WO2023042876A1 - ２'－修飾ヌクレオシドを含むヘテロ核酸 - Google Patents

２'－修飾ヌクレオシドを含むヘテロ核酸 Download PDF

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WO2023042876A1
WO2023042876A1 PCT/JP2022/034531 JP2022034531W WO2023042876A1 WO 2023042876 A1 WO2023042876 A1 WO 2023042876A1 JP 2022034531 W JP2022034531 W JP 2022034531W WO 2023042876 A1 WO2023042876 A1 WO 2023042876A1
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nucleic acid
acid strand
nucleosides
hdo
double
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French (fr)
Japanese (ja)
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隆徳 横田
耕太郎 吉岡
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Tokyo Medical and Dental University NUC
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Tokyo Medical and Dental University NUC
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Priority to US18/690,457 priority Critical patent/US20250179486A1/en
Priority to EP22870023.3A priority patent/EP4403191A4/en
Priority to JP2023548500A priority patent/JPWO2023042876A1/ja
Publication of WO2023042876A1 publication Critical patent/WO2023042876A1/ja
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C12N2310/3341—5-Methylcytosine
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol

Definitions

  • the present invention relates to a double-stranded nucleic acid complex containing a 2'-modified nucleoside, and a pharmaceutical composition containing it as an active ingredient.
  • oligonucleotides In recent years, in the ongoing development of medicines called nucleic acid medicines, attention has been focused on oligonucleotides. is being actively developed.
  • a partial sequence of mRNA or miRNA transcribed from the target gene is used as the target sense strand, and a complementary oligonucleotide (antisense oligonucleotide: in this specification, often referred to as "ASO (Antisense Oligonucleotide)").
  • ASO Antisense Oligonucleotide
  • the present inventors have proposed a double-stranded nucleic acid complex (heteroduplex oligonucleotide, HDO) in which an antisense oligonucleotide and its complementary strand are annealed. developed (Patent Document 1, Non-Patent Documents 1 and 2).
  • the double-stranded nucleic acid complex has a high antisense effect, and is an epoch-making technology that enables control of the central nervous system across the blood-brain barrier.
  • mice intracerebroventricularly administered with single-stranded gapmer-type antisense nucleic acids hypoactivity and motor dysfunction due to central nervous system toxicity are observed within several hours after administration.
  • the present inventors conducted intensive research in search of a new technique that can reduce the central nervous system toxicity of nucleic acid drugs, and introduced 2'-modified nucleosides into double-stranded nucleic acid complexes. As a result, they found that introduction of 2'-modified nucleosides can dramatically reduce or eliminate central nervous system toxicity of double-stranded nucleic acid complexes. This toxicity-suppressing effect is a surprising effect that greatly exceeds conventional expectations.
  • the present invention is based on the above findings, and provides the following.
  • 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 transcript thereof; having an antisense effect on the target gene or its transcript, the second nucleic acid strand comprising a base sequence complementary to the first nucleic acid strand, and containing one or more 2'-modified nucleosides; The double-stranded nucleic acid complex.
  • the second nucleic acid strand has 1 or 2 to 10 consecutive 2'-modified nucleosides located at the 5' end and/or 1 or 2 to 10 consecutive 2'-modified nucleosides located at the 3' end
  • the two strands according to any one of (1) to (6), wherein the second nucleic acid strand contains 1 to 7 2'-modified nucleosides at positions other than the 5' and 3' ends. Stranded nucleic acid complex.
  • the first nucleic acid strand is (1) a central region comprising at least 4 consecutive deoxyribonucleosides; (2) a 5' wing region containing a non-natural nucleoside located at the 5' end of the central region; and (3) a 3' wing region containing a non-natural nucleoside located at the 3' end of the central region.
  • the second nucleic acid strand contains a 2'-modified nucleoside in a region consisting of a nucleotide sequence complementary to the 5' wing region and/or the 3' wing region of the first nucleic acid strand; double-stranded nucleic acid complex of. (10)
  • all nucleosides in a 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, ( The double-stranded nucleic acid complex according to 9).
  • 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,
