACTB.39_POE29), which has phosphate ethyl bonds at the internucleoside positions of +1 and +4 and further has phosphate diester bonds at the internucleoside positions of -3 to -1, 0, +2, +3 and +5 to +9, showed high editing rates. Furthermore, Reference Example Compound 4 (AD1_hACTB.39_POE31), in which all internucleoside bonds in the gRNA are phosphodiester bonds, and Comparative Example Compound 20 (AD1_hACTB.39_POE30), in which there are phosphate ethyl bonds at the +1 and +4 internucleoside positions and the other bonds are phosphodiester bonds, did not exhibit a high editing rate.The reason for this is thought to be the large number of phosphodiester bonds. These findings indicate that, in particular, using ethyl phosphate bonds at the +1 and +4 internucleoside sites in gRNA results in a high editing rate. Furthermore, in addition to the above, using an ethyl phosphate bond at the +2 site results in a high editing rate.

[0354] The results of the RNA editing rate using the example compounds are shown in Figure 10B. Compared to Reference Example Compound 1 (AD1_hACTB.39), in which all bonds between nucleosides in the gRNA are phosphorothioate bonds, Example Compound 36 (AD1_hACTB.39_POE32) has phosphate ethyl bonds at the positions where the internucleosides are +1 and +4, and a phosphodiester bond at the position where the internucleosides are -1, Example Compound 37 (AD1 Example compound 38 (AD1_hACTB.39_POE34) which has phosphate ethyl bonds at the positions where the internucleosides are +1 and +4 and further has phosphate diester bonds at the positions where the internucleosides are -6, +2, and +11; Example compound 39 (AD1_hACTB.39_POE35) which has phosphate ethyl bonds at the positions where the internucleosides are +1 and +4 and further has phosphate diester bonds at the positions where the internucleosides are -6, +2, +6, and +11; Example compound 40 (AD1_hACTB.39_POE36) has ethyl phosphate bonds at the positions where the internucleosides are +1 and +4 and further has phosphodiester bonds at the positions where the internucleosides are -6, +2, +8, and +11; Example compound 41 (AD1_hACTB.39_POE37) has ethyl phosphate bonds at the positions where the internucleosides are +1 and +4 and further has phosphodiester bonds at the positions where the internucleosides are -6, +2, +10, and +11; Further, Example Compound 42 (AD1_hACTB.39_POE38) has phosphate diester bonds at the positions where the internucleosides are -6, -5, +2, and +11; Example Compound 43 (AD1_hACTB.39_POE39) has phosphate ethyl bonds at the positions where the internucleosides are +1 and +4, and has phosphate diester bonds at the positions where the internucleosides are -6, -5, +2, +10, and +11;Example compound 44 (AD1_hACTB.39_POE40) has phosphate diester bonds at the positions where +6 and +11 are internucleoside, Example compound 45 (AD1_hACTB.39_POE41) has phosphate ethyl bonds at the positions where +1 and +4 are internucleoside, and further has phosphate diester bonds at the positions where -6, -3, +2, +8, and +11 are internucleoside, Example compound 46 (AD1_hACTB.39_POE46) has phosphate ethyl bonds at the positions where +1 and +4 are internucleoside, and further has phosphate diester bonds at the positions where -6, -3, +2, +10, and +11 are internucleoside D1_hACTB. 39_POE42), having an ethyl phosphate bond at the site where the nucleosides are +1 and +4, and further having a phosphate diester bond at the site where the nucleosides are -6, -5, -3, +2 and +11 Example compound 47 (AD1_hACTB. 39_POE43), and having an ethyl phosphate bond at the site where the nucleosides are +1 and +4, and further having a phosphate diester bond at the site where the nucleosides are -6, -5, -3, +2, +10 and +11 Example compound 48 (AD1_hACTB. 39_POE44) showed a high editing rate. These indicate that, in particular, in gRNA, using an ethyl phosphate bond at the site where the nucleosides are +1 and +4 shows a high editing rate. Furthermore, in addition to the above, it has been shown that a high editing rate is achieved when phosphate ethyl bonds are present at the internucleoside positions +2, -6, and +11.

