Heteronucleic acid including 2'-modified nucleoside

Abstract

A problem addressed is to provide a double-stranded nucleic acid complex with reduced central nervous system toxicity.　Provided is a double-stranded nucleic acid complex including a first nucleic acid strand and a second nucleic acid strand, wherein the first nucleic acid strand can hybridize with at least a part of a target gene or a transcript thereof and has an antisense effect on the target gene or transcript thereof, and the second nucleic acid strand includes a base sequence complementary to that of the first nucleic acid strand and includes one or more 2'-modified nucleosides.

Description

Heterogeneous nucleic acids containing 2'-modified nucleosides

[0001] The present invention relates to a double-stranded nucleic acid complex containing a 2'-modified nucleoside, and a pharmaceutical composition containing the same as an active ingredient.

[0002] In recent years, oligonucleotides have attracted attention in the ongoing development of pharmaceuticals known as nucleic acid drugs, and in particular, the development of nucleic acid drugs using antisense methods is being actively pursued due to their high selectivity for target genes and low toxicity. The antisense method involves selectively modifying or inhibiting the expression of proteins encoded by target genes or the activity of miRNAs by introducing complementary oligonucleotides (antisense oligonucleotides; often referred to herein as "ASOs (Antisense Oligonucleotides)") into cells, using a partial sequence of mRNA or miRNA transcribed from a target gene as the target sense strand.

[0003] As a nucleic acid utilizing the antisense method, the present inventors have developed a double-stranded nucleic acid complex (heteroduplex oligonucleotide (HDO)) in which an antisense oligonucleotide is annealed to its complementary strand (Patent Document 1, Non-Patent Documents 1 and 2). The double-stranded nucleic acid complex has a high antisense effect and is a groundbreaking technology that enables control of the central nervous system across the blood-brain barrier.

[0004] Meanwhile, clinical development of nucleic acid drugs has progressed in recent years, and the results of preclinical trials have been accumulating. As a result, it has become clear that toxicity and adverse events are often problems with nucleic acid drugs such as ASOs. For example, toxicity and adverse events accounted for nearly half of the reasons for the discontinuation of nucleic acid drug development between 2013 and 2016 (Non-Patent Document 3). Therefore, technologies to avoid the toxicity of nucleic acid drugs are needed.

[0005] International Publication No. 2013 / 089283 International Publication No. 2014 / 192310

[0006] Nishina K, et. al., "DNA / RNA heteroduplex oligonucleotide for highly gene silencing", Nature Communication, 2015, 6:7969.Asami Y, et al., "Drug efficient delivery system of therapeutic oligonucleotides", Drug Discoveries & Therapeutics. 2016; 10(5):256-262.Harrison RK, "Phase II and phase III failures: 2013-2015", Nature Reviews Drug Discovery, 2016; 15:817-818.

[0007] The object is to provide a double-stranded nucleic acid complex with reduced central nervous system toxicity.

[0008] Mice administered single-stranded gapmer antisense nucleic acids intracerebroventricularly exhibit reduced activity and motor dysfunction due to central neurotoxicity within a few hours of administration. The present inventors conducted extensive research to find a new technology that can reduce the central neurotoxicity of nucleic acid drugs, and introduced 2'-modified nucleosides into double-stranded nucleic acid complexes. As a result, they found that the introduction of 2'-modified nucleosides can dramatically reduce or eliminate the central neurotoxicity of double-stranded nucleic acid complexes. This toxicity-suppressing effect is a surprising effect that far exceeds previous expectations. The present invention is based on the above findings and provides the following:

[0009] (1) A double-stranded nucleic acid complex comprising a first nucleic acid strand and a second nucleic acid strand, wherein the first nucleic acid strand is capable of hybridizing to at least a portion of a target gene or its transcription product and has an antisense effect on the target gene or its transcription product, and the second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand and one or more 2'-modified nucleosides. (2) The double-stranded nucleic acid complex according to (1), wherein the first nucleic acid strand is a gapmer. (3) The double-stranded nucleic acid complex according to (1), wherein all of the nucleosides in the first nucleic acid strand are modified nucleosides. (4) The double-stranded nucleic acid complex according to (3), wherein all of the nucleosides in the first nucleic acid strand are 2'-modified nucleosides. (5) The double-stranded nucleic acid complex according to (4), wherein all of the nucleosides in the first nucleic acid strand are 2'-O-methoxyethyl modified nucleosides. (6) The double-stranded nucleic acid complex according to any one of (1) to (5), wherein the second nucleic acid strand comprises one or two to ten consecutive 2'-modified nucleosides located at the 5'-end and / or one or two to ten consecutive 2'-modified nucleosides located at the 3'-end. (7) The double-stranded nucleic acid complex according to any one of (1) to (6), wherein the second nucleic acid strand comprises one to seven 2'-modified nucleosides at a position other than the 5'-end and the 3'-end. (8) The double-stranded nucleic acid complex according to (2), wherein the first nucleic acid strand comprises: (1) a central region comprising at least four consecutive deoxyribonucleosides; (2) a 5'-wing region located on the 5'-end of the central region and comprising an unnatural nucleoside; and (3) a 3'-wing region located on the 3'-end of the central region and comprising an unnatural nucleoside. (9) The double-stranded nucleic acid complex according to (8), wherein the second nucleic acid strand contains 2'-modified nucleosides in a region consisting of a base sequence complementary to a 5' wing region and / or a 3' wing region of the first nucleic acid strand. (10) The double-stranded nucleic acid complex according to (9), wherein all nucleosides in the region consisting of a base sequence complementary to a 5' wing region and / or a 3' wing region of the first nucleic acid strand in the second nucleic acid strand are 2'-modified nucleosides.(11) The double-stranded nucleic acid complex according to (10), wherein in a region of the second nucleic acid strand consisting of a base sequence complementary to a central region of the first nucleic acid strand, all nucleosides containing purine bases are ribonucleosides. (12) The double-stranded nucleic acid complex according to (11), wherein in a region of the second nucleic acid strand consisting of a base sequence complementary to a central region of the first nucleic acid strand, all nucleosides containing pyrimidine bases are deoxyribonucleosides. (13) The double-stranded nucleic acid complex according to (12), wherein in the second nucleic acid strand, the 2'-modified nucleosides are 2'-O-methoxyethyl-modified nucleosides or 2'-O-methyl-modified nucleosides. (14) The double-stranded nucleic acid complex according to any one of (8) to (10), wherein all nucleosides in the region of the second nucleic acid strand consisting of a base sequence complementary to the central region of the first nucleic acid strand are (a) deoxyribonucleosides, (b) deoxyribonucleosides and ribonucleosides, (c) deoxyribonucleosides and 2'-modified nucleosides, (d) ribonucleosides and 2'-modified nucleosides, or (e) deoxyribonucleosides, ribonucleosides, and 2'-modified nucleosides. (15) The double-stranded nucleic acid complex according to (14), wherein in the second nucleic acid strand, all nucleosides in a region consisting of base sequences complementary to the 5' wing region and the 3' wing region of the first nucleic acid strand are 2'-modified nucleosides, and all nucleosides in a region consisting of a base sequence complementary to the central region of the first nucleic acid strand are deoxyribonucleosides. (16) The double-stranded nucleic acid complex according to any of (8) to (10), wherein the second nucleic acid strand comprises at least four consecutive ribonucleosides complementary to at least four consecutive deoxyribonucleosides in the central region of the first nucleic acid strand. (17) The double-stranded nucleic acid complex according to any one of (1) to (16), wherein the nucleoside in the second nucleic acid strand that is complementary to (i) at least one guanosine nucleoside in the first nucleic acid strand, (ii) the nucleoside adjacent to the 5'-terminal side of the guanosine nucleoside, (iii) the nucleoside adjacent to the 3'-terminal side of the guanosine nucleoside, or (iv) any combination of (i) to (iii) is a 2'-modified nucleoside.(18) The double-stranded nucleic acid complex according to any one of (1) to (17), wherein nucleosides in the second nucleic acid strand that are complementary to (i) at least one guanosine nucleoside in the 5' wing region of the first nucleic acid strand, (ii) the nucleoside adjacent to the 5' terminal side of the guanosine nucleoside, (iii) the nucleoside adjacent to the 3' terminal side of the guanosine nucleoside, or (iv) any combination of (i) to (iii) are 2'-modified nucleosides. (19) The double-stranded nucleic acid complex according to any one of (1) to (18), wherein at least one nucleoside containing an adenine base or a pyrimidine base in the second nucleic acid strand is a 2'-modified nucleoside and / or a deoxyribonucleoside. (20) The double-stranded nucleic acid complex according to any one of (1) to (19), wherein at least one of the nucleosides containing a pyrimidine base in the second nucleic acid strand is a 2'-modified nucleoside and / or a deoxyribonucleoside. (21) The double-stranded nucleic acid complex according to any one of (1) to (20), wherein the second nucleic acid strand does not contain a natural ribonucleoside containing a pyrimidine base. (22) The double-stranded nucleic acid complex according to (21), wherein all of the nucleosides containing a pyrimidine base in the second nucleic acid strand are 2'-modified nucleosides and / or deoxyribonucleosides. (23) The double-stranded nucleic acid complex according to any one of (1) to (22), wherein 20% or more of the nucleosides in the second nucleic acid strand are 2'-modified nucleosides. (24) The double-stranded nucleic acid complex according to any one of (1) to (23), wherein in the second nucleic acid strand, all nucleosides other than 2'-modified nucleosides are deoxyribonucleosides. (25) The double-stranded nucleic acid complex according to any one of (1) to (24), wherein in the second nucleic acid strand, all nucleosides in a region consisting of a base sequence complementary to that of the first nucleic acid strand are 2'-modified nucleosides. (26) The double-stranded nucleic acid complex according to any one of (1) to (25), wherein in the second nucleic acid strand, the 2'-modified nucleosides are 2'-O-methoxyethyl-modified nucleosides and / or 2'-O-methyl-modified nucleosides.(27) The double-stranded nucleic acid complex according to any one of (1) to (26), wherein the second nucleic acid strand comprises at least one 2'-O-methyl modified nucleoside, scpBNA nucleoside, AmNA nucleoside, or BNA-NC. (28) The double-stranded nucleic acid complex according to (1), wherein the first nucleic acid strand is a mixmer. (29) The double-stranded nucleic acid complex according to any one of (1) to (28), wherein the second nucleic acid strand comprises at least one bulge structure consisting of a base sequence non-complementary to the first nucleic acid strand. (30) The double-stranded nucleic acid complex according to any one of (1) to (29), wherein the second nucleic acid strand comprises a non-complementary base and / or an insertion sequence and / or deletion of one or more bases relative to the first nucleic acid strand. (31) The double-stranded nucleic acid complex according to (30), wherein the second nucleic acid strand comprises 1 to 3 non-complementary bases. (32) The double-stranded nucleic acid complex according to (30) or (31), wherein the insertion sequence consists of 1 to 8 bases. (33) The double-stranded nucleic acid complex according to any one of (30) to (32), wherein the deletion consists of 1 to 4 consecutive bases. (34) The double-stranded nucleic acid complex according to any one of (30) to (33), wherein the non-complementary bases of the second nucleic acid strand form a bulge, or wherein the deletion position of the second nucleic acid strand contains a bulge consisting of a base sequence non-complementary to the first nucleic acid strand. (35) The double-stranded nucleic acid complex according to (29) or (34), wherein the bulge contains sugar-unmodified nucleosides, or wherein all nucleosides in the bulge are sugar-unmodified nucleosides. (36) The double-stranded nucleic acid complex according to (29), (34), or (35), wherein the bulge is 1 to 10 bases long. (37) The double-stranded nucleic acid complex according to any one of (29) and (34) to (36), wherein all nucleosides in the second nucleic acid strand other than those in the bulge structure are 2'-modified nucleosides. (38) The double-stranded nucleic acid complex according to any one of (1) to (37), wherein the second nucleic acid strand is at least 8 bases long. (39) The double-stranded nucleic acid complex according to any one of (1) to (38), wherein the base length of the second nucleic acid strand is shorter than the base length of the first nucleic acid strand. (40) The double-stranded nucleic acid complex according to any one of (1) to (39), wherein the second nucleic acid strand comprises at least one overhang region located on the 5'-end and / or 3'-end of a region consisting of a base sequence complementary to the first nucleic acid strand.(41) The double-stranded nucleic acid complex according to (40), wherein the overhang region is 1 to 20 bases long. (42) The double-stranded nucleic acid complex according to (40) or (41), wherein the overhang region comprises at least one deoxyribonucleoside and / or unnatural nucleoside. (43) The double-stranded nucleic acid complex according to any one of (1) to (42), wherein the first nucleic acid strand and the second nucleic acid strand are linked via a linker. (44) The double-stranded nucleic acid complex according to (43), wherein the linker links the 5'-end of the first nucleic acid strand to the 3'-end of the second nucleic acid strand and / or the 3'-end of the first nucleic acid strand to the 5'-end of the second nucleic acid strand. (45) The double-stranded nucleic acid complex according to (43) or (44), wherein the linker is a cleavable or uncleavable linker. (46) The double-stranded nucleic acid complex according to any one of (43) to (45), wherein the linker is composed of a nucleic acid containing a natural nucleoside and / or a non-natural nucleoside, or polyethylene glycol. (47) The double-stranded nucleic acid complex according to (46), wherein the linker composed of a nucleic acid is 2 to 10 bases long. (48) The double-stranded nucleic acid complex according to any one of (1) to (47), wherein the second nucleic acid strand is bound to a ligand. (49) The double-stranded nucleic acid complex according to (48), wherein the ligand is at least one selected from the group consisting of a small molecule, a peptide, a lipid, and a nucleic acid aptamer. (50) The double-stranded nucleic acid complex according to (49), wherein the lipid is cholesterol or an analog thereof, or tocopherol or an analog thereof. (51) The double-stranded nucleic acid complex according to any one of (48) to (50), wherein the ligand is bound to the 5' end and / or the 3' end of the second nucleic acid strand. (52) The double-stranded nucleic acid complex according to any one of (1) to (47), which is not bound to a ligand. (53) The double-stranded nucleic acid complex according to any one of (1) to (52), in which all or part of the internucleoside linkages of the first nucleic acid strand and / or the second nucleic acid strand are modified internucleoside linkages. (54) The double-stranded nucleic acid complex according to (53), in which the modified internucleoside linkages are phosphorothioate linkages.

[0010] (55) A pharmaceutical composition comprising the double-stranded nucleic acid complex according to any one of (1) to (54) as an active ingredient. (56) The pharmaceutical composition according to (55) for treating a central nervous system disease in a subject. (57) The pharmaceutical composition according to (55) or (56), which is administered intracerebroventricularly or intrathecally. (58) The pharmaceutical composition according to (57), wherein the intrathecal administration is posterior fossa puncture or lumbar puncture. (59) The pharmaceutical composition according to (57) or (58), wherein the double-stranded nucleic acid complex is administered in an amount of 0.1 mg to 200 mg. (60) The pharmaceutical composition according to (55) or (56), which is administered intravenously or subcutaneously. (61) The pharmaceutical composition according to (60), wherein the double-stranded nucleic acid complex is administered in an amount of 0.1 mg / kg to 100 mg / kg. (62) The pharmaceutical composition according to any one of (55) to (61), wherein central nervous system toxicity is reduced. This specification includes the disclosure of Japanese Patent Application No. 2021-150310, from which this application claims priority.

[0011] According to the present invention, a double-stranded nucleic acid complex with reduced central neurotoxicity is provided.

[0012] Figure 1 shows the structures of various cross-linked nucleic acids. Figure 2 shows the structures of various natural and non-natural nucleotides. Figure 3 shows a scoring system for evaluating central neurotoxicity in mice after administration of nucleic acid agents. Figure 4 shows the structures of the nucleic acids used in Example 1. Figure 4A shows the structure of an ASO targeting the Mapt gene. Figure 4B shows the structure of HDO (all RNA). Figure 4C shows the structure of HDO (all DNA). Figure 4D shows the structure of HDO (6 MOE wing). Figure 4E shows the structure of HDO (all MOE). Figure 5 shows the acute tolerability scores 30 minutes to 4 hours after intracerebroventricular administration of various nucleic acid agents in mice. Error bars indicate standard error. Figure 6 shows the motor function 1 hour after intracerebroventricular administration of various nucleic acid agents in mice. Figure 6A shows the total distance traveled over 5 minutes. Figure 6B shows the maximum movement speed. Error bars indicate standard error. Figure 7 shows Mapt mRNA expression levels in the hippocampus of mice administered various nucleic acid agents intracerebroventricularly. Error bars indicate standard error. Figure 8 shows the structure of the nucleic acid used in Example 2. Figure 8A shows the structure of an ASO targeting the BACE1 gene. Figure 8B shows the structure of HDO (all RNA). Figure 8C shows the structure of HDO (all DNA). Figure 8D shows the structure of HDO (5MOE wing). Figure 9 shows the acute tolerability scores 30 minutes to 4 hours after administration of mice administered various nucleic acid agents intracerebroventricularly. Error bars indicate standard error. Figure 10 shows the motor function 1 hour after administration of mice administered various nucleic acid agents intracerebroventricularly. Figure 10A shows the total distance traveled over 5 minutes. Figure 10B shows the maximum movement speed. Error bars indicate standard error. Figure 11 shows the structure of the nucleic acid used in Example 3. Figure 11A shows the structure of an ASO targeting the Malat1 gene. Figure 11B shows the structure of HDO (all RNA). Figure 11C shows the structure of HDO (all DNA). Figure 11D shows the structure of HDO (10 MOE wing). Figure 12 shows the acute tolerability scores of mice intracerebroventricularly administered various nucleic acid agents 30 minutes to 4 hours after administration. Error bars indicate standard error.Figure 13 shows the motor function of mice intracerebroventricularly administered with various nucleic acid agents 1 hour after administration. Figure 13A shows the total distance traveled over 5 minutes. Figure 13B shows the maximum movement speed. Error bars indicate standard error. Figure 14 shows the structures of the nucleic acids used in Example 4. Figure 14A shows the structure of HDO (all DNA) containing an ASO targeting the Mapt gene. Figure 14B shows the structure of HDO (RNA 6MOE wing). Figure 14C shows the structure of HDO (6MOE wing). Figure 14D shows the structure of HDO (6OMe wing). Figure 14E shows the structure of HDO (6F wing). Figure 15 shows the acute tolerability scores 30 minutes to 4 hours after administration of mice intracerebroventricularly administered with various nucleic acid agents. Error bars indicate standard error. Figure 16 shows the motor function of mice intracerebroventricularly administered with various nucleic acid agents 1 hour after administration. Figure 16A shows the total distance traveled over 5 minutes. Figure 16B shows the maximum movement speed. Error bars indicate standard error. Figure 17 shows the motor function of mice 3 hours after intracerebroventricular administration of various nucleic acid agents. Figure 17A shows the total distance traveled over 5 minutes. Figure 17B shows the maximum movement speed. Error bars indicate standard error. Figure 18 shows the structure of the nucleic acid used in Example 5. Figure 18A shows the structure of ASO targeting the Mapt gene. Figure 18B shows the structure of HDO (6MOE wing). Figure 18C shows the structure of HDO (G. MOE ) structure. Figure 18D shows the structure of HDO(G RNA) structure. Figure 18E shows the structure of HDO(inosine). Figure 19 shows the acute tolerability scores 30 minutes to 4 hours after intracerebroventricular administration of various nucleic acid agents to mice. Error bars indicate standard error. Figure 20 shows the structures of the nucleic acids used in Example 6. Figure 20A shows the structure of HDO(all DNA) containing an ASO targeting the Mapt gene. Figure 20B shows the structure of HDO(6MOE-5'&3'). Figure 20C shows the structure of HDO(6MOE-5'). Figure 20D shows the structure of HDO(6MOE-3'). Figure 20E shows the structure of HDO(10MOE-5'). Figure 20F shows the structure of HDO(10MOE-3'). Figure 21 shows the acute tolerability scores for mice administered various nucleic acid agents intracerebroventricularly from 30 minutes to 4 hours after administration. Error bars indicate standard error. Figure 22 shows the motor function of mice administered various nucleic acid agents intracerebroventricularly 1 hour after administration. Figure 22A shows the total distance traveled over 5 minutes. Figure 22B shows the maximum movement speed. Error bars indicate standard error. Figure 23 shows the motor function of mice administered various nucleic acid agents intracerebroventricularly 3 hours after administration. Figure 23A shows the total distance traveled over 5 minutes. Figure 23B shows the maximum movement speed. Error bars indicate standard error. Figure 24 shows the Mapt mRNA expression level in the hippocampus of mice administered various nucleic acid agents intracerebroventricularly. Error bars indicate standard error. Figure 25 shows the structure of the nucleic acid used in Example 7. Figure 25A shows the structure of HDO (all DNA) containing an ASO targeting the BACE1 gene. Figure 25B shows the structure of HDO(5MOE-5'). Figure 25C shows the structure of HDO(5MOE-3'). Figure 25D shows the structure of HDO(8MOE-5'). Figure 25E shows the structure of HDO(8MOE-3'). Figure 25F shows the structure of HDO(10MOE-5'). Figure 25G shows the structure of HDO(10MOE-3'). Figure 26 shows the acute tolerability scores 30 minutes to 4 hours after intracerebroventricular administration of various nucleic acid agents in mice. Error bars indicate standard error. Figure 27 shows the motor function 1 hour after intracerebroventricular administration of various nucleic acid agents in mice.Figure 27A shows the total distance traveled over 5 minutes. Figure 27B shows the maximum migration speed. Error bars indicate standard error. Figure 28 shows the structures of the nucleic acids used in Example 8. Figure 28A shows the structure of HDO (all DNA) containing an ASO targeting the Mapt gene. Figure 28B shows the structure of HDO (6 MOE wing). Figure 28C shows the structure of HDO (9 MOE wing). Figure 28D shows the structure of HDO (11 MOE wing). Figure 28E shows the structure of HDO (13 MOE wing). Figure 28F shows the structure of HDO (15 MOE wing). Figure 29 shows the acute tolerability scores 30 minutes to 4 hours after intracerebroventricular administration of various nucleic acid agents in mice. Error bars indicate standard error. Figure 30 shows the motor function 1 hour after intracerebroventricular administration of various nucleic acid agents in mice. Figure 27A shows the total distance traveled over 5 minutes. Figure 27B shows the maximum migration velocity. Error bars indicate standard error. Figure 31 shows the structure of the nucleic acid used in Example 9. Figure 31A shows the structure of HDO (all DNA) containing ASO targeting the Mapt gene. Figure 31B shows the structure of HDO (A. MOE ) structure. Figure 31C shows the structure of HDO(G MOE ) structure. Figure 31D shows the structure of HDO(C MOE ) structure. Figure 31E shows the structure of HDO(T MOE ) structure. Figure 32 shows the acute tolerability scores 30 minutes to 4 hours after administration of various nucleic acid agents in mice administered intracerebroventricularly. Error bars indicate standard error. Figure 33 shows the motor function 1 hour after administration of various nucleic acid agents in mice administered intracerebroventricularly. Figure 33A shows the total distance traveled over 5 minutes. Figure 33B shows the maximum movement speed. Error bars indicate standard error. Figure 34 shows the structure of the nucleic acid used in Example 10. Figure 34A shows the structure of HDO (all DNA) containing ASO targeting the Mapt gene. Figure 34B shows the structure of HDO (C MOE ) structure. Figure 34C shows the structure of HDO(2C MOE Figure 34D shows the structure of HDO(2C MOE Figure 34E shows the structure of HDO(3C-3). MOE) structure. Figure 35 shows the acute tolerability scores 30 minutes to 4 hours after intracerebroventricular administration of various nucleic acid agents in mice. Error bars indicate standard error. Figure 36 shows the motor function 1 hour after intracerebroventricular administration of various nucleic acid agents in mice. Figure 36A shows the total distance traveled over 5 minutes. Figure 36B shows the maximum movement speed. Error bars indicate standard error. Figure 37 shows the stability of various nucleic acid agents in human cerebrospinal fluid. Figures 37A and 37B show the structures of the nucleic acids used in Example 11. Figure 37A shows the structure of an ASO targeting the Mapt gene. Figure 37B shows the structure of HDO (ASO / cRNA). Figure 37C shows the results of electrophoresis to examine the stability of various nucleic acid agents after mixing them with human cerebrospinal fluid (hCSF) for 10 minutes or 6 hours. Figure 37D shows the results of quantifying the band intensity of the HDO double strand in HDO(ASO / cRNA). Figure 38 shows the stability of various nucleic acid agents in human and rat cerebrospinal fluid. Figures 38A and 38B show the structures of the nucleic acids used in Example 11. Figure 38A shows the structure of HDO(ASO / cRNA) containing an ASO targeting the Mapt gene. Figure 38B shows the structure of HDO(ASO / cDNA). Figure 38C shows the results of electrophoresis of stability of various nucleic acid agents mixed with human or rat cerebrospinal fluid for 6 hours. Figure 38D shows the stability of the second nucleic acid strand (cRNA and cDNA) in HDO(ASO / cRNA) and HDO(ASO / cDNA) in human and rat cerebrospinal fluid. Figure 39 shows the stability of various nucleic acid agents in mouse, rat, monkey, and human cerebrospinal fluid. Figures 39A and 39B show the structures of the nucleic acids used in Example 12. Figure 39A shows the structure of HDO (all RNA) containing an ASO targeting the Mapt gene. Figure 39B shows the structure of HDO (all DNA). Figure 39C shows the results of electrophoresis of stability of various nucleic acid agents after mixing with cerebrospinal fluid from mice, rats, monkeys, and humans for 6 hours. Figure 40 shows the stability of various nucleic acid agents in cerebrospinal fluid from mice, rats, monkeys, and humans. Figures 40A and 40B show the structures of the nucleic acids used in Example 12. Figure 40A shows the structure of HDO (cRNA 6MOE wing) containing an ASO targeting the Mapt gene.Figure 40B shows the structure of HDO (cDNA 6MOE wing). Figure 40C shows the results of electrophoresis to examine the stability of various nucleic acid agents after mixing them with mouse, rat, monkey, and human cerebrospinal fluid for 6 hours. Figure 41 shows the structure of the nucleic acid used in Example 13. Figure 41A shows the structure of HDO (all DNA) containing ASO targeting the Mapt gene. Figure 41B shows the structure of HDO (A. RNA ) structure. Figure 41C shows the structure of HDO(G RNA ) structure. Figure 41D shows the structure of HDO(C RNA ) structure. Figure 41E shows the structure of HDO(U RNA ) structure. Figure 42 shows the results of examining the stability of various nucleic acid agents by electrophoresis after mixing them with human cerebrospinal fluid for 6 hours. Figure 43 shows the structure of the nucleic acid used in Example 14. Figure 43A shows the structure of HDO (all DNA) containing ASO targeting the Mapt gene. Figure 43B shows the structure of HDO (GA RNA ) structure. Figure 43C shows the structure of HDO(CU RNA ) structure. Figure 43D shows the structure of HDO(C RNA ) structure. Figure 43E shows the structure of HDO(U RNA ) structure. Figure 44 shows the results of examining the stability of various nucleic acid agents by electrophoresis after mixing them with human cerebrospinal fluid for 1 hour or 6 hours. Figure 45 shows the structure of the nucleic acid used in Example 15. Figure 45A shows the structure of HDO (all DNA) containing ASO targeting the Malat1 gene. Figure 45B shows the structure of HDO (A RNA ) structure. Figure 45C shows the structure of HDO(G RNA ) structure. Figure 45D shows the structure of HDO(C RNA ) structure. Figure 45E shows the structure of HDO(U RNA) structure. Figure 46 shows the results of electrophoresis to examine the stability of various nucleic acid agents after mixing them with human cerebrospinal fluid for 6 hours. Figure 47 shows the results of evaluating the central nervous system toxicity of nucleic acid agents in monkeys. Figures 47A-C show the structures of the nucleic acids used in Example 16. Figure 47A shows the structure of an ASO targeting the Mapt gene. Figure 47B shows the structure of HDO(RNA-MOE). Figure 47C shows the structure of HDO(DNA-MOE). Figure 47D shows the procedure for evaluating central nervous system toxicity in monkeys in Example 16. Figure 47E shows the results of evaluating the central nervous system toxicity of various nucleic acid agents in monkeys. Figure 48 shows the structures of the nucleic acids used in Example 17. Figure 48A shows the structure of HDO(all DNA). Figure 48B shows the structure of HDO(all MOE). Figure 48C shows the structure of HDO(bulge 1). Figure 48D shows the structure of HDO(bulge 2). Figure 49 shows Mapt mRNA expression levels and LDH activity in human neuroblastoma-derived cells transfected with various nucleic acid agents. Figure 49A shows the relative Mapt mRNA levels. Figure 49B shows the relative LDH release levels in the supernatant. Error bars indicate standard error. Figure 50 shows the structures of the nucleic acids used in Example 18. Figure 50A shows the structure of HDO (bulge). Figure 50B shows the structure of HDO (ssHDO). Figure 50C shows the structure of PEG linker ssHDO. Figure 50D shows the structure of bulge plus ssHDO. Figure 51 shows the results of evaluating motor function 1 hour after intracerebroventricular administration of various nucleic acid agents in mice. Figure 51A shows the total distance traveled over 5 minutes. Figure 51B shows the maximum movement speed. Error bars indicate standard error. Figure 52 shows the expression levels of Malat1 RNA in the brains of mice intracerebroventricularly administered with various nucleic acid agents. Figure 52A shows the results for the left frontal cortex. Figure 52B shows the results for the right frontal cortex. Error bars indicate standard error. Figure 53 shows the results of evaluating the efficiency of double-strand dissociation by electrophoresis after incubating various nucleic acid agents in brain tissue homogenate for 7 days. Figure 54 shows the acute tolerability scores for mice intracerebroventricularly administered with various nucleic acid agents 30 minutes to 4 hours after administration. Error bars indicate standard error.Figure 55 shows the results of assessing motor function in mice intracerebroventricularly administered with various nucleic acid agents one hour after administration. Figure 55A shows the total distance traveled over 5 minutes. Figure 55B shows the maximum movement speed. Error bars indicate standard error. Figure 56 shows the acute tolerability scores 30 minutes to 4 hours after administration in mice intracerebroventricularly administered with various nucleic acid agents. Error bars indicate standard error. Figure 57 shows the results of assessing motor function in mice intracerebroventricularly administered with various nucleic acid agents one hour after administration. Figure 57A shows the total distance traveled over 5 minutes. Figure 57B shows the maximum movement speed. Error bars indicate standard error. Figure 58 shows the results of measuring the body weight of mice intracerebroventricularly administered with various nucleic acid agents. Error bars indicate standard error. Error bars indicate standard error. Figure 59 shows the results of assessing motor function in mice intracerebroventricularly administered with various nucleic acid agents one day or more after administration. Figure 60 shows the expression level of Mapt mRNA in the right frontal lobe of mice intracerebroventricularly administered with various nucleic acid agents. Error bars indicate standard error. Figure 61 shows the results of evaluating the double-strand dissociation efficiency by electrophoresis after incubating various nucleic acid agents in brain tissue homogenate for 7 days. Figure 62 shows the acute tolerability scores 30 minutes to 4 hours after administration in mice administered various nucleic acid agents intracerebroventricularly. Error bars indicate standard error. Figure 63 shows the results of evaluating motor function in mice administered various nucleic acid agents intracerebroventricularly 1 hour after administration. Figure 63A shows the total distance traveled over 5 minutes. Figure 63B shows the maximum migration speed. Error bars indicate standard error. Figure 64 shows LDH activity and Bace1 mRNA expression levels in mouse neuroblastoma-derived cells (Neuro2a cell line) transfected with various nucleic acid agents. Figure 64A shows the relative LDH release level in the supernatant. Figure 64B shows the relative Bace1 mRNA level. Error bars indicate standard error. Figure 65 shows the method for evaluating the modified FOB score. Figure 66 shows the results of evaluating the modified FOB score in monkeys administered various nucleic acid agents intrathecally. Figure 67 shows the results of measuring the spontaneous movement time and number of jumps in monkeys administered various nucleic acid agents intrathecally using a 3-minute video.