[0355] The results of the RNA editing rate using the example compounds are shown in Figure 11B. All bonds between nucleosides in the gRNA are phosphorothioate bonds compared to Reference Example Compound 1 (AD1_hACTB.39), Example Compound 29 (AD1_hACTB.39_PE03) in which the site between nucleosides is +4 is an ethyl phosphonate bond, Example Compound 30 (AD1_hACTB.39_PP03) in which the site between nucleosides is +4 is a propyl phosphonate bond, Example Compound 31 (AD1_hACTB.39_PIP03) in which the site between nucleosides is +4 is an isopropyl phosphonate bond, Example Compound 32 (AD1_hACTB.39_PB03) in which the site between nucleosides is +4 is a butyl phosphonate bond, Example Compound 32 (AD1_hACTB.39_PB03) in which the site between nucleosides is +4 is a methoxypropyl phosphonate bond Compound 33 (AD1_hACTB.39_PMOP03), Example compound 49 (AD1_hACTB.39_POP03) in which the internucleoside position is replaced by a propyl phosphate bond, Example compound 50 (AD1_hACTB.39_POB03) in which the internucleoside position is replaced by a butyl phosphate bond, Example compound 51 (AD1_hACTB.39_POMOE03) in which the internucleoside position is replaced by a methoxyethyl phosphate bond, Example compound 52 (AD1_hACTB.39_POcP03) in which the internucleoside position is replaced by a cyclopropyl phosphate bond, and Example compound 53 (AD1_hACTB.39_POcB03) in which the internucleoside position is replaced by a cyclobutyl phosphate bond showed improved editing rates. These findings indicate that a high editing rate is achieved when an ethyl phosphonate bond, a propyl phosphonate bond, an isopropyl phosphonate bond, a butyl phosphonate bond, a methoxypropyl phosphonate bond, a propyl phosphate bond, a butyl phosphate bond, a methoxyethyl phosphate bond, a cyclopropyl phosphate bond, or a cyclobutyl phosphate bond is used at a +4 internucleoside site in a gRNA.

[0356] The results of the editing rate by ADAR1p110 are shown in Figure 11D. Compared to Reference Example Compound 32 (AD1_hACTB.86), in which all bonds between nucleosides in the gRNA are phosphorothioate bonds, Example Compound 73 (AD1_hACTB.86_POE45), in which the positions where the internucleosides are +1 and +4 are phosphate ethyl bonds, Example Compound 74 (AD1_hACTB.86_POE46), in which the positions where the internucleosides are +1 and +4 are phosphate diester bonds, and Example Compound 75 (AD1_hACTB.86_POE46), in which the positions where the internucleosides are +1 and +4 are phosphate diester bonds. Example compound 75 (AD1_hACTB.86_POE47) has a phosphate ethyl bond at the positions where the internucleosides are +2 and +6, and a phosphodiester bond is used at those positions. Example compound 76 (AD1_hACTB.86_POE48) has a phosphate ethyl bond at the positions where the internucleosides are +1 and +4, and a phosphodiester bond is used at those positions where the internucleosides are +2 and +8. Example compound 77 (AD1_hACTB.86_POE49) has a phosphate ethyl bond at the positions where the internucleosides are +1 and +4, and a phosphodiester bond is used at those positions where the internucleosides are +2 and +10. Example compound 77 (AD1_hACTB.86_POE49) having phosphate ethyl bonds at the positions where the internucleosides are +1 and +4 and further having phosphate diester bonds at the positions where the internucleosides are +2, +6, and +8; Example compound 78 (AD1_hACTB.86_POE50) having phosphate ethyl bonds at the positions where the internucleosides are +1 and +4 and further having phosphate diester bonds at the positions where the internucleosides are +2, +6, and +10; Example compound 79 (AD1_hACTB.86_POE51) having phosphate ethyl bonds at the positions where the internucleosides are +1 and +4 and further having phosphate diester bonds at the positions where the internucleosides are +2, +6, and +10; Example compound 80 (AD1_hACTB.86_POE52) has ethyl phosphate bonds at the internucleoside positions +1 and +4 and further has phosphodiester bonds at the internucleoside positions +2, +8, and +10; Example compound 81 (AD1_hACTB.86_POE53) has ethyl phosphate bonds at the internucleoside positions +1 and +4 and further has phosphodiester bonds at the internucleoside positions +2, +6, +8, and +10;Example compound 82 (AD1_hACTB.86_POMOE46) has an ethyl phosphate bond at the site where the nucleoside is +4, and a phosphate diester bond at the site where the nucleoside is +2, and the +1 nucleoside is nebularine, and has an ethyl phosphate bond at the site where the nucleoside is +1 and +4, and a phosphate diester bond at the site where the nucleoside is +2 Example compound 83 (AD1_hACTB.86p_POE46) showed a high editing rate. These indicate that, particularly in gRNA, a phosphate ethyl bond at the site where the nucleoside is +1 and +4, or a methoxyethyl phosphate bond at the site where the nucleoside is +1, and a phosphate ethyl bond at the site where the nucleoside is +4 shows a high editing rate. Furthermore, in addition to the above, it has been shown that using a phosphodiester bond at the +2, +6, +8, and / or +10 internucleoside positions, especially at the +2 position, results in a high editing rate.