[0013] 1. Double-stranded nucleic acid complex 1-1. Overview A first aspect of the present invention is a double-stranded nucleic acid complex. The double-stranded nucleic acid complex of the present invention comprises a first nucleic acid strand and a second nucleic acid strand, and contains one or more 2'-modified nucleosides. The double-stranded nucleic acid complex of the present invention is stable in the cerebrospinal fluid of primates, including humans, and has reduced toxicity, such as central nervous system toxicity.

[0014] 1-2. Definition of Terms As used herein, the term "transcription product" of a target gene refers to any RNA that is a direct target of the nucleic acid complex of the present invention and is synthesized by RNA polymerase. Specifically, it may include mRNA transcribed from the target gene (including mature mRNA, pre-mRNA, and mRNA without base modifications), non-coding RNA (ncRNA) such as miRNA, long non-coding RNA (lncRNA), and natural antisense RNA.

[0015] As used herein, the term "target gene" refers to a gene whose transcription or translation product expression level can be suppressed or enhanced, whose transcription or translation product function can be inhibited, or whose steric blocking, splicing switch, RNA editing, exon skipping, or exon inclusion can be induced by the antisense effect of the double-stranded nucleic acid complex of the present invention. The type of target gene is not particularly limited as long as it is expressed in vivo, but examples include genes derived from an organism into which the double-stranded nucleic acid complex of the present invention is introduced, such as genes whose expression is increased in various diseases. Examples of such genes include the scavenger receptor B1 (often referred to herein as "SR-B1") gene, the metastasis associated lung adenocarcinoma transcript 1 (often referred to herein as "Malat1") gene, the microtubule-associated protein tau (often referred to herein as "Mapt") gene, the beta-secretase 1 (often referred to herein as "BACE1") gene, the DMPK (dystrophia myotonica-protein kinase) gene, and the dystrophin gene.

[0016] As used herein, the term "target transcript" refers to any RNA that is a direct target of the nucleic acid complex of the present invention and is synthesized by RNA polymerase. This generally refers to a "transcription product of a target gene." Specifically, this term may include mRNA (including mature mRNA, pre-mRNA, and mRNA without base modifications) transcribed from the target gene, non-coding RNA (ncRNA) such as miRNA, long non-coding RNA (lncRNA), and natural antisense RNA. Examples of transcription products of target genes include SR-B1 mRNA, a transcription product of the SR-B1 gene; Mapt mRNA, a transcription product of the Mapt gene; BACE1 mRNA, a transcription product of the BACE1 gene; Malat1 non-coding RNA, a transcription product of the Malat1 gene; DMPK mRNA, a transcription product of the DMPK gene; and dystrophin mRNA, a transcription product of the dystrophin gene, or its precursor (pre-mRNA).

[0017] Specific examples of target transcripts include the exon 23 / intron 23 boundary region of dystrophin pre-mRNA (GenBank accession number: NC_000086.7), for example, positions 83803482 to 83803566, such as positions 83803512 to 83803536. As other specific examples of target transcripts, the nucleotide sequence of mouse DMPK mRNA is shown in SEQ ID NO: 7, and the nucleotide sequence of human DMPK mRNA is shown in SEQ ID NO: 8. Note that in both SEQ ID NOs: 7 and 8, the nucleotide sequence of mRNA has been replaced with the nucleotide sequence of DNA. The nucleotide sequence information for these genes and transcripts can be obtained from known databases, such as the NCBI (National Center for Biotechnology Information) database.

[0018] As used herein, the terms "antisense oligonucleotide (ASO)" and "antisense nucleic acid" refer to a single-stranded oligonucleotide that contains a base sequence capable of hybridizing to (i.e., complementary to) at least a portion of a target transcript (primarily a transcript of a target gene) and exerts an antisense effect on the target transcript. In the double-stranded nucleic acid complex of the present invention, the first nucleic acid strand functions as the ASO, and its target region may include the 3' UTR, 5' UTR, exon, intron, coding region, translation initiation region, translation termination region, or any other nucleic acid region. The target region of the target transcript may be at least 8 bases long, e.g., 10-35 bases, 12-25 bases, 13-20 bases, 14-19 bases, or 15-18 bases, or 13-22 bases, 16-22 bases, or 16-20 bases long.

[0019] The term "antisense effect" refers to the effect of ASOs hybridizing to a target transcript (e.g., the RNA sense strand) to modulate the expression or editing of that target transcript. "Modulating the expression or editing of a target transcript" refers to suppression or reduction of the expression of a target gene or the expression level of the target transcript (herein, "expression level of a target transcript" is often referred to as "target transcript level"), translation inhibition, RNA editing, splicing function modification effects (e.g., splicing switch, exon inclusion, exon skipping, etc.), or transcript degradation. For example, in post-transcriptional inhibition of a target gene, when an RNA oligonucleotide is introduced into a cell as an ASO, the ASO forms a partial duplex with the mRNA, the transcript of the target gene. This partial duplex acts as a cover to prevent translation by ribosomes, thereby inhibiting the expression of the target protein encoded by the target gene at the translational level (steric blocking). On the other hand, when an oligonucleotide containing DNA is introduced into a cell as an ASO, a partial DNA-RNA heteroduplex is formed. This heteroduplex structure is recognized by RNase H, resulting in degradation of the target gene's mRNA and inhibition of the expression of the protein encoded by the target gene. Furthermore, the antisense effect can also be achieved by targeting an intron in a pre-mRNA. Furthermore, the antisense effect can also be achieved by targeting an miRNA. In this case, inhibition of the function of the miRNA can increase the expression of the gene whose expression the miRNA normally controls. In one embodiment, the modulation of the expression of the target transcript can be a reduction in the amount of the target transcript.

[0020] The antisense effect can be measured, for example, by administering a test nucleic acid compound to a subject (e.g., a mouse) and measuring, for example, several days later (e.g., 2 to 7 days later), the expression level of a target gene or the level (quantity) of a target transcript (e.g., mRNA amount or RNA amount such as microRNA, cDNA amount, protein amount, etc.) whose expression is regulated by the antisense effect provided by the test nucleic acid compound.

[0021] For example, a decrease in the measured expression level of the target gene or level of the target transcript by at least 10%, at least 20%, at least 25%, at least 30%, or at least 40% compared to a negative control (e.g., vehicle administration) indicates that the test nucleic acid compound can produce an antisense effect (e.g., a reduction in the amount of the target transcript).

[0022] The number, type, and position of non-natural nucleotides in a nucleic acid chain can affect the antisense effect provided by the nucleic acid complex. The choice of modification can vary depending on the sequence of the target gene, etc., but those skilled in the art can determine a suitable embodiment by referring to the descriptions in literature related to antisense methods (e.g., WO 2007 / 143315, WO 2008 / 043753, and WO 2008 / 049085). Furthermore, when the antisense effect of a modified nucleic acid complex is measured, if the measured value thus obtained is not significantly lower than that of the nucleic acid complex before modification (e.g., if the measured value obtained after modification is 70% or more, 80% or more, or 90% or more of that of the nucleic acid complex before modification), the relevant modification can be evaluated.

[0023] As used herein, the term "translation product of a target gene" refers to any polypeptide or protein synthesized by translation of the target transcript or transcript of a target gene that is the direct target of the nucleic acid complex of the present invention.

[0024] As used herein, the term "aptamer" refers to a nucleic acid molecule that specifically binds to a specific target molecule inside a cell, on a cell membrane, or outside a cell, for example, on a cell membrane or outside a cell. Aptamers can be prepared by methods known in the art, for example, in vitro selection using the SELEX (systematic evolution of ligands by exponential enrichment) method.

[0025] As used herein, the term "decoy" refers to a nucleic acid having a sequence of the binding site of a transcription factor (e.g., NF-kB) or a sequence similar thereto, which is introduced into cells as a "decoy" to suppress the action of the transcription factor (if it is a transcription activator, it suppresses transcription, and if it is a transcription repressor, it promotes transcription). Decoy nucleic acids can be easily designed based on information about the binding sequence of the target transcription factor.

[0026] As used herein, "bait" refers to a nucleic acid molecule that specifically binds to a specific target molecule within a cell and modifies the function of the target molecule. A target that interacts with a bait is also called a "prey."

[0027] As used herein, the term "nucleic acid" or "nucleic acid molecule" may refer to a monomeric nucleotide or nucleoside, or may refer to an oligonucleotide consisting of multiple monomers, or, if it is a polymer, to a polynucleotide. "Natural nucleic acid" refers to a nucleic acid that exists in nature. Natural nucleic acids include the natural nucleosides and natural nucleotides described below. "Non-natural nucleic acid" or "artificial nucleic acid" refers to any nucleic acid other than natural nucleic acids. Non-natural nucleic acids or artificial nucleic acids include the non-natural nucleosides and non-natural nucleotides described below.

[0028] As used herein, a "nucleic acid strand" or simply a "strand" refers to two or more nucleosides linked by an internucleoside bond, and may be, for example, an oligonucleotide or a polynucleotide. A nucleic acid strand can be produced in full length or in partial lengths by chemical synthesis, for example, using an automated synthesizer, or by enzymatic processes using polymerases, ligases, or restriction reactions. A nucleic acid strand can include natural and / or non-natural nucleotides.

[0029] "Nucleoside" generally refers to a molecule consisting of a combination of a base and a sugar. The sugar portion of a nucleoside is typically, but not limited to, a pentofuranosyl sugar, specific examples of which include ribose and deoxyribose. The base portion (nucleobase) of a nucleoside is typically a heterocyclic base moiety. Examples include, but are not limited to, adenine, cytosine, guanine, thymine, or uracil, as well as other modified nucleobases (modified bases).

[0030] A "nucleotide" refers to a molecule in which a phosphate group is covalently linked to the sugar portion of a nucleoside. In the case of nucleotides containing a pentofuranosyl sugar, the phosphate group is typically linked to the 2', 3', or 5' hydroxyl group of the sugar.

[0031] An "oligonucleotide" refers to a linear oligomer formed by covalently linking several to several dozen hydroxyl groups in the sugar moieties and phosphate groups of adjacent nucleotides. A "polynucleotide" refers to a linear polymer formed by linking several dozens, preferably several hundred, of nucleotides, more numerous than an oligonucleotide, by such covalent bonds. Within an oligonucleotide or polynucleotide structure, the phosphate groups are generally considered to form internucleoside bonds.

[0032] As used herein, "natural nucleosides" refer to nucleosides that exist in nature. Examples include ribonucleosides consisting of ribose and a base such as adenine, cytosine, guanine, or uracil, and deoxyribonucleosides consisting of deoxyribose and a base such as adenine, cytosine, guanine, or thymine. Ribonucleosides found in RNA and deoxyribonucleosides found in DNA are often referred to herein as "DNA nucleosides" and "RNA nucleosides," respectively.

[0033] As used herein, the term "natural nucleotide" refers to a naturally occurring nucleotide molecule in which a phosphate group is covalently bound to the sugar moiety of the natural nucleoside. Examples include ribonucleotides, which are known as building blocks of RNA and in which a phosphate group is bound to a ribonucleoside, and deoxyribonucleotides, which are known as building blocks of DNA and in which a phosphate group is bound to a deoxyribonucleoside.

[0034] As used herein, "non-natural nucleotide" refers to any nucleotide other than a natural nucleotide, including modified nucleotides and nucleotide mimetics. As used herein, "modified nucleotide" refers to a nucleotide having one or more of a modified sugar moiety, a modified internucleoside linkage, and a modified nucleobase. As used herein, "nucleotide mimetics" include structures used to replace nucleosides and linkages at one or more positions in an oligomeric compound. Examples of nucleotide mimetics include peptide nucleic acids and morpholino nucleic acids (morpholinos linked by -N(H)-C(=O)-O- or other non-phosphodiester linkages). Peptide nucleic acids (PNAs) are nucleotide mimetics with a backbone containing N-(2-aminoethyl)glycine linked by amide bonds in place of sugars. Nucleic acid chains containing non-natural oligonucleotides often possess desirable properties, such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity. Therefore, they are preferred over natural nucleotides.

[0035] As used herein, the term "unnatural nucleoside" refers to any nucleoside other than a naturally occurring nucleoside. For example, this includes modified nucleosides and nucleoside mimetics. As used herein, the term "modified nucleoside" refers to a nucleoside having a modified sugar moiety and / or a modified nucleobase.

[0036] As used herein, the term "mimetic" refers to functional groups that replace sugars, nucleobases, and / or internucleoside linkages. Generally, mimetics are used in place of sugars or sugar-internucleoside linkage combinations, while maintaining the nucleobases for hybridization to a selected target. As used herein, "nucleoside mimetics" includes structures used to replace sugars, or sugars and bases, at one or more positions in an oligomeric compound, or to replace linkages between monomeric subunits that comprise the oligomeric compound. An "oligomeric compound" refers to a polymer of linked monomeric subunits that is at least capable of hybridizing to a region of a nucleic acid molecule. Nucleoside mimetics include, for example, morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclic, or tricyclic sugar mimetics, e.g., nucleoside mimetics having non-furanose sugar units.

[0037] "Modified sugar" refers to a sugar having a substitution and / or any change from a natural sugar moiety (i.e., a sugar moiety found in DNA (2'-H) or RNA (2'-OH)), and "sugar modification" refers to a substitution and / or any change from a natural sugar moiety. A nucleic acid strand may optionally include one or more modified nucleosides, including modified sugars. "Sugar-modified nucleoside" refers to a nucleoside having a modified sugar moiety. Such sugar-modified nucleosides may confer enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to a nucleic acid strand. In certain embodiments, the nucleoside includes a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (including 5' and 2' substituents), bridging of non-geminal ring atoms to form bicyclic nucleic acids (bridged nucleic acids, BNAs), and the modification of ribosyl ring oxygen atoms to S, N(R), or C(R1)(R2) (where R, R1, and R2 are each independently H, C1-C 12 alkyl, or protecting groups), and combinations thereof.

[0038] Examples of sugar-modified nucleosides include, but are not limited to, nucleosides containing 5'-vinyl, 5'-methyl (R or S), 5'-allyl (R or S), 4'-S, 2'-F (2'-fluoro), 2'-OCH (2'-O-Me or 2'-O-methyl), 2'-O-[2-(N-methylcarbamoyl)ethyl] (2'-O-MCE), and 2'-O-methoxyethyl (2'-O-MOE or 2-O(CH)OCH) substituents. The 2'-position substituent may also be an allyl, amino, azido, thio, -O-allyl, -O-C-C 10 alkyl, —OCF, —O(CH)SCH, —O(CH)—ON(R)(R), and O—CH—C(═O)—N(R)(R), where each R and R is independently H or a substituted or unsubstituted C—C 10 The term "2'-modified sugar" refers to a furanosyl sugar modified at the 2'-position. A nucleoside containing a 2'-modified sugar is also referred to as a "2'-modified nucleoside" or a "2'-sugar-modified nucleoside."

[0039] "Bicyclic nucleoside" refers to a modified nucleoside containing a bicyclic sugar moiety. Nucleic acids containing a bicyclic sugar moiety are commonly referred to as bridged nucleic acids (BNAs). Nucleosides containing a bicyclic sugar moiety are also sometimes referred to as "bridged nucleosides," "bridged non-natural nucleosides," or "BNA nucleosides." Some examples of bridged nucleic acids are shown in Figure 1.

[0040] A bicyclic sugar may be a sugar in which the 2'- and 4'-carbon atoms are bridged by two or more atoms. Examples of bicyclic sugars are known to those skilled in the art. One subgroup of nucleic acids (BNAs) or BNA nucleosides containing bicyclic sugars is the 4'-(CH2) p -O-2',4'-(CH2) p -CH2-2',4'-(CH2) p -S-2',4'-(CH2) p -OCO-2',4'-(CH2) n -N(R3)-O-(CH2) m-2' [wherein p, m, and n represent an integer of 1 to 4, an integer of 0 to 2, and an integer of 1 to 3, respectively; and R3 represents a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, or a unit substituent (a fluorescent or chemiluminescent labeled molecule, a functional group having nucleic acid cleavage activity, an intracellular or intranuclear localization signal peptide, etc.)]. Furthermore, with respect to BNAs or BNA nucleosides according to certain embodiments, in the OR2 substituent on the 3' carbon atom and the OR1 substituent on the 5' carbon atom, R1 and R2 are typically hydrogen atoms, but may be the same or different from each other and may also be a protecting group for a hydroxyl group for nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, a phosphate group, a phosphate group protected by a protecting group for nucleic acid synthesis, or P(R4)R5 (wherein R4 and R5 may be the same or different from each other and represent, respectively, a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, a mercapto group, a mercapto group protected by a protecting group for nucleic acid synthesis, an amino group, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or an amino group substituted with an alkyl group having 1 to 5 carbon atoms). Non-limiting examples of such BNAs include methyleneoxy (4'-CH2-O-2') BNA (also known as LNA (Locked Nucleic Acid®), 2',4'-BNA) (e.g., α-L-methyleneoxy (4'-CH2-O-2') BNA or β-D-methyleneoxy (4'-CH2-O-2') BNA), ethyleneoxy (4'-(CH2)2-O-2') BNA (also known as ENA), β-D-thio (4'-CH2-S-2') BNA, aminooxy (4'-CH2-ON(R3)-2') BNA, oxyamino (4'-CH2-N(R3)-O-2') BNA (2',4'-BNA NC Also known as ; R=H is 2',4'-BNANC [NH], R=Me is 2',4'-BNA NC [N-Me]), 2',4'-BNA coc , 3'-amino-2',4'-BNA, 5'-methyl BNA, (4'-CH(CH3)-O-2')BNA (also known as cEt BNA), (4'-CH(CH2OCH3)-O-2')BNA (cMOE Examples of BNAs include amide BNAs (amide-bridged nucleic acids) or (4'-C(O)-N(R)-2')BNAs (R = H, Me) (also known as AmNAs; in Figure 1, R = H is AmNA[NH] and R = Me is AmNA[N-Me]), guanidine BNAs (also known as GuNAs (e.g., in Figure 1, R = H is GuNA[NH] and R = Me is GuNA[N-Me])), amine BNAs (also known as 2'-Amino-LNAs) (e.g., 3-(Bis(3-aminopropyl)amino)propanoyl-substituted), 2'-O,4'-C-spirocyclopropylene-bridged nucleic acids (also known as scpBNAs), and other BNAs known to those skilled in the art. Non-limiting examples of such BNA nucleosides include methyleneoxy(4'-CH2-O-2') BNA nucleosides (also known as LNA nucleosides, 2',4'-BNA nucleosides) (e.g., α-L-methyleneoxy(4'-CH2-O-2') BNA nucleosides, β-D-methyleneoxy(4'-CH2-O-2') BNA nucleosides), ethyleneoxy(4'-(CH2)2-O-2') BNA nucleosides (also known as ENA nucleosides), β-D-thio(4'-CH2-S-2') BNA nucleosides, aminooxy(4'-CH2-ON(R3)-2') BNA nucleosides, oxyamino(4'-CH2-N(R3)-O-2') BNA nucleosides (2',4'-BNA NC Also known as nucleosides; R=H is 2',4'-BNA NC [NH] nucleoside, R=Me is 2',4'-BNA NC [N-Me] nucleoside), 2',4'-BNA cocNucleosides include 3'-amino-2',4'-BNA nucleosides, 5'-methyl BNA nucleosides, (4'-CH(CH3)-O-2') BNA nucleosides (also known as cEt nucleosides), (4'-CH(CHOCH3)-O-2') BNA nucleosides (also known as cMOE nucleosides), amide BNA nucleosides, or (4'-C(O)-N(R)-2') BNA nucleosides (R = H, Me) (also known as AmNA nucleosides; in Figure 1, R = H represents AmNA[NH] nucleosides, and R = Me represents AmNA[N-M e]nucleosides), guanidine BNA nucleosides (also known as GuNA nucleosides (e.g., in Figure 1, R = H is a GuNA[NH] nucleoside, and R = Me is a GuNA[N-Me] nucleoside)), amine BNA nucleosides (also known as 2'-Amino-LNA nucleosides) (e.g., 3-(Bis(3-aminopropyl)amino)propanoyl substituted nucleosides), 2'-O,4'-C-spirocyclopropylene bridged nucleosides (also known as scpBNA nucleosides), and other BNA nucleosides known to those of skill in the art.

[0041] As used herein, a "cationic nucleoside" is a modified nucleoside that exists in a cationic form relative to a neutral form (such as the neutral form of a ribonucleoside) at a certain pH (e.g., human physiological pH (about 7.4), the pH of a delivery site (e.g., organelle, cell, tissue, organ, organism, etc.)). A cationic nucleoside may contain one or more cationic modifying groups at any position of the nucleoside. In one embodiment, the cationic nucleoside is a 2'-Amino-LNA nucleoside (e.g., a 3-(Bis(3-aminopropyl)amino)propanoyl-substituted nucleoside), an aminoalkyl-modified nucleoside (e.g., a 2'-O-methyl- and 4'-CHCHCHNH-substituted nucleoside), a GuNA nucleoside (e.g., in Figure 3, R = H represents a GuNA[NH] nucleoside, and R = Me represents a GuNA[N-Me] nucleoside), etc. Bicyclic nucleosides having a methyleneoxy (4'-CH-O-2') bridge are sometimes referred to as LNA nucleosides.

[0042] As used herein, a "modified internucleoside linkage" refers to an internucleoside linkage that has a substitution or any change from a naturally occurring internucleoside linkage (i.e., a phosphodiester linkage). Modified internucleoside linkages include internucleoside linkages that contain a phosphorus atom and internucleoside linkages that do not contain a phosphorus atom. Representative phosphorus-containing internucleoside linkages include phosphodiester linkages, phosphorothioate linkages, phosphorodithioate linkages, phosphotriester linkages (e.g., methyl phosphotriester linkages and ethyl phosphotriester linkages described in U.S. Patent Registration No. 5,955,599), alkyl phosphonate linkages (e.g., methyl phosphonate linkages described in U.S. Patent Registration Nos. 5,264,423 and 5,286,717, and methoxypropyl phosphonate linkages described in WO 2015 / 168172), alkylthiophosphonate linkages, methylthiophosphonate linkages, boranophosphate linkages, and internucleoside linkages containing a cyclic guanidine moiety (e.g., a partial structure represented by the following formula (I): ), 1 to 4 C １～６ and internucleoside linkages (e.g., a moiety represented by the following formula (II): Examples of modified internucleoside linkages include, but are not limited to, the internucleoside linkages and phosphoramidate linkages used in the self-neutralizing nucleic acids (ZONs) described in International Publication No. WO 2016 / 081600. Phosphorothioate linkages refer to internucleoside linkages in which the non-bridging oxygen atom of a phosphodiester bond is replaced with a sulfur atom. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known. Modified internucleoside linkages are preferably those that are more nuclease-resistant than naturally occurring internucleoside linkages.