[0357] Test Example 3 Evaluation of the editing induction ability of target-editing oligonucleotides for RNA having the A1AT gene sequence in a cell-free environment

[0358] (A) RNA synthesis The procedure was the same as that described in (A) ACTB_RNA synthesis in Test Example 1. However, the oligo DNA used was as described below. The A1AT (also known as SERPINA1) gene sequence used here has the base sequence from bases 1129 to 1153 of GenBank accession no. NM_000295.5, and further has a nucleoside mutation called c.1096G>A (rs28929474).

[0359]

[0360] (B) Evaluation of editing induction ability The method for evaluating the editing induction ability of example compounds was the same as in Test Example 1 (B) to (D). (C) Results of editing analysis The results of the editing rate by ADAR1p110 are shown in Figure 12A. Compared to the compound in which all bonds between nucleosides in the gRNA are phosphorothioate bonds (AD1_A1AT.39, Example 136 of the specification of WO 2022 / 124345), Example compound 68 (AD1_A1AT_PM02) in which the site where the internucleoside is +1 is a methylphosphonate bond, Example compound 69 (AD1_A1AT_PM03) in which the site where the internucleoside is +4 is a methylphosphonate bond, showed a high editing rate. Furthermore, Example Compound 67 (AD1_A1AT_PM01), in which the internucleoside positions in gRNA were methylphosphonate bonds, showed even higher editing rates. These results indicate that the use of methylphosphonate bonds at the internucleoside positions in gRNA is associated with a high editing rate.

[0361] Test Example 4 Evaluation of the ability of example compounds to induce editing of RNA having the A1AT gene sequence in cultured cells

[0362] (A) Method for evaluating editing induction ability The editing induction ability of the A1AT gene sequence of the example compound was evaluated using the method described in Test Example 35 of the specification of WO 2022 / 124345. The A1AT (also referred to as SERPINA1) gene sequence used here has the base sequence from 1129 to 1153 of GenBank accession no. NM_000295.5, and further has a nucleoside mutation called c. 1096G>A (rs28929474).

[0363] (B) Results of editing analysis The results of the RNA editing rate using example compounds are shown in Figure 12B. Compared to a compound in which all internucleoside bonds in the gRNA are phosphorothioate bonds (AD1_A1AT.39, the compound described in Example 136 of the specification of WO 2022 / 124345), Example compound 68 (AD1_A1AT_PM02) in which the internucleoside +1 site is a methylphosphonate bond, Example compound 69 (AD1_A1AT_PM03) in which the internucleoside +4 site is a methylphosphonate bond, showed a high editing rate. Furthermore, Example compound 67 (AD1_A1AT_PM01) in which the internucleoside +1 and +4 sites are methylphosphonate bonds in the gRNA showed an even higher editing rate. These results indicate that the use of methylphosphonate bonds at internucleoside positions +1 and / or +4 in gRNA results in a high editing rate.