[0043] When an internucleoside bond has a chiral center, the internucleoside bond may be chiral controlled. "Chiral controlled" refers to a bond existing as a single diastereomer with respect to the chiral center, e.g., chiral phosphorus. A chiral controlled internucleoside bond may be completely chiral pure, or may have a high chiral purity, e.g., 90% de, 95% de, 98% de, 99% de, 99.5% de, 99.8% de, 99.9% de, or higher. As used herein, "chiral purity" refers to the proportion of one diastereomer in a mixture of diastereomers, expressed as diastereomeric excess (% de), and defined as (target diastereomer - other diastereomers) / (total diastereomers) x 100 (%).

[0044] For example, the internucleoside bond may be a phosphorothioate bond chirally controlled in the Rp or Sp configuration, or 1 to 4 C １～６and / or an internucleoside linkage comprising a cyclic guanidine moiety. Methods for preparing chiral internucleoside bonds are known. For example, phosphorothioate bonds chiral controlled to the Rp or Sp configuration are prepared by the methods described in Naoki Iwamoto et al., Angew. Chem. Int. Ed. Engl. 2009, 48(3), 496-9; Natsuhisa Oka et al., J. Am. Chem. Soc. 2003, 125, 8307-8317; Natsuhisa Oka et al., J. Am. Chem. Soc. 2008, 130, 16031-16037; Yohei Nukaga et al., J. Org. Chem. 2016, 81, 2753-2762; Yohei Nukaga et al., J. Org. Chem. 2012, 77, They can be synthesized according to the method described in [Publication ID No. 7913-7922]. Chiral phosphorothioate bonds in the Rp or Sp configuration are also known and are known to have the effects described, for example, in Naoki Iwamoto et al., Nat. Biotechnol., 2017, 35(9), 845-851 and Anastasia Khvorova et al., Nat. Biotechnol., 2017, 35(3), 238-248. For example, in one embodiment, phosphorothioate bonds in the Sp configuration are more stable than those in the Rp configuration, and / or ASOs in the Sp configuration are chiral to promote target RNA cleavage by RNase H1, resulting in a more sustained response in vivo. １～６Methods for preparing internucleoside linkages containing a guanidine moiety (e.g., a TMG moiety) substituted with an alkyl group are known, and the linkages can be synthesized, for example, according to the method described in Alexander A. Lomzov et al., Biochem Biophys Res Commun., 2019, 513(4), 807-811.

[0045] As used herein, the term "nucleobase" or "base" refers to a base component (heterocyclic moiety) constituting a nucleic acid, and the most commonly known are adenine, guanine, cytosine, thymine, and uracil. Unless otherwise specified, "nucleobase" or "base" herein encompasses both modified and unmodified nucleic acid bases (bases). Therefore, unless otherwise specified, a purine base may be either a modified or unmodified purine base. Furthermore, unless otherwise specified, a pyrimidine base may be either a modified or unmodified pyrimidine base.

[0046] "Modified nucleobase" or "modified base" refers to any nucleobase other than adenine, cytosine, guanine, thymine, or uracil. "Unmodified nucleobase" or "unmodified base" (natural nucleobase) refers to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Examples of modified nucleobases include, but are not limited to, hypoxanthine, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, or N4-methylcytosine; N6-methyladenine or 8-bromoadenine; 2-thiothymine; and N2-methylguanine or 8-bromoguanine. The modified nucleobase is preferably 5-methylcytosine.

[0047] The term "complementary" as used herein refers to a relationship in which nucleic acid bases can form so-called Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen base pairs, etc.) through hydrogen bonds. In the present invention, the antisense oligonucleotide region in the first nucleic acid strand does not necessarily need to be completely complementary to at least a portion of the target transcript (e.g., the transcript of the target gene), but it is acceptable as long as the base sequence has at least 70%, preferably at least 80%, and even more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity. The antisense oligonucleotide region in the first nucleic acid strand can hybridize to the target transcript when the base sequence is complementary (typically, when the base sequence is complementary to at least a portion of the base sequence of the target transcript). Similarly, the complementary region in the second nucleic acid strand does not necessarily need to be completely complementary to at least a portion of the first nucleic acid strand; it is acceptable as long as the base sequence is at least 70%, preferably at least 80%, and even more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementary. The complementary region in the second nucleic acid strand can anneal to at least a portion of the first nucleic acid strand if the base sequence is complementary to that of the first nucleic acid strand. The complementarity of the base sequences can be determined using a BLAST program or the like. Those skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which the two strands can anneal or hybridize, taking into account the degree of complementarity between the strands. Furthermore, those skilled in the art can easily design antisense nucleic acids complementary to target transcription products, for example, based on information about the base sequence of the target gene.

[0048] Hybridization conditions may be of various stringent conditions, such as low stringency and high stringency. Low stringency conditions may be conditions of relatively low temperature and high salt concentration, for example, 30°C, 2xSSC, 0.1% SDS. High stringency conditions may be conditions of relatively high temperature and low salt concentration, for example, 65°C, 0.1xSSC, 0.1% SDS. Hybridization stringency can be adjusted by changing conditions such as temperature and salt concentration. Here, 1xSSC contains 150 mM sodium chloride and 15 mM sodium citrate.

[0049] As used herein, "toxicity" refers to an effect that causes undesirable objective or subjective symptoms or functional abnormalities in a subject, such as death, pain, tremors, convulsions, movement disorders, cognitive dysfunction, impaired consciousness, general malaise, fatigue, nausea, vomiting, dizziness, numbness, and unsteadiness. The toxicity may be toxicity in any organ. The toxicity may be neurotoxicity.

[0050] As used herein, "neurotoxicity" refers to an effect that causes damage to nervous tissue, including central and peripheral nervous tissue, and interferes with the normal activity of the nervous system. Neurotoxicity can cause symptoms selected from death, respiratory abnormalities, cardiovascular abnormalities, headache, nausea or vomiting, unresponsiveness or hyporesponsiveness, impaired consciousness, mental disorders, personality changes, hallucinations, delusions, cognitive dysfunction, abnormal posture, involuntary movements, tremors, convulsions, hyperactive motor dysfunction, paralysis, sensory disturbances, or autonomic dysfunction. Neurotoxicity may be acute neurotoxicity. Acute neurotoxicity may occur within 1, 3, 6, 9, 12, 24, or 48 hours after administration. Toxicity can be evaluated, for example, by an acute tolerability score, adverse event rate, or mortality rate, as described in the Examples below.

[0051] As used herein, "central neurotoxicity" refers to an effect that causes damage to at least the central nervous tissue among nervous tissues and interferes with the normal activity of the nervous system.

[0052] As used herein, the term "subject" refers to a target to which the double-stranded nucleic acid complex or pharmaceutical composition of the present invention is applied. Subjects include individuals as well as organs, tissues, and cells. When the subject is an individual, it can be any animal, including humans. Examples of non-human subjects include various livestock, poultry, pets, and laboratory animals. The subject may be, but is not limited to, an individual in need of reducing the expression level of a target transcript or an individual in need of disease treatment or prevention.

[0053] The double-stranded nucleic acid complex of the present invention comprises a first nucleic acid strand and a second nucleic acid strand. The specific structure of each nucleic acid strand is shown below.

[0054] In the double-stranded nucleic acid complex of the present invention, the first nucleic acid strand can hybridize to at least a portion of a target gene or its transcription product and has an antisense effect on the target gene or its transcription product, and the second nucleic acid strand contains a base sequence complementary to the first nucleic acid strand and contains one or more 2'-modified nucleosides.

[0055] The number of 2'-modified nucleosides contained in the second nucleic acid strand is at least 1 and is equal to or less than the total number of nucleosides constituting the second nucleic acid strand (i.e., the base length of the second nucleic acid strand). The specific number of 2'-modified nucleosides contained in the second nucleic acid strand may be, for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, or may be 30 or less, 25 or less, 20 or less, 15 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. For example, the number of 2'-modified nucleosides in the second nucleic acid strand can be 1 to 30, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, or 1 to 6. For example, it can be 1, 2, 3, 4, 5, or 6.

[0056] In one embodiment, 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more of the nucleosides in the second nucleic acid strand are 2'-modified nucleosides. For example, 10% to 90%, 20% to 80%, 30% to 70%, or 40% to 60% may be 2'-modified nucleosides.

[0057] Nucleosides other than 2'-modified nucleosides contained in the second nucleic acid strand may be natural nucleosides, unnatural nucleosides such as bridged nucleosides, or any combination thereof. The number of nucleosides other than 2'-modified nucleosides contained in the second nucleic acid strand is not limited, and may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, for example, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 12, 1 to 10, 1 to 8, or 1 to 6, and may be, for example, 1 to 5, for example, 1, 2, 3, 4, or 5.

[0058] In one embodiment, the second nucleic acid strand contains one 2'-modified nucleoside. In another embodiment, all of the nucleosides constituting the second nucleic acid strand are 2'-modified nucleosides. For example, in the second nucleic acid strand, all of the nucleosides in the region consisting of a base sequence complementary to the first nucleic acid strand may be 2'-modified nucleosides. In a further embodiment, the second nucleic acid strand contains one or more but less than all 2'-modified nucleosides. As used herein, the phrase "containing less than all" 2'-modified nucleosides in the second nucleic acid strand means that the second nucleic acid strand contains at least one nucleoside of any kind other than a 2'-modified nucleoside.

[0059] In one embodiment, the second nucleic acid strand comprises one or more contiguous 2'-modified nucleosides located at the 5'-terminus. As used herein, "comprising a plurality of contiguous 2'-modified nucleosides" means comprising a plurality of 2'-modified nucleosides linked by any internucleoside linkage. For example, the second nucleic acid strand may comprise one or 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3, e.g., 2, 3, or 4, 2'-modified nucleosides located at the 5'-terminus.

[0060] In one embodiment, the second nucleic acid strand comprises one or multiple consecutive 2'-modified nucleosides located at the 3'-terminus. For example, the second nucleic acid strand may comprise one or 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3, e.g., 2, 3, or 4, 2'-modified nucleosides located at the 3'-terminus.

[0061] In a further embodiment, the second nucleic acid strand comprises one or more contiguous 2'-modified nucleosides located at the 5'-terminus and one or more contiguous 2'-modified nucleosides located at the 3'-terminus.

[0062] In one embodiment, the second nucleic acid strand comprises 2'-modified nucleosides at positions other than the 5'-end and the 3'-end. As used herein, the phrase "comprises 2'-modified nucleosides at positions other than the 5'-end and the 3'-end" means that the second nucleic acid strand comprises 2'-modified nucleosides at positions other than the one or more contiguous 2'-modified nucleosides at the 5'-end and the one or more contiguous 2'-modified nucleosides at the 3'-end. For example, the second nucleic acid strand comprises 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 12, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, or 1 to 3, e.g., 1 or 2, 2'-modified nucleosides at positions other than the 5'-end and the 3'-end.

[0063] In one embodiment, the second nucleic acid strand includes one or more nucleosides other than 2'-modified nucleosides. In a further embodiment, all of the nucleosides other than 2'-modified nucleosides in the second nucleic acid strand (e.g., in the region consisting of the base sequence complementary to the first nucleic acid strand) can be deoxyribonucleosides.

[0064] In one embodiment, the 2'-modified nucleoside is a 2'-O-methoxyethyl modified nucleoside and / or a 2'-O-methyl modified nucleoside. The 2'-O-methoxyethyl modified nucleoside has the following formula (III): The 2'-O-methyl modified nucleoside is represented by the following formula (IV): In one embodiment, the 2'-modified nucleoside is a 2'-O-methyl modified nucleoside. In a more preferred embodiment, the 2'-modified nucleoside is a 2'-O-methoxyethyl modified nucleoside.

[0065] In one embodiment, all of the nucleosides in the first nucleic acid strand may be unnatural or modified nucleosides. In a further embodiment, all of the nucleosides in the first nucleic acid strand may be 2'-modified nucleosides. In yet a further embodiment, all of the nucleosides in the first nucleic acid strand may be 2'-O-methoxyethyl modified nucleosides.

[0066] The first nucleic acid strand, when hybridized to a target transcript, can contain at least 4, at least 5, at least 6, or at least 7 consecutive nucleosides recognized by RNase H. Typically, the region can be any region containing 4 to 20, 5 to 16, or 6 to 12 consecutive nucleosides. Nucleosides recognized by RNase H can be, for example, natural deoxyribonucleosides. Modified deoxyribonucleosides and suitable nucleosides containing other bases are well known in the art. It is also known that nucleosides containing a hydroxy group at the 2' position, such as ribonucleosides, are unsuitable for use as such nucleosides. The suitability of nucleosides for use in this region containing "at least 4 consecutive nucleosides" can be easily determined. In one embodiment, the first nucleic acid strand can contain at least 4 consecutive deoxyribonucleosides.

[0067] In one embodiment, the nucleosides of the first nucleic acid strand comprise or consist of deoxyribonucleosides, for example, 70% or more, 80% or more, 90% or more, or 95% or more of the nucleosides of the first nucleic acid strand are deoxyribonucleosides.

[0068] In one embodiment, the first nucleic acid strand may be a gapmer. As used herein, the term "gapmer" generally refers to a single-stranded nucleic acid consisting of a central region (DNA gap region) and wing regions (referred to as the 5' wing region and the 3' wing region, respectively) located directly at the 5' end and the 3' end of the central region. The length of the DNA gap region may be 13 to 22 bases, 16 to 22 bases, or 16 to 20 bases, or 4 to 20 bases, 5 to 18 bases, 6 to 16 bases, 7 to 14 bases, or 8 to 12 bases. In a gapmer, the central region contains at least three or at least four consecutive deoxyribonucleosides, and the wing regions contain at least one unnatural nucleoside. Although not limited thereto, unnatural nucleosides contained in the wing regions typically have stronger RNA-binding affinity and higher resistance to nucleases (such as nucleases) than natural nucleosides. When the non-natural nucleosides comprising the wing regions comprise or consist of bridged nucleosides, the gapmer is specifically referred to as a "BNA / DNA gapmer." The number of bridged nucleosides in the 5' and 3' wing regions is at least one, and may be, for example, two or three. The bridged nucleosides in the 5' and 3' wing regions may be contiguous or non-contiguous within the 5' and 3' wing regions. The bridged nucleoside may further comprise a modified nucleobase (e.g., 5-methylcytosine). When the bridged nucleoside is an LNA nucleoside, the gapmer is specifically referred to as an "LNA / DNA gapmer." When the non-natural nucleosides comprising the 5' and 3' wing regions comprise or consist of peptide nucleic acids, the gapmer is specifically referred to as a "peptide nucleic acid gapmer." When the unnatural nucleosides that make up the 5' wing region and the 3' wing region contain or consist of morpholino nucleic acid, the gapmer is specifically referred to as a "morpholino nucleic acid gapmer." The base lengths of the 5' wing region and the 3' wing region may each independently be at least 2 bases long, for example, 2 to 10 bases long, 2 to 7 bases long, or 3 to 5 bases long.In one embodiment, the 5' wing region and / or the 3' wing region may comprise at least one unnatural nucleoside and may further comprise natural nucleosides. The 5' wing region and the 3' wing region may comprise, for example, unnatural nucleosides linked by modified internucleoside linkages such as phosphorothioate linkages, bridged nucleosides such as LNA nucleosides, or 2'-modified nucleosides such as 2'-O-methyl modified nucleosides.

[0069] In a gapmer, the central region (DNA gap region) contains at least three or at least four consecutive deoxyribonucleosides, and the wing regions (5' wing region and 3' wing region) contain at least one unnatural nucleoside. The boundary positions between the central region (DNA gap region) and the wing regions in a gapmer can be easily determined by those skilled in the art from the nucleoside sequence. For example, the central region can be functionally defined as a region that can be recognized by RNase H1. Here, "recognizable by RNase H1" means that when the gapmer binds to a target RNA, the sequence in the target RNA paired with the gapmer can be cleaved by RNase H1. Therefore, the boundary positions can be determined by defining the region that can be recognized by RNase H1 in the gapmer as the central region and the regions that cannot be recognized by RNase H1 as the wing regions (5' wing region and 3' wing region). In this specification, nucleosides in the 5' wing region and 3' wing region adjacent to the central region are considered to be unnatural nucleosides, and nucleosides in the central region adjacent to the 5' wing region or 3' wing region are considered to be natural nucleosides.

[0070] The first nucleic acid strand constituting the gapmer may be composed of, in order from the 5' end, a bridged nucleoside of 2 to 7 bases or 3 to 5 bases (e.g., 2 or 3 bases), a ribonucleoside or deoxyribonucleoside of 4 to 15 bases or 8 to 12 bases (e.g., 8 or 10 bases), and a bridged nucleoside of 2 to 7 bases or 3 to 5 bases (e.g., 2 or 3 bases).

[0071] In addition, a nucleic acid strand having a wing region only on either the 5'-end or the 3'-end is called a "hemigapmer" in the art, and in this specification, hemigapmers are also included in the term "gapmer."

[0072] In one embodiment, the second nucleic acid strand comprises 2'-modified nucleosides in a region of the second nucleic acid strand that is complementary to the 5' wing region and / or the 3' wing region of the first nucleic acid strand. In a further embodiment, all nucleosides in the second nucleic acid strand that is complementary to the 5' wing region and / or the 3' wing region of the first nucleic acid strand can be 2'-modified nucleosides. The 2'-modified nucleosides can be, for example, 2'-O-methoxyethyl-modified nucleosides or 2'-O-methyl-modified nucleosides.

[0073] In one embodiment, in the second nucleic acid strand, in the region consisting of a base sequence complementary to the central region of the first nucleic acid strand, nucleosides containing purine bases may be ribonucleosides. For example, in the second nucleic acid strand, in the region consisting of a base sequence complementary to the central region of the first nucleic acid strand, all nucleosides containing purine bases may be ribonucleosides.

[0074] In one embodiment, in the second nucleic acid strand, in the region consisting of a base sequence complementary to the central region of the first nucleic acid strand, nucleosides containing pyrimidine bases may be deoxyribonucleosides. For example, in the second nucleic acid strand, in the region consisting of a base sequence complementary to the central region of the first nucleic acid strand, all nucleosides containing pyrimidine bases may be deoxyribonucleosides.

[0075] In a further embodiment, in the second nucleic acid strand, in a region consisting of a base sequence complementary to the central region of the first nucleic acid strand, all nucleosides containing purine bases may be ribonucleosides, and all nucleosides containing pyrimidine bases may be deoxyribonucleosides.

[0076] In a preferred embodiment, in the second nucleic acid strand, all nucleosides in the region consisting of a base sequence complementary to the 5' wing region and / or 3' wing region of the first nucleic acid strand are 2'-modified nucleosides (e.g., 2'-O-methoxyethyl-modified nucleosides or 2'-O-methyl-modified nucleosides), and further, in the second nucleic acid strand, all nucleosides containing purine bases are ribonucleosides and all nucleosides containing pyrimidine bases are deoxyribonucleosides in the region consisting of a base sequence complementary to the central region of the first nucleic acid strand.

[0077] In one embodiment, (i) at least one guanosine nucleoside in a first nucleic acid strand, (ii) the nucleoside adjacent to the 5'-terminus of the guanosine nucleoside, (iii) the nucleoside adjacent to the 3'-terminus of the guanosine nucleoside, or (iv) a nucleoside in a second nucleic acid strand complementary to any combination of (i) to (iii) above may be a 2'-modified nucleoside. In a further embodiment, (i) all guanosine nucleosides in a first nucleic acid strand, (ii) the nucleoside adjacent to the 5'-terminus of the guanosine nucleoside, (iii) the nucleoside adjacent to the 3'-terminus of the guanosine nucleoside, or (iv) a nucleoside in a second nucleic acid strand complementary to any combination of (i) to (iii) above may be a 2'-modified nucleoside.

[0078] In one embodiment, (i) at least one guanosine nucleoside in the 3' wing region and / or 5' wing region of the first nucleic acid strand, (ii) the nucleoside adjacent to the 5' terminal side of the guanosine nucleoside, (iii) the nucleoside adjacent to the 3' terminal side of the guanosine nucleoside, or (iv) a nucleoside in the second nucleic acid strand that is complementary to any combination of (i) to (iii) above may be a 2'-modified nucleoside. In further embodiments, (i) all guanosine nucleosides in the 3' wing region and / or 5' wing region of the first nucleic acid strand, (ii) the nucleoside adjacent to the 5' terminal side of the guanosine nucleoside, (iii) the nucleoside adjacent to the 3' terminal side of the guanosine nucleoside, or (iv) the nucleoside in the second nucleic acid strand that is complementary to any combination of (i) to (iii) above may be 2'-modified nucleosides.

[0079] In one embodiment, in the second nucleic acid strand, all nucleosides in the region consisting of a base sequence complementary to the central region of the first nucleic acid strand are (a) deoxyribonucleosides, (b) deoxyribonucleosides and ribonucleosides, (c) deoxyribonucleosides and 2'-modified nucleosides, or (d) ribonucleosides and 2'-modified nucleosides, or (e) deoxyribonucleosides, ribonucleosides, and 2'-modified nucleosides.

[0080] In one embodiment, all nucleosides in the region consisting of base sequences complementary to the 5' wing region and 3' wing region of the first nucleic acid strand may be 2'-modified nucleosides, and all nucleosides in the region consisting of base sequences complementary to the central region of the first nucleic acid strand may be deoxyribonucleosides.

[0081] In one embodiment, at least one of the nucleosides containing a pyrimidine base in the second nucleic acid strand may be a 2'-modified nucleoside and / or a deoxyribonucleoside. In a further embodiment, the second nucleic acid strand does not contain natural ribonucleosides containing a pyrimidine base, for example, all of the nucleosides containing a pyrimidine base in the second nucleic acid strand may be 2'-modified nucleosides and / or deoxyribonucleosides.

[0082] In one embodiment, the second nucleic acid strand may include, in addition to the 2'-modified nucleosides, modified nucleosides other than the 2'-modified nucleosides. For example, the second nucleic acid strand may include a 2'-O-methyl modified nucleoside represented by the above formula (IV), a scpBNA nucleoside represented by the following formula (V), an AmNA nucleoside represented by the following formula (VI), or an oxyamino (4'-CH2-N(R3)-O-2') BNA nucleoside (2',4'-BNA NC Also known as nucleosides; R=H is 2',4'-BNA NC [NH] nucleoside, R=Me is 2',4'-BNA NC It may contain at least one [N-Me] nucleoside.

[0083] (In the formula, R represents a hydrogen atom or a methyl group.)

[0084] The bridged unnatural nucleoside represented by the formula (V) above is a 2'-O,4'-C-spirocyclopropylene-bridged nucleic acid, and is mainly referred to herein as "scpBNA." The bridged unnatural nucleoside represented by the formula (VI) above is an amide BNA (amide-bridged nucleic acid), and can also be represented as (4'-C(O)-N(R)-2')BNA (R = H, Me), but is mainly referred to herein as "AmNA." In the formula (VI) above, R may be either a hydrogen atom or a methyl group. Unless otherwise specified herein, R may be either a hydrogen atom or a methyl group. However, when distinguishing between the two, when R is a hydrogen atom, it can be represented as AmNA[NH], and when R is a methyl group, it can be represented as AmNA[N-Me].

[0085] In the double-stranded nucleic acid complex of the present invention, the first nucleic acid strand may be a mixmer. As used herein, a "mixmer" refers to a nucleic acid strand that contains alternating natural and unnatural nucleosides of periodic or random segment lengths, but does not contain four or more consecutive deoxyribonucleosides and ribonucleosides. A mixmer in which the unnatural nucleoside is a bridged nucleoside and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "BNA / DNA mixmer." The bridged nucleoside may be a bridged unnatural nucleoside represented by formula (V) or (VI) above. A mixmer in which the unnatural nucleoside is a peptide nucleic acid and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "peptide nucleic acid / DNA mixmer." In a mixmer, a mixmer in which the unnatural nucleoside is a morpholino nucleic acid and the natural nucleoside is a deoxyribonucleoside is specifically referred to as a "morpholino nucleic acid / DNA mixmer." A mixmer is not limited to containing only two types of nucleosides. A mixmer can contain any number of types of nucleosides, regardless of whether they are natural or modified nucleosides or nucleoside mimics. For example, a mixmer may have one or two consecutive deoxyribonucleosides separated by a bridged nucleoside (e.g., an LNA nucleoside or a bridged unnatural nucleoside represented by formula (V) or (VI) above). The bridged nucleoside may further contain a modified nucleobase (e.g., 5-methylcytosine).

[0086] In one embodiment, the second nucleic acid strand may contain at least four consecutive ribonucleosides complementary to the at least four consecutive nucleosides (e.g., deoxyribonucleosides) in the central region of the first nucleic acid strand, such that the second nucleic acid strand forms a partial DNA-RNA heteroduplex with the first nucleic acid strand and is recognized and cleaved by RNase H. The at least four consecutive ribonucleosides in the second nucleic acid strand are preferably linked by naturally occurring internucleoside linkages, i.e., phosphodiester bonds.

[0087] In a further embodiment, the second nucleic acid strand may further comprise at least two consecutive deoxyribonucleosides in addition to the at least four consecutive ribonucleosides. The at least two consecutive deoxyribonucleosides may be complementary to the first nucleic acid strand and may be contained in a region complementary to the central region of the first nucleic acid strand. The at least two consecutive deoxyribonucleosides may be located on either the 5' or 3' side of the at least four consecutive ribonucleosides, or may be located on both the 5' and 3' sides. The at least two consecutive deoxyribonucleosides may also be 2, 3, 4, 5, or six or more consecutive deoxyribonucleosides.

[0088] At least one, at least two (e.g., two), at least three, or at least four nucleosides from the end (5'-end, 3'-end, or both ends) of the second nucleic acid strand may be modified nucleosides. The modified nucleosides may contain a modified sugar and / or a modified nucleobase. The modified sugar may be a 2'-modified sugar (e.g., a sugar containing a 2'-O-methyl group). The modified nucleobase may also be 5-methylcytosine.

[0089] The second nucleic acid strand may be composed of, from the 5'-terminus, 2 to 7 or 3 to 5 (e.g., 2- or 3-base) modified nucleosides (e.g., modified nucleosides containing a 2'-modified sugar), 4 to 15 or 8 to 12 (e.g., 8- or 10-base) ribonucleosides or deoxyribonucleosides (optionally linked via a modified internucleoside bond), and 2 to 7 or 3 to 5 (e.g., 2- or 3-base) modified nucleosides (e.g., modified nucleosides containing a 2'-modified sugar). In this case, the first nucleic acid strand may be a gapmer.

[0090] The first nucleic acid strand and / or the second nucleic acid strand may comprise, in whole or in part, nucleoside or nucleotide mimetics. The nucleotide mimetics may be peptide nucleic acids and / or morpholino nucleic acids.

[0091] In one embodiment, the second nucleic acid strand may contain non-complementary bases and / or one or more inserted and / or deleted bases relative to the first nucleic acid strand. The number of non-complementary bases in the second nucleic acid strand is not limited, and may be, for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 or 2. The sequence consisting of non-complementary bases may form a bulge structure, as described below. The length of the inserted sequence in the second nucleic acid strand is not limited, and may be, for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 or 2. The inserted sequence may form a bulge structure, as described below. The length of the deleted consecutive bases in the second nucleic acid strand is not limited, and may be, for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 or 2. The second nucleic acid strand may contain a bulge structure, as described below, at the deletion position.

[0092] The base lengths of the first and second nucleic acid strands are not particularly limited, but may be at least 8 bases, at least 9 bases, at least 10 bases, at least 11 bases, at least 12 bases, at least 13 bases, at least 14 bases, or at least 15 bases. The base lengths of the first and second nucleic acid strands may be 35 bases or less, 30 bases or less, 25 bases or less, 24 bases or less, 23 bases or less, 22 bases or less, 21 bases or less, 20 bases or less, 19 bases or less, 18 bases or less, 17 bases or less, or 16 bases or less. The first and second nucleic acid strands may be the same length or different lengths (e.g., one of them may be 1 to 3 bases shorter or longer). In one embodiment, the second nucleic acid strand is shorter than the first nucleic acid strand. In this case, the position at which the second nucleic acid strand can bind to the first nucleic acid strand is not important. For example, the second nucleic acid strand may be capable of binding to the 5' region, the central region, or the 3' region of the first nucleic acid strand. In one embodiment, the second nucleic acid strand may be at least 8 bases long. The double-stranded structure formed by the first and second nucleic acid strands may include a bulge. The length can be determined by balancing the strength of the antisense effect and the specificity of the nucleic acid strand for the target, among other factors such as cost and synthesis yield.

[0093] In one embodiment, the second nucleic acid strand may include at least one overhang region located at one or both of its 5'-end and 3'-end. An "overhang region" refers to a region of the second nucleic acid strand adjacent to a region complementary to a first nucleic acid strand, such that when the first and second nucleic acid strands anneal to form a double-stranded structure, the 5'-end of the second nucleic acid strand extends beyond the 3'-end of the first nucleic acid strand and / or the 3'-end of the second nucleic acid strand extends beyond the 5'-end of the first nucleic acid strand, i.e., a nucleotide region in the second nucleic acid strand that protrudes from the double-stranded structure. The overhang region in the second nucleic acid strand may be located at either the 5'-end or the 3'-end of the complementary region. The overhang region in the second nucleic acid strand may be located at either the 5'-end or the 3'-end of the complementary region.

[0094] The length of the overhang region is not limited, but may be 1 to 20 bases, for example, 2 to 15 bases, 2 to 12 bases, 2 to 10 bases, 2 to 8 bases, 2 to 6 bases, 2 to 5 bases, 2 to 4 bases, or 2 to 3 bases. The type of nucleosides constituting the overhang region is also not limited. For example, the overhang region may be composed of natural nucleosides (e.g., deoxyribonucleosides) or non-natural nucleosides (e.g., bridged nucleosides such as LNA nucleosides). Furthermore, all or part of the internucleoside linkages in the overhang region may be modified internucleoside linkages. The modified internucleoside linkages may be, for example, phosphorothioate linkages. The overhang region preferably has protein-binding properties, lipid solubility, and / or nuclease resistance, and may be composed of deoxyribonucleosides or LNA nucleosides linked by phosphorothioate linkages, for example. The base sequence of the overhang region may be a sequence unrelated to the base sequence of the target gene.

[0095] At least one, at least two (e.g., two), at least three, or at least four nucleosides from the end (5'-end, 3'-end, or both ends) of the second nucleic acid strand may be unnatural nucleosides (modified nucleosides). The modified nucleosides may contain a modified sugar and / or a modified nucleobase. The modified sugar may be a 2'-modified sugar (e.g., a sugar containing a 2'-O-methyl group). The modified nucleobase may also be 5-methylcytosine.

[0096] In one embodiment, the second nucleic acid strand may have 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 (e.g., 1 to 2 or 1) non-complementary bases, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 (e.g., 1 to 2 or 1) deleted bases, and / or 1 to 20 (e.g., 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 4, 1 to 3, or 1) inserted bases relative to the first nucleic acid strand, so long as the second nucleic acid strand can form a duplex with the first nucleic acid strand. The sequence region consisting of the inserted bases may form a bulge structure.

[0097] In one embodiment, the second nucleic acid strand contains at least one bulge structure consisting of a base sequence non-complementary to the first nucleic acid strand. As used herein, the term "bulge structure" refers to a portion of a double-stranded nucleic acid in which a portion of nucleic acid in one of the nucleic acid strands constituting the double strand protrudes from the double-stranded structure without base pairing. The base length of the bulge structure is not limited. For example, it may be 1 to 50 bases long, 1 to 40 bases long, 1 to 30 bases long, 1 to 20 bases long, or 1 to 15 bases long, preferably 1 to 10 bases long.

[0098] In one embodiment, the bulge structure comprises sugar-unmodified nucleosides. In a further embodiment, all nucleosides in the bulge structure are sugar-unmodified nucleosides.

[0099] In one embodiment, when the second nucleic acid strand includes a bulge structure, all of the nucleosides other than the bulge structure may be 2'-modified nucleosides.

[0100] The internucleoside linkages in the first and second nucleic acid strands may be naturally occurring internucleoside linkages and / or modified internucleoside linkages. While not limited thereto, it is preferred that at least one, at least two, or at least three internucleoside linkages from the termini (5'-terminus, 3'-terminus, or both) of the first and / or second nucleic acid strands are modified internucleoside linkages. Here, for example, the two internucleoside linkages from the terminus of the nucleic acid strand refer to the internucleoside linkage closest to the terminus of the nucleic acid strand and the adjacent internucleoside linkage located on the opposite side of the terminus. Modified internucleoside linkages in the terminal regions of the nucleic acid strand are preferred because they can suppress or inhibit undesired degradation of the nucleic acid strand.

[0101] In one embodiment, all or some of the internucleoside linkages of the first nucleic acid strand and / or the second nucleic acid strand may be modified internucleoside linkages, and in one embodiment, the first nucleic acid strand and / or the second nucleic acid strand may each contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more modified internucleoside linkages. In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand may each contain at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 93%, at least 95%, at least 98%, or 100% modified internucleoside linkages. In one embodiment, the modified internucleoside linkages may be phosphorothioate linkages or boranophosphate linkages. In one embodiment, when the nucleic acid in the first nucleic acid strand consists of morpholino nucleic acid, all or some of the internucleoside linkages in the first nucleic acid strand may be phosphorothioate linkages.

[0102] In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand may each contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more chiral controlled internucleoside linkages. In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand may each contain at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more chiral controlled internucleoside linkages.

[0103] In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand may each contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more non-negatively charged internucleoside linkages (preferably neutral internucleoside linkages). In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand may each comprise at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more non-negatively charged internucleoside linkages.

[0104] In one embodiment, at least one, at least two, or at least three internucleoside linkages from the 5'-end of the second nucleic acid strand may be modified internucleoside linkages. At least one, at least two, or at least three internucleoside linkages from the 3'-end of the second nucleic acid strand may be modified internucleoside linkages, such as phosphorothioate linkages, 1 to 4 C １～６ The modified internucleoside linkage may be an internucleoside linkage containing a guanidine moiety (e.g., a TMG moiety) substituted with an alkyl group such as the above and / or a cyclic guanidine moiety. The modified internucleoside linkage may be chiral controlled to the Rp or Sp configuration.

[0105] At least one (e.g., three) internucleoside linkages from the 3'-end of the second nucleic acid strand may be a modified internucleoside linkage such as a phosphorothioate linkage, which has high RNase resistance. Inclusion of a modified internucleoside linkage such as a phosphorothioate modification at the 3'-end of the second nucleic acid strand is preferred because it improves the gene silencing activity of the double-stranded nucleic acid complex.

[0106] In one embodiment, the modified internucleoside linkage of the first nucleic acid strand and / or the second nucleic acid strand is such that at a certain pH (e.g., human physiological pH (about 7.4), the pH of a delivery site (e.g., organelle, cell, tissue, organ, organism, etc.), the modified internucleoside linkage is in an anionic form (e.g., —OP(O)(O - )-O- (anionic form of the natural phosphate bond), -OP(O)(S - The modified internucleoside linkages of the first and / or second nucleic acid strands comprise non-negatively charged (neutral or cationic, respectively) internucleoside linkages that exist in a neutral or cationic form compared to the non-negatively charged internucleoside linkages of the first and / or second nucleic acid strands (e.g., the anionic form of a phosphorothioate linkage). In one embodiment, the modified internucleoside linkages of the first and / or second nucleic acid strands comprise neutral internucleoside linkages. In one embodiment, the non-negatively charged internucleoside linkages (e.g., neutral internucleoside linkages), when in their neutral form, do not have moieties with a pKa of less than 8, less than 9, less than 10, less than 11, less than 12, less than 13, or less than 14. In one embodiment, the non-negatively charged internucleoside linkage is, for example, a methyl phosphonate linkage as described in U.S. Patent Registration Nos. 5,264,423 and 5,286,717, a methyl phosphotriester linkage as described in U.S. Patent Registration No. 5,955,599, an ethyl phosphotriester linkage, a methoxypropyl phosphonate linkage as described in WO 2015 / 168172, or an internucleoside linkage used in self-neutralizing nucleic acids (ZON) as described in WO 2016 / 081600. In one embodiment, the non-negatively charged internucleoside linkage comprises a triazole moiety or an alkyne moiety. In one embodiment, the non-negatively charged internucleoside linkage comprises a cyclic guanidine moiety and / or 1 to 4 C １～６ In one embodiment, the modified internucleoside linkage comprising a cyclic guanidine moiety has a moiety represented by formula (I): １～６ The alkyl-substituted guanidine moiety has a moiety represented by formula (II): １～６Neutral internucleoside linkages comprising a guanidine moiety substituted with an alkyl group of the formula (I) are chiral controlled. In one embodiment, the present disclosure relates to a composition comprising an oligonucleotide comprising at least one neutral internucleoside linkage and at least one phosphorothioate internucleoside linkage. Without wishing to be bound by any particular theory, in at least some cases, the neutral internucleoside linkage can improve properties and / or activity compared to a comparable nucleic acid that does not comprise a neutral internucleoside linkage, such as improved delivery, improved resistance to exonucleases and endonucleases, improved cellular uptake, improved endosomal escape, and / or improved nuclear uptake.

[0107] In one embodiment, the second nucleic acid strand may be bound to a ligand (sometimes referred to herein as a binder), which may be a small molecule (small molecule ligand), a medium molecule (medium molecule ligand), a polymer (polymer ligand), a peptide (peptide ligand), a lipid (lipid ligand), or an aptamer (e.g., a nucleic acid aptamer).

[0108] As used herein, "peptide" refers to an amino acid polymer having one or more peptide bonds. The term "peptide" is not limited by the number of amino acid residues contained therein. Therefore, "peptide" encompasses everything from oligopeptides containing a few amino acid residues, such as dipeptides and tripeptides, to polypeptides (proteins) containing many amino acid residues. Peptides can be linear, branched, or cyclic.

[0109] The peptide ligand may bind to a molecule present on the surface of or within a cell, or in a body fluid.

[0110] In one embodiment, the peptide may be an antibody or an active fragment thereof. Examples of antibodies include monoclonal antibodies, polyclonal antibodies, recombinant antibodies such as chimeric antibodies and humanized antibodies, Fab, F(ab')2, Fab', VHH, etc. Examples of active antibody fragments include scFv (single chain fragment of variable region), diabody, triabody, tetrabody, etc.

[0111] Examples of lipids (lipid ligands) include, but are not limited to, tocopherol, cholesterol, fatty acids, phospholipids and their analogs; folic acid, vitamin C, vitamin B1, vitamin B2; estradiol, androstane and their analogs; steroids and their analogs; ligands of LDLR, SRBI, or LRP1 / 2; FK-506, and cyclosporine; and lipids described in PCT / JP2019 / 012077, PCT / JP2019 / 010392, and PCT / JP2020 / 035117. Furthermore, examples of lipids (lipid ligands) include tocopherol or its analogs and / or cholesterol or its analogs, substituted or unsubstituted C １ ~ ３０ an alkyl group of the formula ２ ~ ３０ or a substituted or unsubstituted alkenyl group of １ ~ ３０ It may also be an alkoxy group of the formula:

[0112] As used herein, "tocopherol" refers to a methylated derivative of tocorol, a fat-soluble vitamin (vitamin E) with a ring structure called chroman. Tocorol has strong antioxidant properties and therefore functions in vivo as an antioxidant to eliminate free radicals generated by metabolism and protect cells from damage.

[0113] Several different types of tocopherol are known, consisting of α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol, based on the position of the methyl group bound to the chroman. The tocopherol referred to herein may be any tocopherol. Examples of tocopherol analogs include various unsaturated analogs of tocopherol, such as α-tocotrienol, β-tocotrienol, γ-tocotrienol, and δ-tocotrienol. Preferably, the tocopherol is α-tocopherol.

[0114] As used herein, "cholesterol" refers to a type of sterol, also known as steroid alcohol, which is particularly abundant in animals. Cholesterol plays an important role in metabolic processes in vivo, and is also a major component of the cell membrane system in animal cells, along with phospholipids. Cholesterol analogs refer to various cholesterol metabolites and analogs, which are alcohols having a sterol skeleton, and include, but are not limited to, cholestanol, lanosterol, cerebrosterol, dehydrocholesterol, and coprostanol.

[0115] As used herein, the term "analog" refers to a compound having a similar structure and properties, which has the same or a similar basic skeleton. Analogs include, for example, biosynthetic intermediates, metabolic products, compounds with substituents, etc. Whether a compound is an analog of another compound can be determined by one skilled in the art based on common general technical knowledge.

[0116] Cholesterol analogs refer to various cholesterol metabolites and analogs, which are alcohols having a sterol skeleton, including, but not limited to, cholestanol, lanosterol, cerebrosterol, dehydrocholesterol, and coprostanol.

[0117] In one embodiment, the second nucleic acid strand may be bound to tocopherol, cholesterol, or an analog thereof. The second nucleic acid strand bound to cholesterol or an analog thereof may have a group represented by the following general formula (VII).

[0118] [In the formula, R c represents an alkylene group having 4 to 18 carbon atoms, preferably 5 to 16 carbon atoms, which may have a substituent (wherein the substituent is an alkyl group having 1 to 3 carbon atoms which may be substituted with a halogen atom or a hydroxy group, such as a hydroxymethyl group, and non-adjacent carbon atoms in the alkylene group may be substituted with an oxygen atom).

[0119] R c may be, but is not limited to, -(CH2)3-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-(CH2)2-, -(CH2)3-O-(CH2)2-O-(CH2)2-O-(CH2)2-O-CH2-CH(CH2OH)-, or -(CH2)6-.

[0120] The group represented by the above general formula (VII) can be bound to the 5'-end or 3'-end of the second nucleic acid strand via a phosphate ester bond.

[0121] The ligand such as cholesterol or an analog thereof may be bound to the 5'-end, 3'-end, or both ends of the second nucleic acid strand, or may be bound to an internal nucleotide of the second nucleic acid strand.

[0122] When the second nucleic acid strand contains multiple cholesterols or their analogs, they may be the same or different. For example, one cholesterol may be bound to the 5'-end of the second nucleic acid strand and one other cholesterol analog may be bound to the 3'-end. Regarding the binding positions, cholesterol or its analogs may be bound to multiple positions on the second nucleic acid strand and / or may be bound as a group to one position. One cholesterol or its analog may be linked to each of the 5'-end and 3'-end of the second nucleic acid strand.

[0123] The bond between the second nucleic acid strand and the ligand may be a direct bond or an indirect bond mediated by another substance.

[0124] When the second nucleic acid strand and the ligand are directly bound to each other, the ligand may be bound to the second nucleic acid strand via, for example, a covalent bond, an ionic bond, a hydrogen bond, etc. In view of the fact that a more stable bond can be obtained, a covalent bond is preferred.

[0125] In one embodiment, the second nucleic acid strand is not bound to a ligand. As used herein, "not bound to a ligand" refers to the absence of a ligand such as tocopherol or cholesterol. In a further embodiment, the double-stranded nucleic acid complex of the present invention is not bound to a ligand, i.e., neither the first nor the second nucleic acid strand is bound to a ligand.

[0126] When the second nucleic acid strand and the ligand are indirectly bound, they may be bound via a linking group (often referred to as a "linker" herein). The linker may be either a cleavable linker or an uncleavable linker.

[0127] "Cleavable linker" refers to a linker that can be cleaved under physiological conditions, for example, within a cell or an animal body (e.g., within a human body). Cleavable linkers are selectively cleaved by endogenous enzymes such as nucleases. Cleavable linkers include, but are not limited to, amides, esters, one or both phosphodiesters, phosphate esters, carbamates, and disulfide bonds, as well as natural DNA linkers. As an example, cholesterol or an analog thereof may be linked via a disulfide bond.

[0128] The term "non-cleavable linker" refers to a linker that is not cleaved under physiological conditions, for example, within a cell or an animal body (e.g., within the human body). Examples of non-cleavable linkers include, but are not limited to, linkers consisting of phosphorothioate bonds, and modified or unmodified deoxyribonucleosides or modified or unmodified ribonucleosides linked by phosphorothioate bonds. When the linker is a nucleic acid such as DNA or an oligonucleotide, the chain length is not particularly limited, but may typically be 2 to 20 bases, 3 to 10 bases, or 4 to 6 bases. A specific example of a linker is a linker represented by the following formula (VIII):

[0129] [In the formula, L 2 is a substituted or unsubstituted C1 to C 12 (e.g., propylene, hexylene, dodecylene), a substituted or unsubstituted C3 to C8 cycloalkylene group (e.g., cyclohexylene), —(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)3-, —(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)3-, or CH(CH2-OH)—CH2-O—(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)3-; L 3 represents -NH- or a bond, and L 4 is a substituted or unsubstituted C1 to C 12 alkylene groups (e.g., ethylene, pentylene, heptylene, undecylene), substituted or unsubstituted C3-8 cycloalkylene groups (e.g., cyclohexylene), -(CH2)2-[O-(CH2)2] m - or a bond, where m is an integer from 1 to 25; L 5 represents -NH-(C=O)-, -(C=O)-, or a bond (wherein the substitution is preferably by a halogen atom).

[0130] In one embodiment, the linker of formula (VIII) is L 2is an unsubstituted C3 to C6 alkylene group (e.g., propylene, hexylene), —(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)3-, or —(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)2-O—(CH2)3-, and L 3 is -NH-, and L 4 and L 5 is a bond.

[0131] Another specific example of the linker is a linker represented by the following general formula (IX):

[0132] [In the formula, n represents 0 or 1.]

[0133] The first nucleic acid strand and / or the second nucleic acid strand (preferably the second nucleic acid strand) may further comprise at least one functional moiety bound to a polynucleotide constituting the nucleic acid strand. The term "functional moiety" refers to a moiety that confers a desired function to the double-stranded nucleic acid complex and / or the nucleic acid strand to which the functional moiety is bound. Examples of the desired function include labeling and purification. Examples of moieties that confer labeling include compounds such as fluorescent proteins and luciferase. Examples of moieties that confer purification include compounds such as biotin, avidin, His-tag peptides, GST-tag peptides, and FLAG-tag peptides. In one embodiment, from the viewpoint of highly specific and efficient delivery of the first nucleic acid strand to a target site and highly effective suppression of target gene expression by the nucleic acid, it is preferred that the second nucleic acid strand be bound to a molecule that has the activity of delivering the double-stranded nucleic acid complex to a target site in certain embodiments. Examples of moieties that confer target delivery function include lipids, antibodies, aptamers, and ligands for specific receptors. In one embodiment, the first nucleic acid strand and / or the second nucleic acid strand (preferably the second nucleic acid strand) is bound to a functional moiety. The bond between the second nucleic acid strand and the functional moiety may be direct or indirect via another substance. In one embodiment, however, the second nucleic acid strand is preferably directly bound to the functional moiety via a covalent bond, an ionic bond, a hydrogen bond, or the like, and a covalent bond is more preferred from the viewpoint of obtaining a more stable bond.

[0134] The first and second nucleic acid strands may be linked via a linker. In this case, the first and second nucleic acid strands may be linked via the linker to form a single strand. In this case, the double-stranded nucleic acid complex may be called a hinge nucleic acid, single-stranded HDO, ssHDO, or the like. However, since the functional region in this case is the same as that in the double-stranded nucleic acid complex, this specification also encompasses such single-stranded nucleic acids as an embodiment of the double-stranded nucleic acid complex of the present invention.

[0135] In one embodiment, a linker connects the 5' end of a first nucleic acid strand to the 3' end of a second nucleic acid strand. In another embodiment, a linker connects the 3' end of a first nucleic acid strand to the 5' end of the second nucleic acid strand. In a further embodiment, a linker connects the 5' end of a first nucleic acid strand to the 3' end of a second nucleic acid strand and also connects the 3' end of a first nucleic acid strand to the 5' end of the second nucleic acid strand. In this case, the double-stranded nucleic acid complex has a circular structure.

[0136] The linker can be any polymer. For example, in addition to polynucleotides, polypeptides, alkylenes, etc., linkers that link the second nucleic acid strand and the ligand as described above can also be used. Specifically, the linker can be composed of natural nucleotides or nucleosides such as DNA or RNA, or unnatural nucleotides or nucleosides such as peptide nucleic acids or morpholino nucleic acids. It can also be composed of polyethers such as polyethylene glycol. When the linker is composed of a nucleic acid, the chain length of the linker can be at least one base, for example, 2 to 50 bases, 2 to 40 bases, 2 to 30 bases, 2 to 20 bases, 2 to 15 bases, 2 to 12 bases, 3 to 10 bases, or 4 to 6 bases. A chain length of 4 bases is preferred. The linker can be located on either the 5' or 3' end of the first nucleic acid strand. For example, in a configuration in which cholesterol or its analog is bound to the 5' end of the second nucleic acid strand, the 5' end of the first nucleic acid strand and the 3' end of the second nucleic acid strand are linked via the linker. The linker may be either cleavable or uncleavable.

[0137] In a further embodiment, the first nucleic acid strand and the second nucleic acid strand are linked via a linker, and the second nucleic acid strand can include at least one bulge structure consisting of a base sequence that is non-complementary to the first nucleic acid strand.

[0138] In the double-stranded nucleic acid complex of the present invention, the antisense effect of the first nucleic acid strand on a target transcript can be measured by methods known in the art. For example, after introducing the double-stranded nucleic acid complex into cells, measurement can be performed using known techniques such as Northern blotting, quantitative PCR, or Western blotting. By measuring the expression level of a target gene or the level of a target transcript (e.g., mRNA level or RNA level such as microRNA, cDNA level, protein level, etc.) in a specific tissue, it can be determined whether or not target gene expression is suppressed by the double-stranded nucleic acid complex at those sites. In addition, in the case of exon skipping, for example, the effect can be determined by comparing the product produced by exon skipping with the product produced without exon skipping.

[0139] As described above, exemplary embodiments of the double-stranded nucleic acid complex of the present invention have been described, but the double-stranded nucleic acid complex of the present invention is not limited to the above exemplary embodiments.

[0140] 1-4. Method for Producing Double-Stranded Nucleic Acid Complexes Those skilled in the art can produce the double-stranded nucleic acid complexes of the present invention by appropriately selecting known methods. While not limited thereto, this typically begins with the design and production of each of the first and second nucleic acid strands that make up the double-stranded nucleic acid complex. For example, the first nucleic acid strand is designed based on the base sequence information of the target transcription product (e.g., the base sequence of the target gene), and the second nucleic acid strand is designed as its complementary strand. Next, based on the designed base sequence information, each nucleic acid strand can be synthesized using a commercially available automated nucleic acid synthesizer, for example, from GE Healthcare, Thermo Fisher Scientific, Beckman Coulter, or the like. The resulting oligonucleotides can then be purified using a reverse-phase column or the like.

[0141] Furthermore, in the case of a double-stranded nucleic acid complex to which a functional moiety is bound, the first nucleic acid strand may be prepared according to the above-described method. Meanwhile, the second nucleic acid strand to which a functional moiety is bound can be prepared by carrying out the above-described synthesis and purification using a nucleic acid species to which a functional moiety has already been bound. For example, the second nucleic acid strand may be prepared by carrying out the above-described synthesis and purification using a nucleic acid species to which cholesterol or its analog has already been bound. Alternatively, cholesterol or its analog may be bound to the second nucleic acid strand prepared by carrying out the above-described synthesis and purification using a known method. After preparing each nucleic acid strand, the first and second nucleic acid strands are annealed to produce a double-stranded nucleic acid complex to which the desired functional moiety is bound. Specifically, nucleic acids are mixed in an appropriate buffer solution and denatured at approximately 90°C to 98°C for several minutes (e.g., 5 minutes), and then the nucleic acids are annealed at approximately 30°C to 70°C for approximately 1 to 8 hours to produce one of the double-stranded nucleic acid complexes of the present invention. Methods for linking functional moieties to nucleic acids are well known in the art. Alternatively, nucleic acid chains can be ordered and obtained from various manufacturers (for example, Gene Design Inc.) by specifying the base sequence and the site and type of modification.

[0142] 1-5. Uses of Double-Stranded Nucleic Acid Complexes In one embodiment, the double-stranded nucleic acid complexes of the present invention may be used for at least one of the following purposes: suppressing or enhancing the expression level of a transcription product or translation product of a target gene, inhibiting the function of a transcription product or translation product of a target gene, regulating RNA splicing, and inhibiting the binding of a target gene to a protein, for example, for exon skipping. For example, the double-stranded nucleic acid complexes of the present invention may be used for at least one of the following purposes: exon skipping, exon inclusion, steric blocking, and enhanced RNA expression. The double-stranded nucleic acid complexes of the present invention may be used to exert the above-mentioned effects in specific tissues, such as the brain, spinal cord, kidney, liver, lung, intestinal tract, spleen, adrenal gland, eye, retina, skin, or peripheral nerves, including the brain. The brain may be any of the cerebrum, diencephalon, brainstem, and cerebellum, and may be, for example, one or more of the cerebrum (such as the cerebral cortex), brainstem, cerebellum, hippocampus, and striatum. The double-stranded nucleic acid complex of the present invention may also be one that exerts the above-mentioned effects in muscle tissues, including cardiac and skeletal muscles.

[0143] In one embodiment, the double-stranded nucleic acid complex of the present invention is used to treat or prevent a disease. The disease may be skeletal muscle dysfunction or cardiac dysfunction. Examples of diseases include muscular dystrophies (Duchenne muscular dystrophy, myotonic dystrophy type 1 (DM1), Fukuyama muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, etc.), congenital myopathy, primary age-related tauopathy (PART), Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration / corticobasal syndrome (CBD), Pick's disease, frontotemporal dementia, neuronal inclusion body disease, spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, hereditary spinocerebellar degeneration (SCA), multiple system atrophy, hereditary spastic paraplegia, multiple sclerosis, cerebral infarction, brain tumor, epilepsy, and encephalitis.

[0144] 1-6. Effects The double-stranded nucleic acid complex of the present invention can reduce or eliminate the toxicity, such as central nervous system toxicity, of the double-stranded nucleic acid complex without impairing the efficacy of the double-stranded nucleic acid complex. In other words, compared to conventional double-stranded nucleic acid complexes, central nervous system toxicity is reduced without impairing the antisense effect on the target gene.

[0145] 2. Pharmaceutical Composition 2-1. Overview A second aspect of the present invention is a pharmaceutical composition. The pharmaceutical composition of the present invention contains the double-stranded nucleic acid complex of the first aspect as an active ingredient. The pharmaceutical composition of the present invention has reduced central neurotoxicity and can be administered intrathecally or intracerebroventricularly without causing side effects. Each component that may be contained in the pharmaceutical composition of the present invention will be specifically described below.