[0364] Test Example 5 Evaluation of the editing induction ability of target-editing oligonucleotides for RNA having the mouse GAPDH gene sequence in a cell-free environment

[0365] (A) RNA synthesis The procedure was the same as that described in (A) ACTB_RNA synthesis in Test Example 1. However, the oligo DNA used was as described below. The mouse GAPDH gene sequence used here has the base sequence from bases 1129 to 1153 of GenBank accession no. NM_000295.5.

[0366]

[0367] (B) Evaluation of editing induction ability The editing induction ability of the example compounds was evaluated using the same methods as those in (B) to (D) of Test Example 1.

[0368] (C) Results of editing analysis The results of the editing rate by ADAR1p110 are shown in Figure 13A. The RNA editing efficiency by ADAR1p110 is compared to Reference Example Compound 3 (AD1g_03_mGAPDH_A1222_39), in which all bonds between nucleosides in the gRNA are phosphorothioate bonds, Example Compound 70 (AD1g_03_mGAPDH_A1222_PM02), in which the site where the internucleoside is +1 is a methylphosphonate bond, showed a high editing rate. These indicate that in the gRNA, using a methylphosphonate bond at the site where the internucleoside is +1 shows a high editing rate.

[0369] Test Example 6: Evaluation of the ability of target-editing oligonucleotides to induce editing of RNA having the mouse GAPDH gene sequence in cultured cells

[0370] (A) Method for evaluating editing induction ability CD-1-derived mouse primary hepatocytes (Thermo Fisher Scientific) were placed in a 96-well plate at 2.0 × 10 ４ After 24 hours, the example compound was added to a final concentration of 2 μM. Total RNA was extracted from the cells cultured in a 96-well plate using RNeasy Plus Micro Kit (QIAGEN). ＴＭ IV VILO ＴＭ Master Mix with ezDNase ＴＭ A reverse transcription reaction was performed using Enzyme (Thermo Fisher Scientific) and 0.5 μg of total RNA to amplify the cDNA. （Ｒ）The fragment was amplified by PCR (denaturation: 98°C, 10 seconds, annealing: 55°C, 5 seconds, extension: 72°C, 5 seconds) using Max DNA Polymerase (TaKaRa), endoGapdh_Fw primer (SEQ ID NO: 13), and endoGapdh_Rv (SEQ ID NO: 14). Amplification was confirmed using an E-Gel precast agarose electrophoresis system (Thermo Fisher Scientific). The amplified product was purified using a QIAquick PCR Purification Kit (QIAGEN), and sequence analysis was performed using the endoGapdh_Fw primer (SEQ ID NO: 13).

[0371]

[0372] (B) Results of editing analysis The results of the RNA editing rate using the example compounds are shown in Figure 13 (B). All bonds between nucleosides in the gRNA are phosphorothioate bonds Reference Example Compound 5 (AD1g_03_mGAPDH_A1222_39e) compared to the example compound 70 (AD1g_03_mGAPDH_A1222_PM02) in which the site between nucleosides is +1 is a methylphosphonate bond, Example Compound 71 (AD1g_03_mGAPDH_A1222_POE01) and Example Compound 72 (AD1g_03_mGAPDH_A1222_POE02) showed a high editing rate. These results indicate that a high editing rate is achieved when a methylphosphonate bond is used at the +1 internucleoside site in gRNA, an ethyl phosphate bond is used at the +1 and +4 internucleoside sites, or an ethyl phosphate bond is used at the +1 internucleoside site.

[0373] Examples 84 to 88 The target-editing oligonucleotides of Examples 84 to 88, like the target-editing oligonucleotides of Examples 1 to 83, were obtained by introducing modified oligonucleotides and phosphorus-containing linking groups as shown in the modification patterns in Table 12, based on the base sequences of the target-editing oligonucleotides shown in Table 1.