[0146] 2-2. Composition 2-2-1. Active ingredient The pharmaceutical composition of the present invention contains, as an active ingredient, at least the double-stranded nucleic acid complex described in aspect 1. The pharmaceutical composition of the present invention may contain two or more types of double-stranded nucleic acid complexes.

[0147] The amount (content) of the double-stranded nucleic acid complex contained in a pharmaceutical composition varies depending on the type of double-stranded nucleic acid complex, the delivery site, the dosage form of the pharmaceutical composition, the dosage of the pharmaceutical composition, and the type of carrier (described below). Therefore, it can be determined appropriately taking into account each condition. Typically, a single dose of the pharmaceutical composition is adjusted to contain an effective amount of the double-stranded nucleic acid complex. The term "effective amount" refers to the amount of the double-stranded nucleic acid complex necessary to exert its function as an active ingredient and to cause little or no harmful side effects in the living body to which it is applied. This effective amount may vary depending on various conditions, such as information about the subject, the route of administration, and the number of administrations. Ultimately, it is determined by the judgment of a physician, veterinarian, or pharmacist. "Subject information" refers to various individual information about the living body to which the pharmaceutical composition is applied. For example, if the subject is a human, this information includes age, weight, sex, diet, health condition, disease progression and severity, drug sensitivity, and the presence or absence of concomitant medications.

[0148] 2-2-2. Carrier The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to an additive commonly used in the pharmaceutical technology field. Examples include solvents, vegetable oils, bases, emulsifiers, suspending agents, surfactants, pH adjusters, stabilizers, flavors, fragrances, excipients, vehicles, preservatives, binders, diluents, isotonicity agents, sedatives, bulking agents, disintegrants, buffers, coating agents, lubricants, colorants, sweeteners, thickeners, flavoring agents, solubilizers, and other additives.

[0149] The solvent may be, for example, water or any other pharmaceutically acceptable aqueous solution, or a pharmaceutically acceptable organic solvent. Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffer, and sodium acetate buffer. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc.

[0150] The above-mentioned carriers are used to avoid or suppress the decomposition of the double-stranded nucleic acid complex, which is the active ingredient, by enzymes and the like in the body, as well as to facilitate formulation and administration methods and maintain the dosage form and medicinal efficacy, and may be used appropriately as needed.

[0151] The dosage form of the pharmaceutical composition of the present invention is not particularly limited as long as it is a form that can deliver the double-stranded nucleic acid complex described in the first aspect, which is the active ingredient, to a target site without inactivating it through degradation or the like, and can exert the pharmacological effect of the active ingredient in vivo (antisense effect on the expression of a target gene).

[0152] The specific dosage form varies depending on the administration method and / or formulation conditions. The administration method can be roughly divided into parenteral administration and oral administration, and therefore, a dosage form suitable for each administration method may be used.

[0153] If the administration method is parenteral administration, the preferred dosage form is a liquid that can be administered directly to the target site or systemically via the circulatory system. Examples of liquids include injections. Injections can be formulated by appropriately combining the above-mentioned excipients, elixirs, emulsifiers, suspending agents, surfactants, stabilizers, pH adjusters, etc., and mixing them in a unit dosage form required for generally accepted pharmaceutical practice. Other forms include ointments, plasters, cataplasms, transdermal agents, lotions, inhalants, aerosols, eye drops, and suppositories.

[0154] If the administration method is oral administration, preferred dosage forms may be solid or liquid, such as tablets, capsules, drops, troches, pills, granules, powders, powders, oral solutions, emulsions, syrups, pellets, sublingual tablets, peptizers, buccal tablets, pastes, suspensions, elixirs, coatings, ointments, plasters, cataplasms, transdermal preparations, lotions, inhalants, aerosols, eye drops, injections, and suppositories. If the solid formulation is used, it may be made into a dosage form coated with a coating known in the art, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets, or multi-layer tablets, as needed.

[0155] The specific shape and size of each of the above dosage forms are not particularly limited as long as they are within the range of dosage forms known in the art. The pharmaceutical composition of the present invention may be formulated according to a conventional method in the art.

[0156] In certain embodiments, the double-stranded nucleic acid complex of the present invention has excellent properties as a pharmaceutical, such as excellent solubility in water, Japanese Pharmacopoeia Dissolution Test Fluid 2, or Japanese Pharmacopoeia Disintegration Test Fluid 2, excellent pharmacokinetics (e.g., drug half-life in blood, brain transferability, metabolic stability, CYP inhibition), low toxicity (e.g., superior as a pharmaceutical in terms of acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, drug interactions, carcinogenicity, phototoxicity, etc.), and few side effects (e.g., suppression of excessive sedation, avoidance of lamellar necrosis).

[0157] 2-3. Dosage Form and Dose As used herein, there are no particular limitations on the preferred administration form of the pharmaceutical composition. For example, oral administration or parenteral administration may be used. Specific examples of parenteral administration include intramuscular administration, intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration (including implantable continuous subcutaneous administration), intradermal administration, tracheal / bronchial administration, rectal administration, administration by transfusion, intraventricular administration, intrathecal administration, intranasal administration, and intramuscular administration. Intrathecal administration may be, for example, posterior fossa puncture or lumbar puncture.

[0158] When the pharmaceutical composition is administered or ingested, the dosage or intake may be, for example, such that the amount of the double-stranded nucleic acid complex contained therein is 0.00001 mg / kg / day to 10,000 mg / kg / day, or 0.001 mg / kg / day to 100 mg / kg / day. The pharmaceutical composition may be administered in a single dose or multiple doses. In the case of multiple doses, the composition may be administered daily or at appropriate intervals (e.g., at intervals of 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month), for example, 2 to 20 times. The single dose of the double-stranded nucleic acid complex may be, for example, 0.001 mg / kg or more, 0.005 mg / kg or more, 0.01 mg / kg or more, 0.25 mg / kg or more, 0.5 mg / kg or more, 1 mg / kg or more, 2.5 mg / kg or more, 0.5 mg / kg or more, 1.0 mg / kg or more, 2.0 mg / kg or more, 3.0 mg / kg or more, 4.0 mg / kg or more, 5 mg / kg or more, 10 mg / kg or more, 20 mg / kg or more, 30 mg / kg or more, 40 mg / kg or more, 50 mg / kg or more, 75 mg / kg or more, 80 mg / kg or more, 90 mg / kg or more, 100 mg / kg or more, 120 mg / kg or more, 140 mg / kg or more, 150 mg / kg or more, 160 mg / kg or more, 170 mg / kg or more, 180 mg / kg or more, 190 mg / kg or more, 210 mg / kg or more, 220 mg / kg or more, 230 mg / kg or more, 240 mg / kg or more, 250 mg / kg or more, 260 mg / kg or more, 270 mg / kg or more, 280 mg / kg or more, 290 mg / kg or more, 300 mg / kg or more, 310 mg / kg or more, 320 mg / kg or more, 330 mg / kg or more, 340 mg / kg or more, 350 mg / kg or more, 360 mg / kg or more, 370 mg / kg or more, 380 mg / kg or more, 390 mg / kg or more, 400 mg / kg or more, 410 mg / kg or The dose can be 100 mg / kg or more, 150 mg / kg or more, 200 mg / kg or more, 300 mg / kg or more, 400 mg / kg or more, or 500 mg / kg or more, and can be appropriately selected from any amount within the range of, for example, 0.001 mg / kg to 500 mg / kg (e.g., 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg, 100 mg / kg, or 200 mg / kg).

[0159] The double-stranded nucleic acid complex of the present invention may be administered twice a week for four doses at a dose of 0.01 to 10 mg / kg (e.g., about 6.25 mg / kg). Alternatively, the double-stranded nucleic acid complex may be administered once or twice a week for two to four doses at a dose of 0.05 to 30 mg / kg (e.g., about 25 mg / kg), for example, twice a week for two doses. The use of such a dosing regimen (divided administration) can reduce toxicity (e.g., avoid platelet reduction) and the burden on the subject compared to a single administration of a higher dose.

[0160] The pharmaceutical composition exerts an additive inhibitory effect within cells even when administered repeatedly. Furthermore, when administering repeatedly, the efficacy can be improved by leaving a certain interval between administrations (e.g., half a day or more).

[0161] In one embodiment, the pharmaceutical composition of this aspect is administered intracerebroventricularly or intrathecally. When the pharmaceutical composition of this aspect is administered intracerebroventricularly or intrathecally, the amount administered to monkeys or humans may be 0.01 mg or more, 0.1 mg or more, or 1 mg or more, for example, 2 mg or more, 3 mg or more, 4 mg or more, 5 mg or more, 10 mg or more, 20 mg or more, 30 mg or more, 40 mg or more, 50 mg or more, 75 mg or more, 100 mg or more, 200 mg or more, 300 mg or more, 400 mg or more, or 500 mg or more, or 0.01 mg to 1000 mg, 0.1 mg to 200 mg, or 1 mg to 20 mg, and in the case of mice, 1 μg or more may be administered.

[0162] In one embodiment, the pharmaceutical composition of this aspect is administered intravenously or subcutaneously. When administered intravenously or subcutaneously, the pharmaceutical composition of this aspect may be administered at a dose of 0.01 mg / kg or more, 0.1 mg / kg or more, or 1 mg / kg or more, for example, 2 mg / kg or more, 3 mg / kg or more, 4 mg / kg or more, 5 mg / kg or more, 10 mg / kg or more, 20 mg / kg or more, 30 mg / kg or more, 40 mg / kg or more, 50 mg / kg or more, 75 mg / kg or more, 100 mg / kg or more, 150 mg / kg or more, 200 mg / kg or more, 300 mg / kg or more, 400 mg / kg or more, or 500 mg / kg or more, or at a dose of 0.01 mg / kg to 1000 mg / kg, 0.1 mg / kg to 100 mg / kg, or 1 mg / kg to 10 mg / kg.

[0163] 2-4. Target Diseases There are no limitations on the diseases to which the pharmaceutical composition can be applied. The target diseases include those that may be associated with genes whose transcription or translation products may be suppressed or enhanced in expression, whose transcription or translation products may have their functions inhibited, or whose steric blocking, splicing switch, RNA editing, exon skipping, or exon inclusion may be induced by the antisense effect of the double-stranded nucleic acid complex of the present invention. Specific examples of diseases are as described in "1-5. Uses of the double-stranded nucleic acid complex."

[0164] In one embodiment, the pharmaceutical composition of this aspect can be used to treat a central nervous system disease in a subject. Central nervous system diseases to which the pharmaceutical composition of this aspect can be applied include, but are not limited to, brain tumors, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, etc.

[0165] 2-5. Effects The pharmaceutical composition of the present invention has reduced central nervous system toxicity. Therefore, the pharmaceutical composition of the present invention can achieve preventive or therapeutic effects without side effects when administered intracerebroventricularly or intrathecally.

[0166] In particular, the treatment of neurological diseases such as Alzheimer's disease requires the administration of high doses of nucleic acid agents, which carries the risk of side effects, but the pharmaceutical composition of the present invention can significantly reduce such side effects.

[0167] There is also provided a method for treating and / or preventing a disease such as a central nervous system disease, which comprises administering the above-described double-stranded nucleic acid complex or pharmaceutical composition to a subject.

[0168] Also provided is the use of the double-stranded nucleic acid complex of the present invention in the manufacture of a medicament for treating and / or preventing a disease.

[0169] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0170] Example 1 MOE Modification of HDO Targeting the Mapt Gene (Objective) To verify the toxicity-reducing effect of MOE modification on central nervous system toxicity observed when a heteroduplex oligonucleotide (hereinafter referred to as "HDO") containing a first nucleic acid strand consisting of an antisense nucleic acid (hereinafter referred to as "ASO") targeting the Mapt gene and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand is intracerebroventricularly administered through in vivo experiments.

[0171] (Method) (1) Preparation of Nucleic Acids The base sequences and chemical modifications of the ASO and the first and second nucleic acid strands constituting the HDO used in this example are shown in Table 1 and Figure 4.

[0172]

[0173] The ASO used in this example is an LNA / DNA gapmer antisense nucleic acid targeting mouse microtubule-associated protein tau (Mapt) mRNA. It has a base sequence complementary to a portion of Mapt mRNA, with three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and ten DNA nucleosides between them linked by phosphorothioate bonds. The HDO(all RNA), HDO(all DNA), HDO(6MOE wing), and HDO(all MOE) used in this example all contain the above-mentioned ASO as the first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand (c(all RNA)) of HDO(all RNA) has a structure in which RNA nucleosides are linked by phosphodiester bonds. The second nucleic acid strand of HDO(all DNA) (c(all DNA)) has a structure in which DNA nucleosides are linked by phosphodiester bonds. The second nucleic acid strand of HDO(6MOE wing) (c(6MOE wing)) has a structure in which three 2'-O-MOE-RNA nucleosides at the 5' end, three 2'-O-MOE-RNA nucleosides at the 3' end, and 10 DNA nucleosides between them are linked by phosphodiester bonds. The second nucleic acid strand of HDO(all MOE) (c(all MOE)) has a structure in which 2'-O-MOE-RNA nucleosides are linked by phosphodiester bonds.

[0174] The 2'-O-MOE-RNA nucleoside used in the examples of the present specification is represented by the following formula (III): It is an unnatural nucleoside represented by the formula:

[0175] To prepare the double-stranded nucleic acid complexes listed in Table 1, the first and second nucleic acid strands were mixed in equimolar amounts, and the solution was heated to 95°C for 5 minutes, then cooled to 37°C and maintained for 1 hour, thereby annealing the nucleic acid strands to prepare double-stranded nucleic acid complexes. The annealed nucleic acids were stored at 4°C or on ice. All oligonucleotides were custom synthesized by Gene Design Co., Ltd. (Osaka, Japan).

[0176] (2) In vivo experiments. Seven-week-old female ICR mice were anesthetized with 2.5-4% isoflurane and placed in a stereotaxic apparatus. A 2-3 cm incision was made in the skin between the ears, and a 1 mm drill hole was created 1 mm left and 0.2 mm posterior to the bregma. A Hamilton syringe was filled with the nucleic acid agent. The needle was inserted approximately 3 mm into the hole, and the nucleic acid agent was administered intraventricularly into the left lateral ventricle at a dose of 19 nmol / mouse at a rate of 2-3 μl / min (n = 4-7). The skin was then sutured with nylon thread. A negative control group of mice was also administered PBS alone.

[0177] (3) Evaluation of central neurotoxicity and motor function after administration of nucleic acid agents. After administration of various nucleic acid agents, mice were evaluated for central neurotoxicity and motor function. To evaluate central neurotoxicity, behavioral assessment was performed 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours after administration of the nucleic acid agent using the scoring system shown in Figure 3.

[0178] The scoring system shown in Figure 3 evaluates behaviors belonging to five categories (Figure 3, categories 1 to 5). Each category includes two behavioral evaluation items. Each behavioral evaluation item is scored on a five-point scale from 0 to 5 (Figure 3, scores 0 to 5), with normal being scored as 0 and higher scores indicating higher toxicity. For each category, the higher score of the two behavioral evaluation items is used as the score for that category. The sum of the scores for the five categories represents the acute phase tolerability score (0 to 20 points).

[0179] To assess motor function, an open field test was performed at each time point after administration of each nucleic acid agent. Specifically, mice were placed in the center of a cage (50 cm wide x 50 cm diameter x 40 cm high), and their trajectories were recorded for 5 minutes. Based on the recorded data, total distance traveled (m) and maximum movement speed (m / s) were measured using video tracking software (ANY-maze). Statistical significance between treatment groups was assessed using the Bonferroni test.

[0180] (4) Evaluation of gene silencing effects. The hippocampus was excised from the mice 7 days after administration of various nucleic acid agents. RNA was extracted from the excised left hippocampus using the Isogen I kit (Gene Design Inc.). cDNA was synthesized using Transcriptor Universal cDNA Master, DNase (Roche Diagnostics) according to the protocol.

[0181] Next, quantitative RT-PCR was performed using the resulting cDNA as a template to measure the expression levels of Mapt mRNA and Actb mRNA (internal control genes). Quantitative RT-PCR was performed using TaqMan (Roche Applied Science). Primers used in quantitative RT-PCR were designed and manufactured by Thermo Fisher Scientific (formerly Life Technologies Corp.). The amplification conditions (temperature and time) were as follows: 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 1 second (1 cycle), repeated 40 times.

[0182] The ratio of the expression level of Mapt mRNA to the expression level of Actb mRNA (internal standard gene) was calculated, and the value normalized to the value of the PBS-administered group was used as the relative Mapt mRNA level.

[0183] (Results) Figure 5 shows the results of evaluating central neurotoxicity in mice after intracerebroventricular administration of various nucleic acid agents. At 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours after administration, the HDO (6MOE wing) and HDO (all MOE) groups showed significantly reduced acute tolerability scores compared to the ASO, HDO (all RNA), and HDO (all DNA) groups. These results demonstrate that HDO containing 2'-O-MOE-RNA nucleosides significantly reduces central neurotoxicity.

[0184] Figure 6 shows the results of assessing motor function 1 hour after intracerebroventricular administration of various nucleic acid agents in mice. The HDO (6MOE wing) and HDO (all MOE) groups showed significantly improved total distance traveled (Fig. 6A) and maximum speed (Fig. 6B) compared with the ASO, HDO (all RNA), and HDO (all DNA) groups. These results indicate that HDO containing 2'-O-MOE-RNA nucleosides has minimal inhibitory effect on motor function and is extremely low in toxicity.

[0185] Figure 7 shows the expression level of Mapt mRNA in the hippocampus 7 days after intracerebroventricular administration of various nucleic acid agents. The HDO (all MOE) group showed a reduced gene silencing effect compared to the ASO, HDO (all RNA), and HDO (all DNA) groups. On the other hand, the HDO (6MOE wing) group showed a gene silencing effect equivalent to that of the ASO, HDO (all RNA), and HDO (all DNA) groups. These results demonstrate that HDO (6MOE wing) can achieve a high gene silencing effect without substantial central nervous system toxicity.

[0186] <Example 2: MOE modification of HDO targeting the BACE1 gene> (Purpose) To verify the toxicity reduction effect of MOE modification on the central nervous system toxicity observed when HDO containing a first nucleic acid strand consisting of an ASO targeting the BACE1 gene and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand is administered intracerebroventricularly through in vivo experiments.

[0187] (Method) The base sequences and chemical modifications of the first and second nucleic acid strands constituting the ASO and HDO used in this example are shown in Table 2 and FIG.

[0188]

[0189] The ASO used in this example is an LNA / DNA gapmer antisense nucleic acid targeting mouse beta-secretase 1 (BACE1) mRNA. It has a base sequence complementary to a portion of BACE1 mRNA, with two LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and eight DNA nucleosides between them linked by phosphorothioate bonds. The HDO (all RNA), HDO (all DNA), and HDO (5MOE wing) used in this example all contain the above-mentioned ASO as the first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand (c(all RNA)) of HDO (all RNA) has a structure in which RNA nucleosides are linked by phosphodiester bonds. The second nucleic acid strand of HDO(all DNA) (c(all DNA)) has a structure in which DNA nucleosides are linked by phosphodiester bonds. The second nucleic acid strand of HDO(5MOE wing) (c(5MOE wing)) has a structure in which three 2'-O-MOE-RNA nucleosides at the 5' end, two 2'-O-MOE-RNA nucleosides at the 3' end, and eight DNA nucleosides between them are linked by phosphodiester bonds.

[0190] For the ASO, HDO (all RNA), HDO (all DNA), and HDO (5 MOE wing) listed in Table 2, nucleic acid preparation, in vivo experiments, central neurotoxicity evaluation, and motor function evaluation were performed in the same manner as in Example 1. However, in this example, the dose of the nucleic acid agent administered per mouse was 11.5 nmol / mouse.

[0191] (Results) Figure 9 shows the results of evaluating central neurotoxicity in mice administered various nucleic acid agents intracerebroventricularly. At 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours after administration, the HDO (5MOE wing) administration group showed significantly reduced acute tolerability scores compared to the ASO, HDO (all RNA), and HDO (all DNA) administration groups. These results demonstrate that HDO containing 2'-O-MOE-RNA nucleosides significantly reduces central neurotoxicity.

[0192] Figure 10 shows the results of assessing motor function 1 hour after intracerebroventricular administration of various nucleic acid agents in mice. The HDO (5MOE wing) group showed significantly improved total distance traveled (Figure 10A) and maximum speed (Figure 10B) compared with the ASO, HDO (all RNA), and HDO (all DNA) groups. These results indicate that HDO containing 2'-O-MOE-RNA nucleosides has minimal inhibitory effect on motor function and is extremely low in toxicity.

[0193] <Example 3: MOE modification of HDO targeting the Malat1 gene> (Purpose) To verify the toxicity reduction effect of MOE modification on the central nervous system toxicity observed when HDO containing a first nucleic acid strand consisting of an ASO targeting the Malat1 gene and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand is administered intracerebroventricularly through in vivo experiments.

[0194] (Method) The base sequences and chemical modifications of the ASO and the first and second nucleic acid strands constituting the HDO used in this example are shown in Table 3 and FIG.

[0195]

[0196] The ASO used in this example is a 2'-O-MOE-RNA / DNA gapmer antisense nucleic acid targeting the mouse metastasis-associated lung adenocarcinoma transcript 1 (Malat1) non-coding RNA. It has a base sequence complementary to a portion of Malat1 ncRNA, with five 2'-O-MOE-RNA nucleosides at the 5' end, five 2'-O-MOE-RNA nucleosides at the 3' end, and 10 DNA nucleosides between them linked by phosphorothioate bonds. The HDO(all RNA), HDO(all DNA), and HDO(10MOE wing) used in this example all contain the above ASO as the first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand (c(all RNA)) of HDO(all RNA) has a structure in which RNA nucleosides are linked by phosphodiester bonds. The second nucleic acid strand of HDO(all DNA) (c(all DNA)) has a structure in which DNA nucleosides are linked by phosphodiester bonds. The second nucleic acid strand of HDO(10MOE wing) (c(10MOE wing)) has a structure in which five 2'-O-MOE-RNA nucleosides at the 5' end, five 2'-O-MOE-RNA nucleosides at the 3' end, and 10 DNA nucleosides between them are linked by phosphodiester bonds.

[0197] For the ASO, HDO (all RNA), HDO (all DNA), and HDO (10 MOE wing) listed in Table 3, nucleic acid preparation, in vivo experiments, central neurotoxicity evaluation, and motor function evaluation were performed in the same manner as in Example 1. However, in this example, the dose of nucleic acid agent administered per mouse was 13.86 nmol / mouse.

[0198] (Results) Figure 12 shows the results of evaluating central neurotoxicity in mice administered various nucleic acid agents intracerebroventricularly. At 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours after administration, the HDO (5MOE wing) group showed significantly reduced acute-phase tolerance scores compared to the ASO and HDO (all DNA) groups. Furthermore, at 30 minutes, 1 hour, and 2 hours after administration, the HDO (5MOE wing) group showed lower acute-phase tolerance scores compared to the HDO (all RNA) group. These results demonstrate that HDO containing 2'-O-MOE-RNA nucleosides significantly reduces central neurotoxicity.

[0199] Figure 13 shows the results of assessing motor function 1 hour after intracerebroventricular administration of various nucleic acid agents in mice. The HDO (10MOE wing) group showed significantly improved total distance traveled (Figure 13A) and maximum speed (Figure 13B) compared with the ASO, HDO (all RNA), and HDO (all DNA) groups. These results indicate that HDO containing 2'-O-MOE-RNA nucleosides has minimal inhibitory effect on motor function and is extremely low in toxicity.

[0200] Example 4: Comparison of MOE modification, 2'OMe modification, and 2'F modification (Objective) To compare the toxicity-reducing effects of MOE modification, 2'OMe modification, and 2'F modification on central nervous system toxicity observed when HDO containing a first nucleic acid strand consisting of an ASO targeting the Mapt gene and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand is administered intracerebroventricularly through in vivo experiments.

[0201] (Method) The base sequences and chemical modifications of the ASO and the first and second nucleic acid strands constituting the HDO used in this example are shown in Table 4 and FIG.

[0202]

[0203] The ASO used in this example is an LNA / DNA gapmer antisense nucleic acid targeting Mapt mRNA, and has a base sequence complementary to a portion of Mapt mRNA. It has a structure in which three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and ten DNA nucleosides between them are linked by phosphorothioate bonds. The HDO(all DNA), HDO(RNA 6MOE wing), HDO(6MOE wing), HDO(6OMe wing), and HDO(6F wing) used in this example all contain the above-mentioned ASO as a first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand (c(all DNA)) of HDO(all DNA) has a structure in which DNA nucleosides are linked by phosphodiester bonds. The second nucleic acid strand (c(RNA 6MOE wing)) of HDO(RNA 6MOE wing) has a structure in which three 2'-O-MOE-RNA nucleosides at the 5'-terminus, three 2'-O-MOE-RNA nucleosides at the 3'-terminus, and ten RNA nucleosides between them are linked by phosphodiester bonds. The second nucleic acid strand (c(6MOE wing)) of HDO(6MOE wing) has a structure in which three 2'-O-MOE-RNA nucleosides at the 5'-terminus, three 2'-O-MOE-RNA nucleosides at the 3'-terminus, and ten DNA nucleosides between them are linked by phosphodiester bonds. The second nucleic acid strand (c(6OMe wing)) of HDO(6OMe wing) has a structure in which three 2'-O-Me-RNA nucleosides at the 5'-terminus, three 2'-O-Me-RNA nucleosides at the 3'-terminus, and ten DNA nucleosides between them are linked by phosphodiester bonds. The second nucleic acid strand (c(6F wing)) of HDO(6F wing) has a structure in which three 2'F-RNA nucleosides at the 5'-terminus, three 2'F-RNA nucleosides at the 3'-terminus, and ten DNA nucleosides between them are linked by phosphodiester bonds.

[0204] For HDO (RNA 6MOE wing), HDO (6MOE wing), HDO (6OMe wing), and HDO (6F wing) listed in Table 4, nucleic acid preparation, in vivo experiments, central neurotoxicity evaluation, and motor function evaluation were performed in the same manner as in Example 1. However, in this example, the dose of the nucleic acid agent administered per mouse was 19 nmol / mouse.

[0205] (Results) Figure 15 shows the results of evaluating central neurotoxicity in mice administered various nucleic acid agents intracerebroventricularly. At 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours after administration, the HDO (RNA 6MOE wing) and HDO (6MOE wing) groups showed significantly reduced acute tolerability scores compared to the HDO (all DNA), HDO (6OMe wing), and HDO (6F wing) groups. Furthermore, the HDO (6MOE wing) group showed a lower acute tolerability score compared to the HDO (RNA 6MOE wing) group. These results demonstrate that HDO containing 2'-O-MOE-RNA nucleosides can achieve lower central neurotoxicity than HDO containing 2'-O-Me-RNA or 2'F-RNA nucleosides.

[0206] Figures 16 and 17 show the results of assessing motor function in mice intracerebroventricularly administered with various nucleic acid agents 1 and 3 hours after administration. The HDO (RNA 6MOE wing) and HDO (6MOE wing) groups showed significantly improved total distance traveled (Figures 16A and 17A) and maximum speed (Figures 16B and 17B) compared with the HDO (all DNA), HDO (6OMe wing), and HDO (6F wing) groups. These results indicate that HDO containing 2'-O-MOE-RNA nucleosides has significantly less inhibitory effect on motor function and is significantly less toxic than HDO containing 2'-O-Me-RNA or 2'F-RNA nucleosides.

[0207] Example 5: Substitution of guanosine nucleoside (Objective) The central nervous system toxicity observed when HDO containing a first nucleic acid strand consisting of an ASO targeting the Mapt gene and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand is intracerebroventricularly administered is examined through in vivo experiments to determine the toxicity-reducing effect of substitution of guanosine nucleoside in the second nucleic acid strand.

[0208] (Method) The base sequences and chemical modifications of the ASO and the first and second nucleic acid strands constituting the HDO used in this example are shown in Table 5 and FIG.

[0209]

[0210] The ASO used in this example is an LNA / DNA gapmer antisense nucleic acid that targets Mapt mRNA and has a base sequence complementary to a portion of Mapt mRNA. It has a structure in which three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and ten DNA nucleosides between them are linked by phosphorothioate bonds. The ASOs used in this example were HDO (6MOE wing), HDO (G MOE ), HDO(G RNA Both HDO(6MOE wing) and HDO(inosine) contain the above-mentioned ASO as the first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand (c(6MOE wing)) of HDO(6MOE wing) has a structure in which three 2'-O-MOE-RNA nucleosides at the 5' end, three 2'-O-MOE-RNA nucleosides at the 3' end, and 10 DNA nucleosides between them are linked by phosphodiester bonds. HDO(G MOE ) of the second nucleic acid strand (c(G MOE )) has a c(6MOE wing) in which the DNA nucleoside containing the guanine base is replaced by a 2'-O-MOE-RNA nucleoside. RNA ) of the second nucleic acid strand (c(G RNAIn the c(6MOE wing) DNA nucleoside containing a guanine base is replaced by an RNA nucleoside. In the HDO(inosine) second strand (c(inosine)), the guanine base in the c(6MOE wing) DNA nucleoside is replaced by an inosine base.

[0211] ASO, HDO(6MOE wing), HDO(G MOE ), HDO(G RNA For HCl (H2O3), HCl (H2O4), and HDO (inosine), nucleic acid preparation, in vivo experiments, and evaluation of central neurotoxicity and motor function were performed in the same manner as in Example 1. However, in this example, the dose of the nucleic acid agent administered per mouse was 18.86 nmol / mouse.

[0212] (Results) Figure 19 shows the results of evaluating central neurotoxicity in mice after intracerebroventricular administration of various nucleic acid agents. HDO(G MOE ) and HDO(G RNA ) group showed acute tolerability scores that were equivalent to or lower than those of the HDO (6MOE wing) group.

[0213] Example 6: Various MOE modifications in HDO targeting the Mapt gene (Objective) To verify the toxicity-reducing effects of various MOE modifications on central nervous system toxicity observed when HDO containing a first nucleic acid strand consisting of an ASO targeting the Mapt gene and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand is intracerebroventricularly administered, through in vivo experiments.

[0214] (Method) The base sequences and chemical modifications of the first and second nucleic acid strands constituting the HDO used in this example are shown in Table 6 and FIG.

[0215]

[0216] The ASO used in this example is an LNA / DNA gapmer antisense nucleic acid targeting Mapt mRNA. It has a base sequence complementary to a portion of Mapt mRNA, with three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and ten DNA nucleosides between them linked by phosphorothioate bonds. The HDO(all DNA), HDO(6MOE-5'&3'), HDO(6MOE-5'), HDO(6MOE-3'), HDO(10MOE-5'), and HDO(10MOE-3') used in this example all contain the above ASO as the first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand (c(all DNA)) of HDO(all DNA) has a structure in which DNA nucleosides are linked by phosphodiester bonds. The second nucleic acid strand of HDO(6MOE-5'&3') (c(6MOE-5'&3')) has a structure in which three 2'-O-MOE-RNA nucleosides at the 5' end, three 2'-O-MOE-RNA nucleosides at the 3' end, and ten DNA nucleosides between them are linked by phosphodiester bonds. The second nucleic acid strand of HDO(6MOE-5') (c(6MOE-5')) has a structure in which six 2'-O-MOE-RNA nucleosides at the 5' end and ten DNA nucleosides at the 3' end are linked by phosphodiester bonds. The second nucleic acid strand of HDO(6MOE-3') (c(6MOE-3')) has a structure in which ten DNA nucleosides at the 5' end and six 2'-O-MOE-RNA nucleosides at the 3' end are linked by phosphodiester bonds. The second nucleic acid strand (c(10MOE-5')) of HDO(10MOE-5') has a structure in which ten 2'-O-MOE-RNA nucleosides at the 5' end and six DNA nucleosides at the 3' end are linked by phosphodiester bonds. The second nucleic acid strand (c(10MOE-3')) of HDO(10MOE-3') has a structure in which six DNA nucleosides at the 5' end and ten 2'-O-MOE-RNA nucleosides at the 3' end are linked by phosphodiester bonds.

[0217] For HDO (all DNA), HDO (6MOE-5'&3'), HDO (6MOE-5'), HDO (6MOE-3'), HDO (10MOE-5'), and HDO (10MOE-3') listed in Table 6, nucleic acid preparation, in vivo experiments, central neurotoxicity evaluation, motor function evaluation, and gene suppression effect evaluation were performed in the same manner as in Example 1. However, in this example, the dose of the nucleic acid agent administered per mouse was 18.86 nmol / mouse.

[0218] (Results) Figure 21 shows the results of evaluating central neurotoxicity in mice administered various nucleic acid agents intracerebroventricularly. At 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours after administration of the nucleic acid agent, the acute phase tolerance scores were significantly reduced in the HDO(6MOE-5'&3'), HDO(6MOE-5'), HDO(6MOE-3'), HDO(10MOE-5'), and HDO(10MOE-3') groups compared to the HDO(all DNA) group. In particular, the acute phase tolerance scores in the HDO(10MOE-5') and HDO(10MOE-3') groups were the lowest.

[0219] Figures 22 and 23 show the results of assessing motor function in mice intracerebroventricularly administered with various nucleic acid agents 1 and 3 hours after administration. The HDO(6MOE-5'&3'), HDO(6MOE-5'), HDO(6MOE-3'), HDO(10MOE-5'), and HDO(10MOE-3') groups showed significant improvements in total distance traveled (Figures 22A and 23A) and maximum speed (Figures 22B and 23B) compared with the HDO(all DNA) group. At 1 hour after administration, the improvement in the HDO(10MOE-5') and HDO(10MOE-3') groups was greatest in the HDO group. At 3 hours after administration, the improvement in the HDO(6MOE-5'&3') group was greatest.

[0220] Figure 24 shows the expression level of Mapt mRNA in the hippocampus 7 days after intracerebroventricular administration of various nucleic acid agents. The groups treated with HDO (all DNA), HDO (6MOE-5'&3'), HDO (6MOE-5'), HDO (6MOE-3'), HDO (10MOE-5'), and HDO (10MOE-3') showed a significant decrease in Mapt mRNA expression compared with the negative control group treated with PBS only.

[0221] <Example 7: Various MOE modifications in HDO targeting the BACE1 gene> (Objective) To verify the toxicity-reducing effects of various MOE modifications on the central nervous system toxicity observed when HDO containing a first nucleic acid strand consisting of an ASO targeting the BACE1 gene and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand is administered intracerebroventricularly through in vivo experiments.

[0222] (Method) The base sequences and chemical modifications of the first and second nucleic acid strands constituting the HDO used in this example are shown in Table 7 and FIG.

[0223]

[0224] The ASO used in this example is an LNA / DNA gapmer antisense nucleic acid targeting BACE1 mRNA. It has a base sequence complementary to a portion of BACE1 mRNA, with two LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and eight DNA nucleosides between them linked by phosphorothioate bonds. The HDO(all DNA), HDO(5MOE-5'), HDO(5MOE-3'), HDO(8MOE-5'), HDO(8MOE-3'), HDO(10MOE-5'), and HDO(10MOE-3') used in this example all contain the above-mentioned ASO as the first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand (c(all DNA)) of HDO(all DNA) has a structure in which DNA nucleosides are linked by phosphodiester bonds. The second nucleic acid strand of HDO(5MOE-5') (c(5MOE-5')) has a structure in which five 2'-O-MOE-RNA nucleosides at the 5' end and eight DNA nucleosides at the 3' end are linked by phosphodiester bonds. The second nucleic acid strand of HDO(5MOE-3') (c(5MOE-3')) has a structure in which eight DNA nucleosides at the 5' end and five 2'-O-MOE-RNA nucleosides at the 3' end are linked by phosphodiester bonds. The second nucleic acid strand of HDO(8MOE-5') (c(8MOE-5')) has a structure in which eight 2'-O-MOE-RNA nucleosides at the 5' end and five DNA nucleosides at the 3' end are linked by phosphodiester bonds. The second nucleic acid strand of HDO(8MOE-3') (c(8MOE-3')) has a structure in which five DNA nucleosides at the 5' end and eight 2'-O-MOE-RNA nucleosides at the 3' end are linked by phosphodiester bonds. The second nucleic acid strand of HDO(10MOE-5') (c(10MOE-5')) has a structure in which ten 2'-O-MOE-RNA nucleosides at the 5' end and three DNA nucleosides at the 3' end are linked by phosphodiester bonds. The second nucleic acid strand of HDO(10MOE-3') (c(10MOE-3')) has a structure in which three DNA nucleosides at the 5' end and ten 2'-O-MOE-RNA nucleosides at the 3' end are linked by phosphodiester bonds.

[0225] For HDO (all DNA), HDO (5MOE-5'), HDO (5MOE-3'), HDO (8MOE-5'), HDO (8MOE-3'), HDO (10MOE-5'), and HDO (10MOE-3') listed in Table 7, nucleic acid preparation, in vivo experiments, and central neurotoxicity and motor function evaluations were performed in the same manner as in Example 1. However, in this example, the dose of the nucleic acid agent administered per mouse was 11.5 nmol / mouse.

[0226] (Results) Figure 26 shows the results of evaluating central neurotoxicity in mice administered various nucleic acid agents intracerebroventricularly. At 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours after administration of the nucleic acid agent, the groups administered HDO(5MOE-5'), HDO(5MOE-3'), HDO(8MOE-5'), HDO(8MOE-3'), HDO(10MOE-5'), and HDO(10MOE-3') showed significantly reduced acute tolerability scores compared to the group administered HDO(all DNA).

[0227] Figure 27 shows the results of evaluating motor function 1 hour after intracerebroventricular administration of various nucleic acid agents in mice. The HDO(5MOE-5'), HDO(5MOE-3'), HDO(8MOE-5'), HDO(8MOE-3'), HDO(10MOE-5'), and HDO(10MOE-3') groups showed significantly improved total distance traveled (Figure 27A) and maximum movement speed (Figure 27B) compared to the HDO(all DNA) group.

[0228] Example 8: Various MOE modifications in HDO targeting the Mapt gene (Objective) To verify the toxicity-reducing effects of various MOE modifications on central nervous system toxicity observed when HDO containing a first nucleic acid strand consisting of an ASO targeting the Mapt gene and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand is intracerebroventricularly administered, through in vivo experiments.

[0229] (Method) The base sequences and chemical modifications of the first and second nucleic acid strands constituting the HDO used in this example are shown in Table 8 and FIG.

[0230]

[0231] The ASO used in this example is an LNA / DNA gapmer antisense nucleic acid targeting Mapt mRNA. It has a base sequence complementary to a portion of Mapt mRNA, with three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and ten DNA nucleosides between them linked by phosphorothioate bonds. The HDO(all DNA), HDO(6MOE wing), HDO(9MOE wing), HDO(11MOE wing), HDO(13MOE wing), and HDO(15MOE wing) used in this example all contain the above-mentioned ASO as the first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand (c(all DNA)) of HDO(all DNA) has a structure in which DNA nucleosides are linked by phosphodiester bonds. The second nucleic acid strand (c(6MOE wing)) of HDO(6MOE wing) has a structure in which three 2'-O-MOE-RNA nucleosides at the 5'-terminus, three 2'-O-MOE-RNA nucleosides at the 3'-terminus, and ten DNA nucleosides between them are linked by phosphodiester bonds. The second nucleic acid strand (c(9MOE wing)) of HDO(9MOE wing) has a structure in which five 2'-O-MOE-RNA nucleosides at the 5'-terminus, four 2'-O-MOE-RNA nucleosides at the 3'-terminus, and seven DNA nucleosides between them are linked by phosphodiester bonds. The second nucleic acid strand (c(11MOE wing)) of HDO(11MOE wing) has a structure in which six 2'-O-MOE-RNA nucleosides at the 5'-terminus, five 2'-O-MOE-RNA nucleosides at the 3'-terminus, and five DNA nucleosides between them are linked by phosphodiester bonds. The second nucleic acid strand (c(13MOE wing)) of HDO(13MOE wing) has a structure in which seven 2'-O-MOE-RNA nucleosides at the 5'-terminus, six 2'-O-MOE-RNA nucleosides at the 3'-terminus, and three DNA nucleosides between them are linked by phosphodiester bonds.The second nucleic acid strand (c(15MOE wing)) of HDO(15MOE wing) has a structure in which eight 2'-O-MOE-RNA nucleosides at the 5' end, seven 2'-O-MOE-RNA nucleosides at the 3' end, and one DNA nucleoside between them are linked by phosphodiester bonds.

[0232] For HDO (all DNA), HDO (6 MOE wing), HDO (9 MOE wing), HDO (11 MOE wing), HDO (13 MOE wing), and HDO (15 MOE wing) listed in Table 8, nucleic acid preparation, in vivo experiments, central neurotoxicity evaluation, motor function evaluation, and gene suppression effect evaluation were performed in the same manner as in Example 1. However, in this example, the dose of the nucleic acid agent administered per mouse was 18.86 nmol / mouse.

[0233] (Results) Figure 29 shows the results of evaluating central neurotoxicity in mice administered various nucleic acid agents intracerebroventricularly. At 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours after administration of the nucleic acid agent, the acute tolerability scores were significantly reduced in the HDO (6 MOE wing), HDO (9 MOE wing), HDO (11 MOE wing), HDO (13 MOE wing), and HDO (15 MOE wing) groups compared to the HDO (all DNA) group.

[0234] Figure 30 shows the results of evaluating motor function 1 hour after intracerebroventricular administration of various nucleic acid agents in mice. The HDO(6MOE wing), HDO(9MOE wing), HDO(11MOE wing), HDO(13MOE wing), and HDO(15MOE wing) groups showed significantly improved total distance traveled (Figure 30A) and maximum movement speed (Figure 30B) compared to the HDO(all DNA) group.

[0235] <Example 9: Comparison of base types in MOE modification> (Purpose) Regarding central neurotoxicity observed when HDO containing a first nucleic acid strand consisting of an ASO targeting the Mapt gene and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand is administered intracerebroventricularly, the toxicity reduction effect depending on the type of nucleoside (nucleoside containing an adenine base, guanine base, cytosine base, or thymine base) to which MOE modification is introduced in the second nucleic acid strand is compared through in vivo experiments.

[0236] (Method) The base sequences and chemical modifications of the ASO and the first and second nucleic acid strands constituting the HDO used in this example are shown in Table 9 and FIG.

[0237]

[0238] The ASO used in this example is an LNA / DNA gapmer antisense nucleic acid that targets Mapt mRNA and has a base sequence complementary to a portion of Mapt mRNA, with three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and ten DNA nucleosides between them linked by phosphorothioate bonds. MOE ), HDO(G MOE ), HDO(C MOE ), and HDO(T MOE ) each contain the above-mentioned ASO as the first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand (c(all DNA)) of HDO(all DNA) has a structure in which DNA nucleosides are linked by phosphodiester bonds. HDO(A MOE ) of the second nucleic acid strand (a(A MOE )) has a structure in which all DNA nucleosides containing adenine bases are replaced with 2'-O-MOE-RNA nucleosides, and similarly, HDO(G MOE ) is a guanine base, HDO(C MOE ) is a cytosine base, HDO(T MOE ) has a structure in which all DNA nucleosides containing thymine bases are replaced with 2'-O-MOE-RNA nucleosides.

[0239] HDO (all DNA), HDO (A MOE ), HDO(G MOE ), HDO(C MOE ), and HDO(T MOE ) were subjected to nucleic acid preparation, in vivo experiments, and evaluation of central neurotoxicity and motor function in the same manner as in Example 1. However, in this example, the dose of the nucleic acid agent administered per mouse was 19 nmol / mouse.

[0240] (Results) Figure 32 shows the results of evaluating central neurotoxicity in mice after intracerebroventricular administration of various nucleic acid agents. HDO(A) was significantly elevated at 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours after administration of the nucleic acid agent. MOE ), HDO(C MOE ), and HDO(T MOE In the HDO(G) group, acute tolerability scores were significantly reduced compared to the HDO(all DNA) group. MOE The acute phase tolerability score was slightly reduced in the HDO (all DNA) group compared to the HDO (all DNA) group. These results indicate that HDO, in which nucleosides containing adenine, cytosine, or thymine bases in the second nucleic acid strand are replaced with 2'-O-MOE-RNA nucleosides, can reduce central neurotoxicity compared to HDO in which the second nucleic acid strand consists only of DNA nucleosides.

[0241] Figure 33 shows the results of evaluating motor function in mice intracerebroventricularly administered with various nucleic acid agents 1 hour and 3 hours after administration. MOE ), HDO(C MOE ), and HDO(T MOE In the HDO(G)-treated group, the total distance traveled (Fig. 33A) and maximum migration speed (Fig. 33B) were significantly improved compared to the HDO(all DNA)-treated group. MOEThe HDO (all DNA)-treated group showed slightly improved total migration distance (Fig. 33A) and maximum migration speed (Fig. 33B) compared with the HDO (all DNA)-treated group. These results indicate that HDO, in which nucleosides containing adenine, cytosine, or thymine bases in the second nucleic acid strand are replaced with 2'-O-MOE-RNA nucleosides, can reduce central neurotoxicity compared with HDO in which the second nucleic acid strand consists only of DNA nucleosides.

[0242] <Example 10: Comparison of adjacent nucleic acids in MOE modification> (Purpose) Regarding central neurotoxicity observed when HDO containing a first nucleic acid strand consisting of an ASO targeting the Mapt gene and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand is administered intracerebroventricularly, the toxicity-reducing effect of introducing MOE modification to nucleosides containing cytosine bases in the second nucleic acid strand and the nucleosides adjacent to the 5' and / or 3' sides thereof will be compared through in vivo experiments.

[0243] (Method) The base sequences and chemical modifications of the first and second nucleic acid strands constituting the ASO and HDO used in this example are shown in Table 10 and FIG.

[0244]

[0245] The ASO used in this example is an LNA / DNA gapmer antisense nucleic acid that targets Mapt mRNA and has a base sequence complementary to a portion of Mapt mRNA, with three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and ten DNA nucleosides between them linked by phosphorothioate bonds. MOE ), HDO(2C MOE -5), HDO(2C MOE -3) and HDO(3C MOE ) each contain the above-mentioned ASO as the first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand (c(all DNA)) of HDO(all DNA) has a structure in which DNA nucleosides are linked by phosphodiester bonds. HDO(C MOE ) of the second nucleic acid strand (c(C MOEHDO(2C) has a structure in which all DNA nucleosides containing cytosine bases are replaced with 2'-O-MOE-RNA nucleosides. MOE The second nucleic acid strand (c(2C-5) MOE HDO(2C -5)) has a structure in which all DNA nucleosides containing cytosine bases and the DNA nucleosides adjacent to them on the 5' side are replaced with 2'-O-MOE-RNA nucleosides. MOE The second nucleic acid strand (c(2C-3) MOE HDO(3C -3)) has a structure in which all DNA nucleosides containing cytosine bases and the DNA nucleosides adjacent to them at the 3' end are replaced with 2'-O-MOE-RNA nucleosides. MOE ) of the second nucleic acid strand (c(3C MOE )) has a structure in which all DNA nucleosides containing a cytosine base and the DNA nucleosides adjacent to it on the 5' and 3' sides are replaced with 2'-O-MOE-RNA nucleosides.

[0246] HDO (all DNA), HDO (C MOE ), HDO(2C MOE -5), HDO(2C MOE -3) and HDO(3C MOE ) were subjected to nucleic acid preparation, in vivo experiments, and evaluation of central neurotoxicity and motor function in the same manner as in Example 1. However, in this example, the dose of the nucleic acid agent administered per mouse was 19 nmol / mouse.

[0247] (Results) Figure 35 shows the results of evaluating central neurotoxicity in mice after intracerebroventricular administration of various nucleic acid agents. HDO(2C MOE -5), HDO(2C MOE -3) and HDO(3C MOE In the group administered HDO (all DNA) and HDO (C MOEThe acute tolerability score was significantly reduced compared to the group administered 2'-O-MOE-RNA nucleosides. These results indicate that HDO, in which nucleosides containing cytosine bases and their adjacent nucleosides in the second nucleic acid strand are replaced with 2'-O-MOE-RNA nucleosides, has reduced central neurotoxicity compared to HDO in which the second nucleic acid strand is composed of DNA nucleosides.

[0248] Figure 36 shows the results of evaluating motor function in mice intracerebroventricularly administered with various nucleic acid agents 1 hour and 3 hours after administration. MOE -5), HDO(2C MOE -3) and HDO(3C MOE In the group administered HDO (all DNA) and HDO (C MOE The total migration distance (Fig. 36A) and maximum migration speed (Fig. 36B) were significantly improved compared to the group treated with 2'-O-MOE-RNA nucleosides. These results indicate that HDO, in which nucleosides containing cytosine bases and their adjacent nucleosides in the second nucleic acid strand are replaced with 2'-O-MOE-RNA nucleosides, can reduce central neurotoxicity compared to HDO in which the second nucleic acid strand is composed of DNA nucleosides.

[0249] Example 11: Stability of HDO in cerebrospinal fluid (Purpose) An HDO containing a first nucleic acid strand consisting of an ASO targeting the Mapt gene and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand is incubated in human or rat cerebrospinal fluid to examine its stability.

[0250] (Method) (1) Preparation of Nucleic Acid The base sequences and chemical modifications of the first and second nucleic acid strands constituting the HDO used in this example are shown in Table 11, Figures 37A, 37B, 38A, and 38B.

[0251]

[0252] The ASO used in this example is an LNA / DNA gapmer antisense nucleic acid targeting Mapt mRNA, and has a base sequence complementary to a portion of Mapt mRNA, with three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and ten DNA nucleosides between them linked by phosphorothioate bonds. The HDO(ASO / cRNA) and HDO(ASO / cDNA) used in this example both contain the ASO as a first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand (cRNA) of HDO(ASO / cRNA) has a structure in which RNA nucleosides are linked by three phosphorothioate bonds from the 5' end, three phosphorothioate bonds from the 3' end, and nine phosphodiester bonds between them. The second nucleic acid strand (cDNA) of HDO (ASO / cDNA) has a structure in which DNA nucleosides are linked by three phosphorothioate bonds from the 5' end, three phosphorothioate bonds from the 3' end, and nine phosphodiester bonds between them.

[0253] For the HDO (ASO / cRNA) and HDO (ASO / cDNA) listed in Table 11, nucleic acids were prepared in the same manner as in Example 1.

[0254] (2) Evaluation of nucleic acid drug stability in cerebrospinal fluid. Four μL each of 10 μM HDO (ASO / cRNA) and HDO (ASO / cDNA) was prepared. Four μL of HDO was mixed with 16 μL of human or rat cerebrospinal fluid and incubated in a 37°C incubator. After 10 minutes, 1 hour, and 6 hours, the mixture was immersed in liquid nitrogen to terminate the reaction.

[0255] A 16% acrylamide gel (1x TBE) was prepared and run. Six microliters of the above sample was loaded onto the gel and electrophoresed at 100 V for 80 minutes. As controls, ASO alone and cRNA alone were run simultaneously. Next, a solution of GelRed (x10,000) aqueous solution (Biotium) was diluted to a concentration of 1 / 10,000 in 1x TBE to prepare a solution. The gel was then permeated with this solution for 10 minutes. The gel was then photographed using a ChemiDoc Touch imaging system (BioRad).

[0256] (Results) Figure 37C shows the electrophoresis results of HDO(ASO / cRNA) after incubation in human CSF for 10 minutes and 6 hours. Figure 37D shows the results of quantification of the band intensity of the HDO double-stranded band in the electrophoresis results of Figure 37C. HDO(ASO / cRNA) was largely degraded after 10 minutes of incubation in human CSF, and completely degraded after 6 hours.

[0257] Figure 38C shows the electrophoresis results of HDO(ASO / cRNA) or HDO(ASO / cDNA) after incubation in human or rat cerebrospinal fluid for 6 hours. In human cerebrospinal fluid, HDO(ASO / cRNA) was degraded and unstable, whereas HDO(ASO / cDNA) remained stable. In rat cerebrospinal fluid, both HDO(ASO / cRNA) and HDO(ASO / cDNA) remained stable and undegraded (Figure 38D).

[0258] Example 12: Stability of HDO in cerebrospinal fluid (Purpose) An HDO containing a first nucleic acid strand consisting of an ASO targeting the Mapt gene and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand is incubated in the cerebrospinal fluid of a mouse, rat, monkey, or human, and its stability is examined.

[0259] (Method) (1) Preparation of Nucleic Acid The base sequences and chemical modifications of the first and second nucleic acid strands constituting the HDO used in this example are shown in Table 12, Figures 39A, 39B, 40A, and 40B.

[0260]

[0261] The ASO used in this example is an LNA / DNA gapmer antisense nucleic acid targeting Mapt mRNA, with a base sequence complementary to a portion of Mapt mRNA, and a structure in which three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and ten DNA nucleosides between them are linked by phosphorothioate bonds. The HDO (all RNA), HDO (all DNA), HDO (cRNA 6MOE wing), and HDO (cDNA 6MOE wing) used in this example all contain the above-mentioned ASO as a first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand (cRNA) of HDO (all RNA) has a structure in which RNA nucleosides are linked by phosphodiester bonds. The second nucleic acid strand (cDNA) of HDO (all DNA) has a structure in which DNA nucleosides are linked by phosphodiester bonds. The second nucleic acid strand (cRNA(6MOE wing)) of HDO(cRNA 6MOE wing) has a structure in which three 2'-O-MOE-RNA nucleosides at the 5'-terminus, three 2'-O-MOE-RNA nucleosides at the 3'-terminus, and ten RNA nucleosides between them are linked by phosphodiester bonds. The second nucleic acid strand (cDNA(6MOE wing)) of HDO(cDNA 6MOE wing) has a structure in which three 2'-O-MOE-RNA nucleosides at the 5'-terminus, three 2'-O-MOE-RNA nucleosides at the 3'-terminus, and ten DNA nucleosides between them are linked by phosphodiester bonds.

[0262] For HDO (all RNA), HDO (all DNA), HDO (cRNA 6MOE wing), and HDO (cDNA 6MOE wing) listed in Table 12, nucleic acid preparation and stability evaluation in cerebrospinal fluid were performed in the same manner as in Example 11.

[0263] (Results) Figure 39C shows the electrophoresis results of HDO (all RNA) and HDO (all DNA) after incubation for 6 hours in the cerebrospinal fluids of mice, rats, monkeys, and humans. HDO (all RNA) was stable and not degraded in the cerebrospinal fluids of mice and rats, whereas it was degraded and unstable in the cerebrospinal fluids of monkeys and humans. In contrast, HDO (all DNA) was stable and not degraded in the cerebrospinal fluids of mice, rats, monkeys, and humans.