[0374]

[0375] The explanations for "molecular weight" and "sequence" in Table 12 are the same as those in Tables 2 to 5.

[0376] Test Example 7: Evaluation of the ability of target-editing oligonucleotides to induce editing of RNA having the mouse GAPDH gene sequence in cultured cells (2)

[0377] (A) Method for evaluating editing induction ability The editing induction ability of the example compounds was evaluated using the same method as in Test Example 6 (A).

[0378] (B) Results of editing analysis The results of the RNA editing rate using the example compounds are shown in Figure 14. Tests were conducted in two wells, and the values ​​of the RNA editing rate for each well were plotted. Figure 14 shows the average values ​​of the two plotted in a bar graph. Compared to Reference Example Compound 5 (AD1g_03_mGAPDH_A1222_39e), in which all bonds between nucleosides in the gRNA are phosphorothioate bonds, Example Compound 84 (AD1g_03_mGAPDH_A1222_39e_POMOE23) has a phosphate methoxyethyl bond at the site where the internucleoside is +1, a phosphodiester bond at the site where the internucleoside is +2, and a phosphate ethyl bond at the site where the internucleoside is +4. In addition to the modification of Example Compound 84, the sites where the internucleosides are -6 and +11 are phosphodiester bonds. Example compound 85 (AD1g_03_mGAPDH_A1222_39e_POMOE34), in which the 11th nucleoside residue counting in the 3' direction from the target-corresponding nucleoside residue of Example compound 84 was replaced with a 2'-O-methyl nucleoside (Example compound 86 (AD1g_03_mGAPDH_A1222_39e_POMOE46)); and Example compound 86 (AD1g_03_mGAPDH_A1222_39e_POMOE46), in which the +10 internucleoside position of Example compound 86 was replaced with a phosphodiester bond, all showed high editing rates. These results indicate that a high editing rate is achieved when a methoxyethyl phosphate bond is used at the site where the internucleoside spacing is +1, a phosphodiester bond is used at the site where the internucleoside spacing is +2, and an ethyl phosphate bond is used at the site where the internucleoside spacing is +4 in gRNA.

[0379] The disclosure of Japanese Patent Application No. 2023-137467 (filing date: August 25, 2023) is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards mentioned herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard was specifically and individually indicated to be incorporated by reference.

Claims

1. A nucleic acid sequence comprising: a first oligonucleotide for identifying a target RNA; and a second oligonucleotide linked to the 5' side of the first oligonucleotide, wherein the first oligonucleotide comprises: a target-corresponding nucleoside residue corresponding to an adenosine residue in the target RNA; an oligonucleotide of 3 to 6 residues linked to the 5' side of the target-corresponding nucleoside residue and having a base sequence complementary to the target RNA; and an oligonucleotide of 10 to 24 residues linked to the 3' side of the target-corresponding nucleoside residue and having a base sequence complementary to the target RNA; wherein in the oligonucleotide linked to the 3' side of the target-corresponding nucleoside residue, at least one phosphorus-containing linking group selected from the group consisting of a phosphorus-containing linking group linking a first nucleoside residue to a second nucleoside residue and a phosphorus-containing linking group linking a fourth nucleoside residue to a fifth nucleoside residue, counting in the 3' direction from the target-corresponding nucleoside residue, is at least one type selected from the group consisting of an alkylphosphonic acid residue, an alkyl phosphate residue and a substituted phosphoramide residue; The second oligonucleotide lacks a nucleoside residue at its 3' end that corresponds to a nucleoside residue of the target RNA or has a nucleoside residue that does not form a complementary pair with a nucleoside residue of the target RNA; the second oligonucleotide has 2 to 10 residues, and at least the nucleoside residues other than those at the 3' end form a double-stranded structure complementary to the target RNA; at least one of the first oligonucleotide and the second oligonucleotide contains a phosphorothioate bond; and an oligonucleotide that induces site-specific editing of the target RNA, or a pharma- ceutically acceptable salt thereof.

2. The oligonucleotide according to claim 1, or a pharma- ceutically acceptable salt thereof, wherein the nucleoside residue linked to the 3' side of the target-corresponding nucleoside residue is a 2'-deoxynucleoside residue.

3. The oligonucleotide according to claim 1 or 2, or a pharma- ceutically acceptable salt thereof, wherein in the oligonucleotide linked to the 3' side of the target-corresponding nucleoside residue, the third nucleoside residue counting in the 3' direction from the target-corresponding nucleoside residue is a 2'-deoxy-2'-fluoronucleoside residue.

4. The oligonucleotide according to any one of claims 1 to 3, wherein the second oligonucleotide has from 4 to 8 residues, or a pharma- ceutically acceptable salt thereof.

5. An oligonucleotide according to any one of claims 1 to 4, wherein the 3' end of the second oligonucleotide lacks a nucleoside residue corresponding to a nucleoside residue in the target RNA, or a pharma- ceutically acceptable salt thereof.

6. An oligonucleotide according to any one of claims 1 to 5, or a pharma- ceutically acceptable salt thereof, wherein the site-specific editing is caused by an enzymatic reaction by adenosine deaminase 1.

7. The oligonucleotide of any one of claims 1 to 6, or a pharma- ceutically acceptable salt thereof, wherein the first oligonucleotide comprises a phosphorothioate linkage.

8. An oligonucleotide according to any one of claims 1 to 7, or a pharma- ceutically acceptable salt thereof, wherein at least one phosphorus-containing linking group selected from the group consisting of a phosphorus-containing linking group linking the second nucleoside residue to the third nucleoside residue, a phosphorus-containing linking group linking the eighth nucleoside residue to the ninth nucleoside residue, a phosphorus-containing linking group linking the tenth nucleoside residue to the eleventh nucleoside residue, and a phosphorus-containing linking group linking the eleventh nucleoside residue to the twelfth nucleoside residue, counting in the 3' direction from the target-corresponding nucleoside residue, is a phosphate residue.

9. The oligonucleotide according to any one of claims 1 to 8, or a pharma- ceutically acceptable salt thereof, wherein the first oligonucleotide has a second nucleoside residue and a third nucleoside residue linked by a phosphodiester bond, counting in the 3' direction from the target-corresponding nucleoside residue.

10. The oligonucleotide according to any one of claims 1 to 9, wherein the first oligonucleotide comprises at least one modified nucleoside residue selected from the group consisting of 2'-O-alkylribonucleoside residues, 2'-deoxy-2'-fluororibonucleoside residues, bridged nucleoside residues, and 2'-deoxyribonucleoside residues, or a pharma-ceutically acceptable salt thereof.

11. The oligonucleotide according to any one of claims 1 to 10, or a pharma- ceutically acceptable salt thereof, comprising a substructure in which the target-corresponding nucleoside residue is linked to an adjacent nucleoside residue via a phosphorothioate bond.

12. The oligonucleotide according to any one of claims 1 to 11, or a pharma- ceutically acceptable salt thereof, wherein the nucleoside residue linked to the 5' side of the target-corresponding nucleoside residue is a 2'-O-alkylribonucleoside residue.

13. The oligonucleotide according to any one of claims 1 to 12, or a pharma- ceutically acceptable salt thereof, wherein the second oligonucleotide comprises at least one modified nucleoside residue selected from the group consisting of a 2'-O-alkylribonucleoside residue, a 2'-deoxy-2'-fluororibonucleoside residue, and a bridged nucleoside residue.

14. The oligonucleotide according to any one of claims 1 to 13, or a pharma- ceutically acceptable salt thereof, wherein the oligonucleotide linked to the 3' side of the target-corresponding nucleoside residue has a base sequence in which 2'-deoxy-2'-fluoronucleoside residues and 2'-O-alkylribonucleoside residues are alternately linked.