[0264] Figure 40C shows the electrophoresis results of HDO (cRNA 6MOE wing) and HDO (cDNA 6MOE wing) after incubation in mouse, rat, monkey, and human cerebrospinal fluid for 6 hours. HDO (cRNA 6MOE wing) was stable and not degraded in mouse and rat cerebrospinal fluid, but was degraded and unstable in monkey and human cerebrospinal fluid. In contrast, HDO (cDNA 6MOE wing) was stable and not degraded in any of the cerebrospinal fluids of mice, rats, monkeys, and humans.

[0265] Example 13: Stability of HDO in cerebrospinal fluid (Purpose) To examine the stability in cerebrospinal fluid of an HDO containing a first nucleic acid strand consisting of an ASO targeting the Mapt gene and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand, when some of the DNA nucleosides in the second nucleic acid strand are replaced with RNA nucleosides.

[0266] (Method) (1) Preparation of Nucleic Acid The base sequences and chemical modifications of the first and second nucleic acid strands constituting the HDO used in this example are shown in Table 13 and FIG.

[0267]

[0268] The ASO used in this example is an LNA / DNA gapmer antisense nucleic acid that targets Mapt mRNA and has a base sequence complementary to a portion of Mapt mRNA, with three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and ten DNA nucleosides between them linked by phosphorothioate bonds. RNA), HDO(G RNA ), HDO(C RNA ), and HDO(U RNA ) each contain the above-mentioned ASO as the first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand (c(all DNA)) of HDO(all DNA) has a structure in which DNA nucleosides are linked by phosphodiester bonds. HDO(A RNA ) of the second nucleic acid strand (c(A RNA )) has a structure in which all DNA nucleosides containing adenine bases in c(all DNA) are replaced with RNA nucleosides. RNA ) of the second nucleic acid strand (c(G RNA HDO(C) has a structure in which all DNA nucleosides containing guanine bases in c(all DNA) are replaced with RNA nucleosides. RNA ) of the second nucleic acid strand (c(C RNA )) has a structure in which all DNA nucleosides containing cytosine bases in c(all DNA) are replaced with RNA nucleosides. RNA ) of the second nucleic acid strand (c(U RNA )) has a structure in which all DNA nucleosides containing thymine bases in c(all DNA) are replaced with RNA nucleosides containing uracil bases.

[0269] HDO (all DNA), HDO (A RNA ), HDO(G RNA ), HDO(C RNA ), and HDO(U RNA ) was subjected to nucleic acid preparation and stability evaluation in cerebrospinal fluid in the same manner as in Example 11.

[0270] (Results) Figure 42 shows the results of HDO (all DNA), HDO (A RNA ), HDO(G RNA ), HDO(C RNA ), and HDO(U RNA ) in human cerebrospinal fluid for 6 hours. RNA ), and HDO(G RNA) was stable in human cerebrospinal fluid and was not decomposed. RNA ) and HDO(U RNA ) was degraded and unstable in human cerebrospinal fluid.

[0271] Example 14: Stability of HDO in cerebrospinal fluid (Purpose) To examine the stability in cerebrospinal fluid of an HDO containing a first nucleic acid strand consisting of an ASO targeting the Mapt gene and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand, when some of the DNA nucleosides in the second nucleic acid strand are replaced with RNA nucleosides.

[0272] (Method) (1) Preparation of Nucleic Acid The base sequences and chemical modifications of the first and second nucleic acid strands constituting the HDO used in this example are shown in Table 14 and FIG.

[0273]

[0274] The ASO used in this example is an LNA / DNA gapmer antisense nucleic acid that targets Mapt mRNA and has a base sequence complementary to a portion of Mapt mRNA, with three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and ten DNA nucleosides between them linked by phosphorothioate bonds. RNA ), HDO(CU RNA ), HDO(C RNA ), and HDO(U RNA ) each contain the above-mentioned ASO as the first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand (c(all DNA)) of HDO(all DNA) has a structure in which DNA nucleosides are linked by phosphodiester bonds. HDO(GA RNA ) of the second nucleic acid strand (c(GA RNA )) has a structure in which all DNA nucleosides containing guanine bases and DNA nucleosides containing adenine bases in c(all DNA) are replaced with RNA nucleosides. RNA ) of the second nucleic acid strand (c(CU RNAHDO(C) has a structure in which all DNA nucleosides containing cytosine bases are replaced with RNA nucleosides, and all DNA nucleosides containing thymine bases are replaced with RNA nucleosides containing uracil bases. RNA ) of the second nucleic acid strand (c(C RNA )) has a structure in which all DNA nucleosides containing cytosine bases in c(all DNA) are replaced with RNA nucleosides. RNA ) of the second nucleic acid strand (c(U RNA )) has a structure in which all DNA nucleosides containing thymine bases in c(all DNA) are replaced with RNA nucleosides containing uracil bases.

[0275] HDO (all DNA), HDO (GA) RNA ), HDO(CU RNA ), HDO(C RNA ), and HDO(U RNA ) was subjected to nucleic acid preparation and stability evaluation in cerebrospinal fluid in the same manner as in Example 11.

[0276] (Results) Figure 44 shows the results of HDO (all DNA), HDO (GA RNA ), HDO(CU RNA ), HDO(C RNA ), and HDO(U RNA ) in human cerebrospinal fluid for 1 hour and 6 hours. RNA ) was stable in human cerebrospinal fluid and was not decomposed. RNA ), HDO(C RNA ), and HDO(U RNA ) was degraded and unstable in human cerebrospinal fluid.

[0277] Example 15: Stability of HDO in cerebrospinal fluid (Purpose) To examine the stability in cerebrospinal fluid of an HDO containing a first nucleic acid strand consisting of an ASO targeting the Malat1 gene and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand when some of the DNA nucleosides in the second nucleic acid strand are replaced with RNA nucleosides.

[0278] (Method) (1) Preparation of Nucleic Acid The base sequences and chemical modifications of the first and second nucleic acid strands constituting the HDO used in this example are shown in Table 15 and FIG.

[0279]

[0280] The ASO used in this example is a 2'-O-MOE-RNA / DNA gapmer antisense nucleic acid that targets Malat1 ncRNA. It has a base sequence complementary to a portion of Malat1 ncRNA, and has a structure in which five 2'-O-MOE-RNA nucleosides at the 5' end, five 2'-O-MOE-RNA nucleosides at the 3' end, and 10 DNA nucleosides between them are linked by phosphorothioate bonds. The ASOs used in this example were HDO (all DNA), HDO (A RNA ), HDO(G RNA ), HDO(C RNA ), and HDO(U RNA ) each contain the above-mentioned ASO as the first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand (c(all DNA)) of HDO(all DNA) has a structure in which DNA nucleosides are linked by phosphodiester bonds. HDO(A RNA ) of the second nucleic acid strand (c(A RNA )) has a structure in which all DNA nucleosides containing adenine bases in c(all DNA) are replaced with RNA nucleosides. RNA ) of the second nucleic acid strand (c(G RNA HDO(C) has a structure in which all DNA nucleosides containing guanine bases in c(all DNA) are replaced with RNA nucleosides. RNA ) of the second nucleic acid strand (c(C RNA )) has a structure in which all DNA nucleosides containing cytosine bases in c(all DNA) are replaced with RNA nucleosides. RNA ) of the second nucleic acid strand (c(U RNA )) has a structure in which all DNA nucleosides containing thymine bases in c(all DNA) are replaced with RNA nucleosides containing uracil bases.

[0281] HDO (all DNA), HDO (A RNA ), HDO(G RNA ), HDO(C RNA ), and HDO(U RNA ) was subjected to nucleic acid preparation and stability evaluation in cerebrospinal fluid in the same manner as in Example 11.

[0282] (Results) Figure 46 shows the results of HDO (all DNA), HDO (A RNA ), HDO(G RNA ), HDO(C RNA ), and HDO(U RNA ) in human cerebrospinal fluid for 6 hours. RNA ), and HDO(G RNA ) was stable in human cerebrospinal fluid and was not decomposed. RNA ) and HDO(U RNA ) was degraded and unstable in human cerebrospinal fluid.

[0283] Example 16: Evaluation of central neurotoxicity in monkeys (Objective) HDO containing MOE modification is administered intrathecally to monkeys to evaluate central neurotoxicity.

[0284] (Method) (1) Preparation of Nucleic Acids The base sequences and chemical modifications of the ASO and the first and second nucleic acid strands constituting the HDO used in this example are shown in Table 16 and Figures 47A-C.

[0285]

[0286] Nucleic acids were prepared for the ASO, HDO (RNA-MOE), and HDO (DNA-MOE) listed in Table 16 using the same method as in Example 1.

[0287] (2) Evaluation of central neurotoxicity in monkeys. Male cynomolgus monkeys (3 years old, 2 kg) were administered ASO, HDO (RNA-MOE), and HDO (DNA-MOE) intrathecally at 5 mg ASO or 0.94 μmol HDO. Behavioral observations were performed 2 hours after intrathecal administration (Figure 47D).

[0288] (Results) The results are shown in Figure 47E. Monkeys administered intrathecally with ASO exhibited severe limb paralysis and strong central nervous system toxicity. Monkeys administered intrathecally with HDO(RNA-MOE) exhibited moderate paralysis in the lower limbs, but showed reduced central nervous system toxicity compared to intrathecally administered ASO. Monkeys administered intrathecally with HDO(DNA-MOE) did not exhibit paralysis and maintained normal consciousness and motor function.

[0289] Example 17: HDO with a bulge structure (Objective) An HDO containing a first nucleic acid strand consisting of an ASO targeting the Mapt gene and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand is introduced into a neuronal cell line. The toxicity reduction and gene silencing effects of introducing a bulge structure into the second nucleic acid strand are verified through in vitro experiments.

[0290] (Method) (1) Preparation of Nucleic Acid The base sequences and chemical modifications of the first and second nucleic acid strands constituting the HDO used in this example are shown in Table 17 and FIG.

[0291]

[0292] The ASO used in this example is an LNA / DNA gapmer antisense nucleic acid targeting Mapt mRNA. It has a base sequence complementary to a portion of Mapt mRNA, with three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and ten DNA nucleosides between them linked by phosphorothioate bonds. The HDO(all DNA), HDO(all MOE), HDO(bulge 1), and HDO(bulge 2) used in this example all contain the above-mentioned ASO as the first nucleic acid strand, and the second nucleic acid strand contains a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand (c(all DNA)) of HDO(all DNA) has a structure in which DNA nucleosides are linked by phosphodiester bonds. The second nucleic acid strand (c(all MOE)) of HDO(all MOE) has a structure in which 2'-O-MOE-RNA nucleosides are linked by phosphodiester bonds.

[0293] The second nucleic acid strand (c(bulge1)) of HDO(bulge1) contains a complementary region consisting of a base sequence complementary to the entire length of the first nucleic acid strand, and a bulge structure located in the center of the complementary region. The complementary region has a structure in which 2'-O-MOE-RNA nucleosides are linked by phosphodiester bonds. The bulge structure has a structure in which two DNA nucleosides are linked by phosphodiester bonds.

[0294] The second nucleic acid strand (c(bulge2)) of HDO(bulge2) contains a complementary region consisting of a base sequence complementary to the first nucleic acid strand and a bulge structure located in the center of the complementary region, but the complementary region has a one-base deletion compared to the first nucleic acid strand, and a bulge structure is located at the position of this deletion. The complementary region has a structure of 2'-O-MOE-RNA nucleosides linked by phosphodiester bonds. The bulge structure has a structure of three DNA nucleosides linked by phosphodiester bonds.

[0295] For HDO (all DNA), HDO (all MOE), HDO (bulge 1), and HDO (bulge 2) listed in Table 17, nucleic acids were prepared in the same manner as in Example 1.

[0296] (2) Evaluation of gene suppression effect The HDO prepared in (1) was introduced into human neuroblastoma-derived cells (BE(2)-M17 cell line) using the lipofection method (lipofectamine2000).

[0297] Forty-eight hours after HDO induction, RNA was extracted from the cells using the Isogen I kit (Gene Design, Inc.). cDNA was synthesized using the Transcriptor Universal cDNA Master and DNase (Roche Diagnostics) according to the protocol. The expression levels of Mapt mRNA and Actb mRNA (internal control genes) were then measured by quantitative RT-PCR using the resulting cDNA as a template. Quantitative RT-PCR was performed using TaqMan (Roche Applied Science). Primers used in qRT-PCR were designed and manufactured by Thermo Fisher Scientific (formerly Life Technologies Corp.). Amplification was performed by repeating 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 1 second.

[0298] The ratio of the Mapt mRNA expression level to the Actb mRNA expression level (internal standard gene) was calculated, and the value normalized to the value of the PBS-administered group was used as the relative Mapt mRNA level.

[0299] (3) Evaluation of cytotoxicity. Forty-eight hours after transfection, lactate dehydrogenase (LDH) activity in the cell supernatant was measured to assess neuronal cytotoxicity. LDH activity was measured using the Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories) according to the attached protocol. The value normalized to the LDH activity in the PBS-treated group was used as the relative LDH release level.

[0300] (Results) Figure 49A shows the results of evaluating the target gene silencing effects of various nucleic acid agents in human neuroblastoma-derived cells (BE(2)-M17 cell line). The silencing effect was significantly weakened in the HDO(all MOE) administration group compared to HDO(all DNA). In contrast, the HDO(bulge1) and HDO(bulge2) administration groups achieved a strong gene silencing effect equivalent to that of HDO(all DNA).

[0301] Figure 49B shows the results of evaluating LDH activity in the supernatant as a measure of cytotoxicity after intracellular delivery of various nucleic acid agents. The HDO (all DNA) administration group showed increased LDH activity, whereas HDO (all MOE), HDO (bulge 1), and HDO (bulge 2) showed LDH activity at or below that of the PBS administration group, indicating significantly reduced cytotoxicity. These results demonstrate that HDO containing 2'-O-MOE-RNA nucleosides significantly reduced cytotoxicity.

[0302] These results demonstrate that HDO(bulge1) and HDO(bulge2) exert a strong gene suppression effect without neuronal toxicity.

[0303] Example 18: Single-stranded HDO (Objective) Regarding central nervous system toxicity observed when HDO containing a first nucleic acid strand consisting of an ASO targeting the Mapt gene and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand is administered intracerebroventricularly, the toxicity reduction and gene suppression effects of introducing a bulge structure into the second nucleic acid strand, linking the first and second nucleic acid strands with a linker, and both of these will be verified through in vivo experiments.

[0304] (Methods) (1) Preparation of Nucleic Acid The base sequence and chemical modification of HDO used in this example are shown in Table 18 and Figure 50.

[0305]

[0306] The HDO(bulge), ssHDO, PEG linker ssHDO, and Bulge plus ssHDO used in this example all comprise a first nucleic acid strand and a second nucleic acid strand. The first nucleic acid strand of the HDO(bulge), ssHDO, PEG linker ssHDO, and Bulge plus ssHDO is an LNA / DNA gapmer antisense nucleic acid, having a base sequence complementary to a portion of Mapt mRNA, and a structure in which three LNA nucleosides at the 5'-end, three LNA nucleosides at the 3'-end, and ten DNA nucleosides between them are linked by phosphorothioate bonds. While the first and second nucleic acid strands of the HDO(bulge) are not linked by a linker, the 3'-end of the second nucleic acid strand is linked to the 5'-end of the first nucleic acid strand in the ssHDO, PEG linker ssHDO, and Bulge plus ssHDO via a linker.

[0307] The second nucleic acid strand (c(bulge)) of HDO(bulge) contains a complementary region consisting of a base sequence complementary to the first nucleic acid strand, and a bulge structure. The complementary region has a structure in which 2'-O-MOE-RNA nucleosides are linked by phosphodiester bonds. The bulge structure has a structure in which three DNA nucleosides are linked by phosphodiester bonds.

[0308] The second nucleic acid strand of ssHDO has a sequence complementary to that of the first nucleic acid strand, and has a structure in which three 2'-O-MOE-RNA nucleosides, ten DNA nucleosides, and three 2'-O-MOE-RNA nucleosides are linked by phosphodiester bonds in order from the 5' end. The linker connecting the first and second nucleic acid strands in ssHDO consists of three DNA nucleosides linked by phosphodiester bonds.

[0309] The structure of the second nucleic acid strand of the PEG linker ssHDO is the same as that of the second nucleic acid strand of the ssHDO described above. In the PEG linker ssHDO, the linker connecting the first and second nucleic acid strands is composed of PEG (polyethylene glycol).

[0310] The second nucleic acid strand of the Bulge plus ssHDO contains a complementary region consisting of a base sequence complementary to the first nucleic acid strand, and two bulge structures. The complementary region has a structure in which 2'-O-MOE-RNA nucleosides are linked by phosphodiester bonds. Each of the two bulge structures has a structure in which three DNA nucleosides are linked by phosphodiester bonds. The linker connecting the first and second nucleic acid strands in the Bulge plus ssHDO consists of three DNA nucleosides linked by phosphodiester bonds.

[0311] For the ssHDO, PEG linker ssHDO, and Bulge plus ssHDO listed in Table 18, nucleic acid preparation, in vivo experiments, central neurotoxicity evaluation, motor function evaluation, and gene suppression effect evaluation will be performed in the same manner as in Example 1.

[0312] (Results) Mice intracerebroventricularly administered with HDO (bulge), ssHDO, PEG linker ssHDO, and Bulge plus ssHDO showed significantly reduced acute tolerance scores 30 minutes to 4 hours after administration compared to mice administered with HDO (all DNA) used in Example 1. Furthermore, mice intracerebroventricularly administered with HDO (bulge), ssHDO, PEG linker ssHDO, and Bulge plus ssHDO showed significantly improved motor function, such as total movement distance and maximum movement speed, 1 hour after administration compared to mice administered with HDO (all DNA) used in Example 1. Furthermore, mice intracerebroventricularly administered with HDO (bulge), ssHDO, PEG linker ssHDO, and Bulge plus ssHDO showed reduced Mapt mRNA expression levels in the hippocampus 7 days after administration compared to mice administered with HDO (all MOE) used in Example 1, demonstrating improved target gene suppression effects. These results demonstrate that introducing a bulge structure into the second nucleic acid strand and linking the first and second nucleic acid strands of HDO with a linker can both reduce central nervous system toxicity and achieve excellent gene silencing. Furthermore, introducing both a bulge structure and a linker into HDO can both reduce central nervous system toxicity and achieve excellent gene silencing.

[0313] Example 19: Study of nucleic acid species to be introduced into the gap region of the second nucleic acid strand (Objective) To study the central neurotoxicity observed when HDO targeting the Malat1 gene is administered intracerebroventricularly, the effect of nucleic acid species introduced into a region of the second nucleic acid strand consisting of a base sequence complementary to the central region (gap region) of the first nucleic acid strand (hereinafter also referred to as the "gap region" in the second nucleic acid strand) is examined. Specifically, in vivo experiments are conducted to compare the toxicity of HDO in which all nucleosides in the gap region of the second nucleic acid strand are RNA nucleosides, HDO in which all nucleosides are DNA nucleosides, and HDO in which RNA nucleosides are located at the positions of adenine and guanine bases and DNA nucleosides are located at the positions of cytosine and thymine bases.

[0314] (Method) Table 19 shows the base sequences and chemical modifications of the ASO and the first and second nucleic acid strands constituting the HDO used in this example.

[0315]

[0316] The HDO(all DNA), HDO(cRNA 10MOE), HDO(cDNA 10MOE), and HDO(agRNA 10MOE) used in this example all contain the common ASO as the first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand (c(all DNA)) of HDO(all DNA) has a structure in which DNA nucleosides are linked by phosphodiester bonds. The second nucleic acid strand c (RNA 10MOE) of HDO (cRNA 10MOE) has a region consisting of a base sequence complementary to the wing region of the first nucleic acid strand (hereinafter also referred to as the "wing region" in the second nucleic acid strand) composed of 2'-O-MOE-RNA nucleosides, and the gap region composed of RNA nucleosides; the second nucleic acid strand c (DNA 10MOE) of HDO (cDNA 10MOE) has a wing region composed of 2'-O-MOE-RNA nucleosides, and the gap region composed of DNA nucleoside structures; and the second nucleic acid strand c (agRNA 10MOE) of HDO (agRNA 10MOE) has a wing region composed of 2'-O-MOE-RNA nucleosides, and in the gap region, nucleosides having adenine bases and guanine bases are RNA nucleosides, and nucleosides having cytosine bases and thymine bases are DNA nucleosides. For the nucleic acid molecules listed in Table 19, nucleic acid preparation, in vivo experiments, central neurotoxicity evaluation, and motor function evaluation were performed in the same manner as in Example 1. However, in this example, the dose of the nucleic acid agent administered per mouse was 14 nmol / mouse.

[0317] (Results) Figure 51 shows the results of assessing motor function 1 hour after intracerebroventricular administration of various nucleic acid agents in mice. The HDO(all DNA), HDO(cRNA 10MOE), HDO(cDNA 10MOE), and HDO(agRNA 10MOE) groups significantly improved total distance traveled (Figure 51A) and average speed (Figure 51B) compared with the ASO group. Furthermore, the HDO(cRNA 10MOE), HDO(cDNA 10MOE), and HDO(agRNA 10MOE) groups also showed improved total distance traveled and average speed compared with the HDO(all DNA) group. Furthermore, the HDO(agRNA 10MOE) group showed improved total distance traveled and average speed compared with the HDO(cRNA 10MOE) and HDO(cDNA 10MOE) groups. These results indicate that HDOs in which the adenine and guanine bases in the gap region of the second nucleic acid strand are RNA nucleosides and the cytosine and thymine bases in the gap region are DNA nucleosides can significantly reduce central neurotoxicity compared to single-stranded ASOs and other HDOs. Figure 52 shows Malat1 RNA expression levels in the right and left frontal lobes 7 days after intracerebroventricular administration of various nucleic acid agents. The HDO (agRNA 10MOE) group demonstrated significantly greater suppression of Malat1 RNA expression levels than the ASO and other HDO groups. These results indicate that HDOs containing a second nucleic acid strand in which the adenine and guanine bases in the gap region are RNA nucleosides and the cytosine and thymine bases in the gap region are DNA nucleosides can achieve superior gene expression suppression effects compared to HDOs containing a second nucleic acid strand in which all nucleosides in the gap region are DNA nucleosides or RNA nucleosides.

[0318] Example 20: Evaluation of duplex dissociation efficiency in brain tissue (Objective) In target tissue, the first nucleic acid strand dissociates from the second nucleic acid strand, potentially exerting an antisense effect on a target gene or its transcription product. To this end, HDO containing the first and second nucleic acid strands was incubated in mouse brain tissue homogenate, and its duplex dissociation efficiency was examined.

[0319] (Methods) (1) Preparation of Nucleic Acids In this example, the same ASO, HDO (cRNA 10 MOE), HDO (cDNA 10 MOE), and HDO (agRNA 10 MOE) as in Example 19 were used. The ASO and HDO were prepared by the same method as in Example 19.

[0320] (2) Evaluation of double-strand dissociation efficiency in brain tissue homogenate. 5.2 μL each of 25 μM HDO (ASO / cRNA) and HDO (ASO / cDNA) was mixed with 16.7 μL of mouse brain homogenate solution and 28.1 μL of PBS and incubated in a 37°C incubator. After incubation, the reaction was stopped by mixing with protein kinase K. A 16% acrylamide gel (1x TBE) was prepared and prepared. 9.6 μL of the above sample was loaded onto the gel and electrophoresed at 100 V for 120 minutes. ASO alone was run as a control. A solution of GelRed (x10000) aqueous solution (Biotium) was then diluted to a concentration of 1 / 10000 in 1x TBE to prepare a solution. The gel was then permeated with this solution for 10 minutes. The gel was then photographed using a ChemiDoc Touch imaging system (BioRad).

[0321] (Results) Figure 53 shows the electrophoresis results after 7 days of incubation of ASO, HDO(cRNA 10MOE), HDO(cDNA 10MOE), and HDO(agRNA 10MOE) in mouse brain tissue homogenate. HDO(agRNA 10MOE) showed a significant reduction in double-stranded nucleic acids (Figure 53, arrows) after 7 days of incubation compared to HDO(cRNA 10MOE) and HDO(cDNA 10MOE), demonstrating its excellent ability to separate double strands in brain tissue. HDOs containing a second nucleic acid strand in which the adenine and guanine bases are replaced by RNA nucleosides and the cytosine and thymine bases are replaced by DNA nucleosides in the gap region have an extremely high double-strand dissociation efficiency in brain tissue compared to HDOs containing a second nucleic acid strand in which all nucleosides in the gap region are DNA nucleosides or RNA nucleosides, strongly supporting the results of Example 19 above, which showed significant suppression of target gene expression.

[0322] Example 21: Examination of nucleic acid modifications to be introduced into the wing region of the second nucleic acid strand (Objective) Regarding central neurotoxicity observed when HDO targeting the Malat1 gene is administered intracerebroventricularly, the effects of introducing MOE-modified nucleic acids or natural nucleic acids (RNA or DNA) into the wing and gap regions of the second nucleic acid strand are compared through in vivo experiments.

[0323] (Method) Table 20 shows the base sequences and chemical modifications of the first and second nucleic acid strands constituting the HDO used in this example.

[0324]

[0325] The HDO(all DNA), HDO(cRNA MOEwing), HDO(gapMOE RNA), HDO(cDNA MOEwing), HDO(gapDNA MOE), and HDO(agRNA 10MOE) used in this example all contain the ASO as a common first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand of HDO(all DNA) has a structure in which DNA nucleosides are linked by phosphodiester bonds. The second nucleic acid strand of HDO (cRNA MOEwing) has a wing region composed of 2'-O-MOE-RNA nucleosides and a gap region composed of RNA nucleoside structures; the second nucleic acid strand of HDO (gapMOE RNA) has a region corresponding to the wing region composed of RNA nucleosides and a region corresponding to the gap region composed of 2'-O-MOE-RNA nucleoside structures; the second nucleic acid strand of HDO (cDNA MOEwing) has a wing region composed of 2'-O-MOE-RNA nucleosides and a gap region composed of DNA nucleoside structures; the second nucleic acid strand of HDO (gapMOE DNA) has a region corresponding to the wing region composed of DNA nucleosides and a gap region composed of 2'-O-MOE-RNA nucleoside structures; and the second nucleic acid strand of HDO (agRNA The second nucleic acid strand (10MOE) has wing regions composed of 2'-O-MOE-RNA nucleosides, and in the gap region, nucleosides containing adenine and guanine bases are RNA nucleosides, and nucleosides containing cytosine and thymine bases are DNA nucleosides. For the nucleic acid molecules listed in Table 21, nucleic acid preparation, in vivo experiments, central neurotoxicity evaluation, and motor function evaluation were performed in the same manner as in Example 1. However, in this example, the dose of the nucleic acid agent administered per mouse was 28 nmol / mouse.

[0326] (Results) Figure 54 shows the results of evaluating central neurotoxicity in mice administered various nucleic acid agents intracerebroventricularly. The groups administered HDO(cRNA MOEwing), HDO(gapMOE RNA), HDO(cDNA MOEwing), HDO(gapDNA MOE), and HDO(agRNA 10MOE) showed significantly reduced acute-phase tolerance scores compared to the group administered HDO(all DNA). Furthermore, the groups administered HDO(gapMOE RNA), HDO(cDNA MOEwing), HDO(gapDNA MOE), and HDO(agRNA 10MOE) showed even greater reductions in acute-phase tolerance scores compared to the group administered HDO(cDNA MOEwing).