15. The oligonucleotide according to any one of claims 1 to 14, or a pharma- ceutically acceptable salt thereof, wherein the oligonucleotide linked to the 3' side of the target-corresponding nucleoside residue has a base sequence in which a bridged nucleoside residue and a 2'-O-alkylribonucleoside residue are alternately linked.

16. An oligonucleotide according to any one of claims 1 to 15, wherein in the first oligonucleotide, the oligonucleotide linked to the 5' side of the target-corresponding nucleoside residue has a base sequence in which 2'-O-alkylribonucleoside residues are consecutive, or a pharma-ceutically acceptable salt thereof.

17. The oligonucleotide according to any one of claims 1 to 16, or a pharma- ceutically acceptable salt thereof, wherein the second oligonucleotide has a base sequence in which 2'-O-alkylribonucleoside residues and bridged nucleoside residues are linked alternately.

18. The oligonucleotide according to any one of claims 1 to 17, or a pharma- ceutically acceptable salt thereof, wherein the target-corresponding nucleoside residue is an N-alkylpyrimidine nucleoside residue or a 2'-deoxycytidine residue.

19. The second oligonucleotide according to any one of claims 1 to 18, wherein the nucleoside residues are linked by phosphorothioate bonds, or a pharma- ceutically acceptable salt thereof.

20. An oligonucleotide according to any one of claims 1 to 19, or a pharma- ceutically acceptable salt thereof, wherein the first oligonucleotide has a bridged nucleoside residue at any nucleoside residue 10 or later in the 3' direction from the target-corresponding nucleoside residue.

21. The oligonucleotide according to claim 20, or a pharma- ceutically acceptable salt thereof, wherein the first oligonucleotide is an oligonucleotide consisting of nucleoside residues from the 10th nucleoside residue onward counting in the 3' direction from the target-corresponding nucleoside residue, and has a base sequence in which 2'-O-alkylribonucleoside residues and bridged nucleoside residues are alternately linked.

22. The oligonucleotide according to any one of claims 1 to 21, wherein the second oligonucleotide has a bridged nucleoside residue in the second or subsequent nucleoside residue counting in the 5' direction from the 3'-terminal nucleoside residue, or a pharma- ceutically acceptable salt thereof.

23. The oligonucleotide according to claim 22, or a pharma- ceutically acceptable salt thereof, wherein the second oligonucleotide comprises three or more nucleoside residues, and the oligonucleotide consisting of the second and subsequent nucleoside residues counting in the 5' direction from the 3'-terminal nucleoside residue has a base sequence in which 2'-O-alkylribonucleoside residues and bridged nucleoside residues are alternately linked.

24. The oligonucleotide according to any one of claims 1 to 7, comprising a modified oligonucleotide having a chemical modification pattern represented by any one of the following formulas (1) to (22), or a pharma- ceutically acceptable salt thereof: (1) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsLsMsLsM-OH -3' (2) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsMsMsLsM-OH -3' (3) 5'- HO-MsLsMsMsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsLsMsLsM-OH -3' (4) 5'- HO-MsLsMsMsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsMsMsLsM-OH -3' (5) 5'- HO-LsMsMsMsMsFsXFsMsMsdCsDqMsFsMqFsMsFsMsFsMsMsMsLsM-OH -3' (6) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMsLsMsLsM-OH -3' (7) 5'- HO-MsLsMsLsMsFsXFpMsMsdCsDqMpFsMqFsMsFsMsFsMsLsMsLsM-OH -3' (8) 5'- HO-MsLsMsLsMsFsXFsMpMsdCsDqMsFsMqFsMsFsMsFsMsLsMsLsM-OH -3' (9) 5'- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMsLpMsLsM-OH -3' (10) 5'- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMpFsMsFsMsLpMsLsM-OH -3' (11) 5'- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMsFsMpFsMsLpMsLsM-OH -3' (12) 5'- HO-MsLsMsLpMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMpLpMsLsM-OH -3' (13) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMsMsMsLsM-OH -3'(14) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMsFsMsMsMsLsM-OH -3' (15) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMpFsMsMsMsLsM-OH -3' (16) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMsFsMpMsMsLsM-OH -3' (17) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMpFsMsMsMsLsM-OH -3' (18) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMsFsMpMsMsLsM-OH -3' (19) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMsFsMpFsMpMsMsLsM-OH -3' (20) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMqFsMpFsMpFsMpMsMsLsM-OH -3' (21) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMsFsMsFsMsFsMsLsMsLsM-OH -3' (22) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDqMpFsMsFsMsFsMsFsMsMsMsLsM-OH -3' (In each formula, D represents a 2'-deoxynucleoside residue, F represents a 2'-deoxy-2'-fluororibonucleoside residue, L represents a bridged nucleoside residue, M represents a 2'-O-methylribonucleoside residue, and dC represents a 2'-deoxycytidine residue which is a target-corresponding nucleoside residue. X indicates that one nucleoside residue is missing from the target RNA. p represents a phosphate residue, s represents a thiophosphate residue, and q represents an alkylphosphonate residue, an alkyl phosphate residue, or a substituted phosphoramide residue. HO- represents a hydroxyl group at the 5' end, and -OH represents a hydroxyl group at the 3' end.) 25. An oligonucleotide according to any one of claims 1 to 7, comprising a modified oligonucleotide having a chemical modification pattern represented by any one of the following formulas (1a) to (22a), or a pharma- ceutically acceptable salt thereof: (1a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsLsMsLsM-OH -3' (2a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsMsMsLsM-OH -3' (3a) 5'- HO-MsLsMsMsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsLsMsLsM-OH -3' (4a) 5'- HO-MsLsMsMsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsMsMsLsM-OH -3' (5a) 5'- HO-LsMsMsMsMsFsXFsMsMsdCsDpmMsFsMpmFsMsFsMsFsMsMsMsLsM-OH -3' (6a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMsLsMsLsM-OH -3' (7a) 5'- HO-MsLsMsLsMsFsXFpMsMsdCsDrMpFsMrFsMsFsMsFsMsLsMsLsM-OH -3' (8a) 5'- HO-MsLsMsLsMsFsXFsMpMsdCsDrMsFsMrFsMsFsMsFsMsLsMsLsM-OH -3' (9a) 5'- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMsLpMsLsM-OH -3' (10a) 5'- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMpFsMsFsMsLpMsLsM-OH -3' (11a) 5'- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMsFsMpFsMsLpMsLsM-OH -3' (12a) 5'- HO-MsLsMsLpMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMpLpMsLsM-OH -3'(13a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMsMsMsLsM-OH -3' (14a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMsFsMsMsMsLsM-OH -3' (15a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMpFsMsMsMsLsM-OH -3' (16a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMsFsMpMsMsLsM-OH -3' (17a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMpFsMsMsMsLsM-OH -3' (18a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMsFsMpMsMsLsM-OH -3' (19a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMsFsMpFsMpMsMsLsM-OH -3' (20a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMrFsMpFsMpFsMpMsMsLsM-OH -3' (21a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMsFsMsFsMsFsMsLsMsLsM-OH -3' (22a) 5'- HO-MsLsMsLsMsFsXFsMsMsdCsDrMpFsMsFsMsFsMsFsMsMsMsLsM-OH -3' (In each formula, D represents a 2'-deoxynucleoside residue, F represents a 2'-deoxy-2'-fluororibonucleoside residue, L represents a bridged nucleoside residue, M represents a 2'-O-methylribonucleoside residue, dC represents a 2'-deoxycytidine residue which is a target-corresponding nucleoside residue. X indicates that one nucleoside residue for the target RNA is deleted. p represents a phosphate residue, s represents a thiophosphate residue, pm represents a methylphosphonic acid residue, and r represents an ethyl phosphate residue or a methoxyethyl phosphate residue. HO- represents a hydroxyl group at the 5' end, and -OH represents a hydroxyl group at the 3' end.)

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