[0327] Figure 55 shows the results of assessing motor function 1 hour after intracerebroventricular administration of various nucleic acid agents in mice. The HDO(cRNA MOEwing), HDO(gapMOE RNA), HDO(cDNA MOEwing), HDO(gapDNA MOE), and HDO(agRNA 10MOE) groups significantly improved total distance traveled (Figure 55A) and average speed (Figure 55B) compared with the HDO(all DNA) group. Furthermore, the HDO(gapMOE RNA), HDO(cDNA MOEwing), HDO(gapDNA MOE), and HDO(agRNA 10MOE) groups also improved total distance traveled and average speed compared with the HDO(cDNA MOEwing) group. These results suggest that HDO containing MOE modifications in the wing or gap region of the second nucleic acid strand may reduce central neurotoxicity compared with HDO whose second nucleic acid strand is composed of DNA nucleosides.

[0328] Example 22: Examination of nucleic acid species to be introduced into the gap region of the second nucleic acid strand: Comparison of acute and delayed neurotoxicity (Objective) When HDO containing a first nucleic acid strand consisting of an ASO targeting the Mapt gene and an MOE wing-modified second nucleic acid strand having a base sequence complementary to the first nucleic acid strand is intracerebroventricularly administered, the toxicity reduction effects observed within one day after administration and delayed neurotoxicity observed one day or later after administration are compared through in vivo experiments.

[0329] (Method) Table 21 shows the base sequences and chemical modifications of the ASO and the first and second nucleic acid strands constituting the HDO used in this example.

[0330] The HDO(all DNA), HDO(cRNA 6MOE), HDO(cDNA 6MOE), and HDO(agRNA 6MOE) used in this example all contain the ASO as a common first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand (c(all DNA)) of HDO(all DNA) has a structure in which DNA nucleosides are linked by phosphodiester bonds. The second nucleic acid strand of HDO(cRNA 6MOE) has a structure in which three 2'-O-MOE-RNA nucleosides at the 5' end, three 2'-O-MOE-RNA nucleosides at the 3' end, and 10 RNA nucleosides between them are linked by phosphodiester bonds. The second nucleic acid strand of HDO (cDNA 6MOE) has a structure in which three 2'-O-MOE-RNA nucleosides at the 5' end, three 2'-O-MOE-RNA nucleosides at the 3' end, and ten DNA nucleosides between them are linked by phosphodiester bonds. The second nucleic acid strand of HDO (agRNA 6MOE) is composed of three 2'-O-Me-RNA nucleosides at the 5' end, three 2'-O-Me-RNA nucleosides at the 3' end, and a gap region between them, where nucleosides containing adenine and guanine bases are RNA nucleosides and nucleosides containing cytosine and thymine bases are DNA nucleosides. For the ASO, HDO (all DNA), HDO (cRNA 6MOE), HDO (cDNA 6MOE), and HDO (agRNA 6MOE) listed in Table 21, nucleic acid preparation, in vivo experiments, and central neurotoxicity and motor function evaluations were performed in the same manner as in Example 1. However, in this example, the dose of the nucleic acid agent administered per mouse was 28 nmol / mouse.

[0331] (Result 1: Acute Neurotoxicity) Figure 56 shows the results of evaluating acute central neurotoxicity within one day of intracerebroventricular administration of various nucleic acid agents in mice. At 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours after administration, the HDO(cRNA 6MOE), HDO(cDNA 6MOE), and HDO(agRNA 6MOE) groups showed significantly reduced acute tolerability scores compared to the ASO and HDO(all DNA) groups. Furthermore, the HDO(agRNA 6MOE) group showed a lower acute tolerability score compared to the HDO(cRNA 6MOE) and HDO(cDNA 6MOE) groups. These results indicate that HDO, in which nucleosides containing adenine and guanine bases in the gap region of the second nucleic acid strand are RNA nucleosides and nucleosides containing cytosine and thymine bases are DNA nucleosides, can significantly reduce central neurotoxicity compared with other HDOs. Figure 57 shows the results of motor function assessment 1 hour after intracerebroventricular administration of various nucleic acid agents in mice. The HDO(cRNA 6MOE), HDO(cDNA 6MOE), and HDO(agRNA 6MOE) groups showed improved total distance traveled (Figure 57A) and maximum speed (Figure 57B) compared with the ASO and HDO(all DNA) groups. Furthermore, the HDO(agRNA 6MOE) group showed significantly improved motor function compared with the HDO(cRNA 6MOE) and HDO(cDNA 6MOE) groups. These results showed that HDOs in which nucleosides having adenine and guanine bases in the gap region of the second nucleic acid strand are RNA nucleosides and nucleosides having cytosine and thymine bases are DNA nucleosides have extremely small effects on motor function and are extremely low in toxicity compared to other HDOs.

[0332] (Result 2: Delayed Neurotoxicity) Figure 58 shows the results of evaluating body weight loss in mice administered various nucleic acid agents intracerebroventricularly, as an indicator of delayed central neurotoxicity, starting one day after administration. Body weight was measured 0, 7, 14, and 21 days after administration. The ASO and HDO (all DNA) groups showed weight loss compared to the PBS group at all times, including days 7, 14, and 21. The HDO (cRNA 6MOE), HDO (cDNA 6MOE), and HDO (agRNA 6MOE) groups showed reduced weight loss compared to the ASO and HDO (all DNA) groups. Figure 59 shows the results of evaluating motor function in mice administered various nucleic acid agents intracerebroventricularly, as an indicator of delayed central neurotoxicity, starting one day after administration. At 7, 14, and 21 days after administration, the HDO(cRNA 6MOE), HDO(cDNA 6MOE), and HDO(agRNA 6MOE) administration groups showed a reduced decrease in maximum migration speed compared to the ASO and HDO(all DNA) administration groups.

[0333] (Result 3: Target gene suppression effect) Figure 60 shows the Mapt mRNA expression level in the right frontal lobe 21 days after intracerebroventricular administration of various nucleic acid agents. The HDO (agRNA 6MOE) administration group showed a significant suppression effect on Mapt mRNA expression level compared to the HDO (cRNA 6MOE) and HDO (cDNA 6MOE) administration groups. These results demonstrated that HDO, in which nucleosides containing adenine and guanine bases in the gap region of the second nucleic acid strand are RNA nucleosides and nucleosides containing cytosine and thymine bases are DNA nucleosides, can achieve a superior gene expression suppression effect compared to HDO in which the second nucleic acid strand is composed of DNA nucleosides or RNA nucleosides.

[0334] (Result 4: Evaluation of double-strand dissociation efficiency in brain tissue homogenate) Figure 61 shows the results of electrophoresis after incubating HDO(cRNA 6MOE), HDO(cDNA 6MOE), and HDO(agRNA 6MOE) in mouse brain tissue for 7 days using a method similar to that used in Example 20 to evaluate the double-strand dissociation efficiency of nucleic acid agents in brain tissue homogenate. HDO(agRNA 6MOE) showed higher double-strand dissociation efficiency in brain tissue homogenate than HDO(cRNA 6MOE) and HDO(cDNA 6MOE). These results strongly support the finding that HDOs in which nucleosides having adenine and guanine bases in the gap region of the second nucleic acid strand are RNA nucleosides and nucleosides having cytosine and thymine bases are DNA nucleosides have an extremely high double-strand dissociation efficiency in brain tissue compared to HDOs in which the second nucleic acid strand is composed of DNA nucleosides or RNA nucleosides, significantly suppressing the expression of the target gene as shown in Result 3 above.

[0335] Example 23: Introduction of MOE modification into the second nucleic acid strand of an HDO containing an all-MOE-modified ASO (Objective) This example focuses on an HDO containing, as the first nucleic acid strand, an ASO in which all nucleosides are MOE-modified (hereinafter referred to as an "all-MOE-modified ASO"), which can target the Mecp2 gene and control splicing. In vivo experiments were conducted to verify whether the central nervous system toxicity observed when the HDO was administered intracerebroventricularly could be reduced by introducing an MOE modification into the second nucleic acid strand.

[0336] (Method) Table 22 shows the base sequences and chemical modifications of the ASO and the first and second nucleic acid strands constituting the HDO used in this example.

[0337]

[0338] The ASO used in this example is a non-gapmer antisense nucleic acid targeting MeCP2 (methyl-CpG binding protein 2) pre-mRNA. It has a base sequence complementary to a portion of MECP2 pre-mRNA and is a 2'-O-MOE-RNA nucleoside in which all nucleosides are linked by phosphorothioate bonds. The HDO (all DNA) and HDO (cRDNA MOE) used in this example contain the above-mentioned ASO as a common first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand. Specifically, the second nucleic acid strand of HDO (all DNA) has a structure in which DNA nucleosides are linked by phosphodiester bonds. The second nucleic acid strand of HDO (cRDNA MOE) is composed of three 2'-O-MOE-RNA nucleosides at the 5' end, three 2'-O-MOE-RNA nucleosides at the 3' end, and a gap region between them. In the gap region, nucleosides containing adenine and guanine bases are RNA nucleosides, and nucleosides containing cytosine and thymine bases are DNA nucleosides. For the ASO, HDO (all DNA), and HDO (cRDNA MOE) listed in Table 22, nucleic acid preparation, in vivo experiments, central neurotoxicity evaluation, and motor function evaluation were performed using the same methods as in Example 1. However, in this example, the nucleic acid agent dose per mouse was 28 nmol / mouse.

[0339] (Results) Figure 62 shows the results of acute central neurotoxicity evaluation in mice after intracerebroventricular administration of various nucleic acid agents. At 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours after administration, the HDO (all DNA) and HDO (cRDNA MOE) groups showed significantly reduced acute tolerability scores compared to the ASO group. Furthermore, the HDO (cRDNA MOE) group showed a lower acute tolerability score compared to the HDO (all DNA) group. These results indicate that HDO, in which the wing regions of the second nucleic acid strand are composed of 2'-O-MOE-RNA nucleosides and the nucleosides containing adenine and guanine bases in the gap region are RNA nucleosides and the nucleosides containing cytosine and thymine bases are DNA nucleosides, can achieve lower central neurotoxicity than other HDOs. Figure 63 shows the results of assessing motor function 1 hour after intracerebroventricular administration of various nucleic acid agents in mice. The HDO (all DNA) and HDO (cRDNA MOE) groups showed improved total distance traveled (Figure 63A) and maximum speed (Figure 63B) compared with the ASO group. Furthermore, the HDO (cRDNA MOE) group showed improved total distance traveled and maximum speed compared with the HDO (all DNA) group. These results indicate that HDO, in which the wing regions of the second nucleic acid strand are composed of 2'-O-MOE-RNA nucleosides and the nucleosides containing adenine and guanine bases in the gap region are RNA nucleosides and the nucleosides containing cytosine and thymine bases are DNA nucleosides, has a significantly smaller inhibitory effect on motor function and is significantly less toxic than other HDOs.

[0340] Example 24: Single-stranded HDO (Objective) To verify the neurotoxicity of single-stranded HDO (ssHDO), which is composed of a first nucleic acid strand consisting of an ASO targeting the Bace1 gene and a second nucleic acid strand having a base sequence complementary to the first nucleic acid strand, linked by a linker, through in vitro experiments.

[0341] (Methods) (1) Preparation of Nucleic Acids Table 23 shows the base sequences and chemical modifications of the HDO and ssHDO used in this example.

[0342]

[0343] (2) Evaluation of gene silencing effects. The HDO prepared in (1) was transfected into human neuroblastoma-derived cells (Neuro2a cell line) using lipofection (lipofectamine 2000). Forty-eight hours after HDO transfection, RNA was extracted from the cells using the Isogen I kit (Gene Design, Inc.). cDNA was synthesized using Transcriptor Universal cDNA Master, DNase (Roche Diagnostics) according to the protocol. Next, quantitative RT-PCR was performed using the resulting cDNA as a template to measure the expression levels of Bace1 mRNA and Actb mRNA (internal control genes). Quantitative RT-PCR was performed using TaqMan (Roche Applied Science). Primers used in qRT-PCR were designed and manufactured by Thermo Fisher Scientific (formerly Life Technologies Corp.). Amplification was performed by repeating 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 1 second. The ratio of Bace1 mRNA expression to Actb mRNA (internal control gene) expression was calculated, and the value normalized to the value in the PBS-treated group was used as the relative Bace1 mRNA level.

[0344] (3) Evaluation of cytotoxicity. Forty-eight hours after transfection, lactate dehydrogenase (LDH) activity in the cell supernatant was measured to assess neuronal cytotoxicity. LDH activity was measured using the Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories) according to the attached protocol. The value normalized to the LDH activity in the PBS-treated group was used as the relative LDH release level.

[0345] (Results) Figure 64B shows the results of evaluating the target gene silencing effects of various nucleic acid agents in mouse neuroblastoma-derived cells (Neuro2a cell line). The ssHDO treatment group achieved a strong gene silencing effect equivalent to that of HDO (all DNA). Figure 64A shows the results of evaluating LDH activity in the supernatant as a measure of cytotoxicity upon intracellular administration of various nucleic acid agents. Cells treated with ssHDO at 5 nM and 25 nM showed a suppressed increase in LDH activity compared to cells treated with HDO (all DNA) at 5 nM and 25 nM, respectively, demonstrating a significant reduction in cytotoxicity. These results demonstrate that ssHDO containing 2'-O-MOE-RNA nucleosides significantly reduces cytotoxicity. These results demonstrate that ssHDO achieves both reduced neuronal toxicity and potent gene silencing effects.

[0346] Example 25: Evaluation of central neurotoxicity in monkeys (Objective) MOE-modified HDO is intrathecally administered to monkeys to evaluate central neurotoxicity.

[0347] (Method) (1) Preparation of Nucleic Acids Table 24 shows the base sequences and chemical modifications of the ASO and the first and second nucleic acid strands constituting the HDO used in this example.

[0348]

[0349] Nucleic acids were prepared in the same manner as in Example 1 for the ASO, HDO (all DNA), HDO (DNA MOE), HDO (RNA MOE), and HDO (DNARNA MOE) listed in Table 24.

[0350] (2) Evaluation of central neurotoxicity in monkeys. Two male cynomolgus monkeys (3 years old, 2 kg) were administered ASO, HDO (all DNA), HDO (DNA MOE), HDO (RNA MOE), and HDO (DNARNA MOE). ASO or HDO (0.94 μmol) was administered intrathecally via lumbar puncture. To evaluate central neurotoxicity, blinded evaluators evaluated the monkeys' behavior using the modified FOB score (Figure 65) and measurements of spontaneous movement time or jump counts using a 3-minute video recording.

[0351] (Results) Figure 66 shows the results of acute central neurotoxicity evaluation in monkeys administered various nucleic acid agents intrathecally. At 1, 2, and 3 hours after nucleic acid administration, the HDO (all DNA), HDO (DNA MOE), HDO (RNA MOE), and HDO (DNARNA MOE) groups showed significantly reduced modified FOB scores compared to the ASO group. Furthermore, the HDO (DNA MOE) and HDO (DNARNA MOE) groups showed reduced modified FOB scores compared to the HDO (all DNA) group. Figure 67 also shows the results of spontaneous motor activity time and jump count measured using a 3-minute video. The HDO (all DNA), HDO (DNA MOE), HDO (RNA MOE), and HDO (DNARNA MOE) groups showed reduced decreases in spontaneous motor activity time and jump count compared to the ASO group. Furthermore, the HDO(DNA MOE) and HDO(DNARNA MOE) administration groups showed a suppressed decrease in spontaneous exercise time or jump count compared to the HDO(all DNA) administration group. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.

Claims

1. A double-stranded nucleic acid complex comprising a first nucleic acid strand and a second nucleic acid strand, the first nucleic acid strand is capable of hybridizing to at least a part of a target gene or a transcription product thereof and has an antisense effect on the target gene or a transcription product thereof; The double-stranded nucleic acid complex, wherein the second nucleic acid strand comprises a base sequence complementary to that of the first nucleic acid strand and comprises one or more 2'-modified nucleosides.

2. The double-stranded nucleic acid complex of claim 1 , wherein the first nucleic acid strand is a gapmer.

3. The double-stranded nucleic acid complex of claim 1, wherein all of the nucleosides in the first nucleic acid strand are modified nucleosides.

4. The double-stranded nucleic acid complex of claim 3, wherein all of the nucleosides in the first nucleic acid strand are 2'-modified nucleosides.

5. The double-stranded nucleic acid complex of claim 4, wherein all of the nucleosides in the first nucleic acid strand are 2'-O-methoxyethyl modified nucleosides.

6. The double-stranded nucleic acid complex of claim 1, wherein the second nucleic acid strand comprises one or two to ten consecutive 2'-modified nucleosides located at the 5'-terminus and / or one or two to ten consecutive 2'-modified nucleosides located at the 3'-terminus.

7. The double-stranded nucleic acid complex of claim 1, wherein the second nucleic acid strand comprises 1 to 7 2'-modified nucleosides at a position other than the 5' end and the 3' end.

8. the first nucleic acid strand is (1) a central region containing at least four consecutive deoxyribonucleosides; (2) a 5' wing region including an unnatural nucleoside, located on the 5' end of the central region; and (3) The double-stranded nucleic acid complex according to claim 2, further comprising a 3' wing region containing an unnatural nucleoside located on the 3' end side of the central region.

9. The double-stranded nucleic acid complex of claim 8, wherein the second nucleic acid strand contains a 2'-modified nucleoside in a region consisting of a base sequence complementary to the 5' wing region and / or the 3' wing region of the first nucleic acid strand.

10. The double-stranded nucleic acid complex of claim 9, wherein in the second nucleic acid strand, all nucleosides in a region consisting of a base sequence complementary to the 5' wing region and / or the 3' wing region of the first nucleic acid strand are 2'-modified nucleosides.

11. The double-stranded nucleic acid complex according to claim 10, wherein in the region of the second nucleic acid strand consisting of a base sequence complementary to the central region of the first nucleic acid strand, all nucleosides containing purine bases are ribonucleosides.

12. The double-stranded nucleic acid complex of claim 11, wherein in the region of the second nucleic acid strand consisting of a base sequence complementary to the central region of the first nucleic acid strand, all nucleosides containing pyrimidine bases are deoxyribonucleosides.

13. The double-stranded nucleic acid complex of claim 12, wherein in the second nucleic acid strand, the 2'-modified nucleoside is a 2'-O-methoxyethyl modified nucleoside or a 2'-O-methyl modified nucleoside.

14. In the second nucleic acid strand, all nucleosides in a region consisting of a base sequence complementary to the central region of the first nucleic acid strand are (a) deoxyribonucleosides, (b) deoxyribonucleosides and ribonucleosides, (c) deoxyribonucleosides and 2'-modified nucleosides, (d) ribonucleosides and 2'-modified nucleosides, or (e) Deoxyribonucleosides, ribonucleosides, and 2'-modified nucleosides The double-stranded nucleic acid complex of claim 8, wherein

15. In the second nucleic acid strand, all nucleosides in a region consisting of base sequences complementary to the 5' wing region and the 3' wing region of the first nucleic acid strand are 2'-modified nucleosides; and The double-stranded nucleic acid complex according to claim 14, wherein all nucleosides in the region consisting of a base sequence complementary to the central region of the first nucleic acid strand are deoxyribonucleosides.

16. 9. The double-stranded nucleic acid complex of claim 8, wherein the second nucleic acid strand comprises at least four consecutive ribonucleosides complementary to at least four consecutive deoxyribonucleosides in the central region of the first nucleic acid strand.

17. (i) at least one guanosine nucleoside in the first nucleic acid strand; (ii) a nucleoside adjacent to the 5'-terminus of the guanosine nucleoside; (iii) a nucleoside adjacent to the 3'-terminus of the guanosine nucleoside, or (iv) Any combination of (i) to (iii). The double-stranded nucleic acid complex of claim 1, wherein the nucleoside in the second nucleic acid strand that is complementary to is a 2'-modified nucleoside.

18. (i) at least one guanosine nucleoside in the 5' wing region of the first nucleic acid strand; (ii) a nucleoside adjacent to the 5'-terminus of the guanosine nucleoside; (iii) a nucleoside adjacent to the 3'-terminus of the guanosine nucleoside, or (iv) Any combination of (i) to (iii). The double-stranded nucleic acid complex of claim 1, wherein the nucleoside in the second nucleic acid strand that is complementary to is a 2'-modified nucleoside.

19. The double-stranded nucleic acid complex according to claim 1, wherein in the second nucleic acid strand, at least one nucleoside containing an adenine base or a pyrimidine base is a 2'-modified nucleoside and / or a deoxyribonucleoside.

20. The double-stranded nucleic acid complex according to claim 1, wherein at least one of the nucleosides containing a pyrimidine base in the second nucleic acid strand is a 2'-modified nucleoside and / or a deoxyribonucleoside.

21. The double-stranded nucleic acid complex of claim 1 , wherein the second nucleic acid strand does not contain any natural ribonucleosides containing pyrimidine bases.

22. The double-stranded nucleic acid complex according to claim 18, wherein all of the nucleosides containing pyrimidine bases in the second nucleic acid strand are 2'-modified nucleosides and / or deoxyribonucleosides.

23. The double-stranded nucleic acid complex of claim 1, wherein 20% or more of the nucleosides in the second nucleic acid strand are 2'-modified nucleosides.

24. The double-stranded nucleic acid complex of claim 1, wherein in the second nucleic acid strand, all nucleosides other than 2'-modified nucleosides are deoxyribonucleosides.

25. The double-stranded nucleic acid complex according to claim 1, wherein in the second nucleic acid strand, all nucleosides in the region consisting of a base sequence complementary to that of the first nucleic acid strand are 2'-modified nucleosides.

26. The double-stranded nucleic acid complex of claim 1, wherein in the second nucleic acid strand, the 2'-modified nucleosides are 2'-O-methoxyethyl modified nucleosides and / or 2'-O-methyl modified nucleosides.

27. The double-stranded nucleic acid complex of claim 1, wherein the second nucleic acid strand comprises at least one 2'-O-methyl modified nucleoside, scpBNA nucleoside, AmNA nucleoside, or BNA-NC.

28. The double-stranded nucleic acid complex of claim 1 , wherein the first nucleic acid strand is a mixmer.

29. The double-stranded nucleic acid complex according to claim 1, wherein the second nucleic acid strand contains at least one bulge structure consisting of a base sequence that is non-complementary to the first nucleic acid strand.

30. The double-stranded nucleic acid complex of claim 1, wherein the second nucleic acid strand contains non-complementary bases and / or one or more inserted sequences and / or deletions relative to the first nucleic acid strand.

31. The double-stranded nucleic acid complex of claim 27, wherein the second nucleic acid strand comprises 1 to 3 non-complementary bases.

32. The double-stranded nucleic acid complex according to claim 30, wherein the insertion sequence consists of 1 to 8 bases.

33. The double-stranded nucleic acid complex according to claim 30, wherein the deletion consists of 1 to 4 consecutive bases.

34. the non-complementary bases of the second nucleic acid strand form a bulge structure; or The double-stranded nucleic acid complex of claim 30, wherein the second nucleic acid strand comprises a bulge structure at the deleted position, the bulge structure consisting of a base sequence that is non-complementary to the first nucleic acid strand.

35. The double-stranded nucleic acid complex of claim 29, wherein the bulge structure comprises sugar-unmodified nucleosides, or all nucleosides in the bulge structure are sugar-unmodified nucleosides.

36. The double-stranded nucleic acid complex according to claim 29, wherein the bulge structure is 1 to 10 bases long.

37. 30. The double-stranded nucleic acid complex of claim 29, wherein in the second nucleic acid strand, all nucleosides other than those in the bulge structure are 2'-modified nucleosides.

38. The double-stranded nucleic acid complex of claim 1 , wherein the second nucleic acid strand is at least 8 bases in length.

39. The double-stranded nucleic acid complex according to claim 1 , wherein the base length of the second nucleic acid strand is shorter than the base length of the first nucleic acid strand.

40. 2. The double-stranded nucleic acid complex according to claim 1, wherein the second nucleic acid strand comprises at least one overhang region located on the 5'-end side and / or the 3'-end side of a region consisting of a base sequence complementary to the first nucleic acid strand.

41. The double-stranded nucleic acid complex of claim 40, wherein the overhang region is 1 to 20 bases in length.

42. The double-stranded nucleic acid complex of claim 40, wherein the overhang region comprises at least one deoxyribonucleoside and / or unnatural nucleoside.

43. The double-stranded nucleic acid complex according to claim 1 , wherein the first nucleic acid strand and the second nucleic acid strand are linked via a linker.

44. The linker is linking the 5' end of the first nucleic acid strand to the 3' end of the second nucleic acid strand; and / or 44. The double-stranded nucleic acid complex of claim 43, which links the 3' end of the first nucleic acid strand and the 5' end of the second nucleic acid strand.

45. The double-stranded nucleic acid complex of claim 43, wherein the linker is a cleavable or uncleavable linker.

46. The double-stranded nucleic acid complex of claim 43, wherein the linker is composed of a nucleic acid containing natural nucleosides and / or unnatural nucleosides, or polyethylene glycol.

47. The double-stranded nucleic acid complex according to claim 46, wherein the linker made of nucleic acid is 2 to 10 bases in length.

48. The double-stranded nucleic acid complex of claim 1 , wherein the second nucleic acid strand is bound to a ligand.

49. The double-stranded nucleic acid complex of claim 48, wherein the ligand is any one or more selected from the group consisting of a small molecule, a peptide, a lipid, and a nucleic acid aptamer.

50. 50. The double-stranded nucleic acid complex of claim 49, wherein the lipid is cholesterol or an analog thereof, or tocopherol or an analog thereof.

51. 46. ​​The double-stranded nucleic acid complex of claim 45, wherein the ligand is attached to the 5' end and / or the 3' end of the second nucleic acid strand.

52. The double-stranded nucleic acid complex of claim 1, which is not bound to a ligand.

53. The double-stranded nucleic acid complex according to claim 1, wherein all or part of the internucleoside linkages of the first nucleic acid strand and / or the second nucleic acid strand are modified internucleoside linkages.

54. 54. The double-stranded nucleic acid complex of claim 53, wherein the modified internucleoside linkage is a phosphorothioate linkage.

55. A pharmaceutical composition comprising the double-stranded nucleic acid complex according to any one of claims 1 to 54 as an active ingredient.

56. 56. The pharmaceutical composition of claim 55 for treating a central nervous system disorder in a subject.

57. 56. The pharmaceutical composition of claim 55, which is administered intracerebroventricularly or intrathecally.

58. 58. The pharmaceutical composition of claim 57, wherein the intrathecal administration is a posterior fossa puncture or a lumbar puncture.

59. 58. The pharmaceutical composition of claim 57, wherein the double-stranded nucleic acid complex is administered in an amount of 0.1 mg to 200 mg.

60. 56. The pharmaceutical composition of claim 55, which is administered intravenously or subcutaneously.

61. 61. The pharmaceutical composition of claim 60, wherein the double-stranded nucleic acid complex is administered in an amount of 0.1 mg / kg to 100 mg / kg.

62. 56. The pharmaceutical composition of claim 55, having reduced central neurotoxicity.