  • the double-stranded nucleic acid complex according to any one of (8) to (10), which is (d) a ribonucleoside and a 2'-modified nucleoside, or (e) a deoxyribonucleoside, a ribonucleoside, and a 2'-modified nucleoside.
  • 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
  • the double-stranded nucleic acid complex according to (14), wherein all nucleosides in the region consisting of a nucleotide sequence complementary to the central region of one nucleic acid strand are deoxyribonucleosides.
  • said second nucleic acid strand comprises at least 4 contiguous ribonucleosides complementary to at least 4 contiguous deoxyribonucleosides in said central region of said first nucleic acid strand, (8)-(10) ), the double-stranded nucleic acid complex according to any one of (17) (i) at least one guanosine nucleoside in said first nucleic acid strand; (ii) a nucleoside adjacent to the 5' end of the guanosine nucleoside; (iii) a 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) is a 2'-modified nucleoside
  • Stranded nucleic acid complex (20) The double-stranded nucleic acid complex according to any one of (1) to (19), wherein at least one nucleoside containing a pyrimidine base in the second nucleic acid strand is a 2'-modified nucleoside and/or a deoxyribonucleoside. body. (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 nucleosides containing pyrimidine bases 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 all nucleosides other than 2'-modified nucleosides in the second nucleic acid strand are deoxyribonucleosides.
  • the second nucleic acid strand according to any one of (1) to (24), wherein all nucleosides in a region consisting of a base sequence complementary to the first nucleic acid strand are 2'-modified nucleosides. Stranded nucleic acid complex.
  • the 2'-modified nucleoside is a 2'-O-methoxyethyl modified nucleoside and/or a 2'-O-methyl modified nucleoside.
  • the double-stranded nucleic acid complex according to 1.
  • the non-complementary bases of the second nucleic acid strand form a bulge structure, or the deletion position of the second nucleic acid strand consists of a base sequence that is non-complementary to the first nucleic acid strand.
  • the double-stranded nucleic acid complex according to any one of (30) to (33), which contains a bulge structure.
  • (35) The double-stranded nucleic acid complex according to (29) or (34), wherein the bulge structure contains sugar-unmodified nucleosides, or all nucleosides in the bulge structure are sugar-unmodified nucleosides.
  • the second nucleic acid strand comprises at least one overhang region located on the 5'-terminal side and/or 3'-terminal side of a region consisting of a base sequence complementary to the first nucleic acid strand, (1 ) to the double-stranded nucleic acid complex according to any one of (39).
  • (41) The double-stranded nucleic acid complex according to (40), wherein the overhang region has a length of 1 to 20 bases.
  • the linker connects the 5' end of the first nucleic acid strand and the 3' end of the second nucleic acid strand, and/or the 3' end of the first nucleic acid strand and the second nucleic acid strand.
  • a pharmaceutical composition comprising the double-stranded nucleic acid complex according to any one of (1) to (54) as an active ingredient.
  • a double-stranded nucleic acid complex with reduced central nervous system toxicity is provided.
  • FIG. 1 shows the structures of various crosslinked nucleic acids.
  • Figure 2 shows the structures of various natural or non-natural nucleotides.
  • FIG. 3 shows a scoring system for evaluating central neurotoxicity in mice after administration of nucleic acid agents.
  • 4 shows the structure of the nucleic acid used in Example 1.
  • FIG. 4A shows the structure of ASO targeting the Mapt gene.
  • FIG. 4B shows the structure of HDO (all RNA).
  • FIG. 4C shows the structure of HDO (all DNA).
  • FIG. 4D shows the structure of HDO (6MOE wing).
  • FIG. 4E shows the structure of HDO (all MOE).
  • FIG. 5 shows acute tolerability scores 30 minutes to 4 hours after administration of mice intracerebroventricularly administered with various nucleic acid agents. Error bars indicate standard error.
  • FIG. 6 shows the motor function of mice intracerebroventricularly administered with various nucleic acid agents 1 hour after administration.
  • Figure 6A shows the total distance traveled for 5 minutes.
  • FIG. 6B shows the maximum movement speed. Error bars indicate standard error.
  • FIG. 7 shows Mapt mRNA expression levels in the hippocampus of mice to which various nucleic acid agents were intracerebroventricularly administered. Error bars indicate standard error.
  • 8 shows the structure of the nucleic acid used in Example 2.
  • FIG. FIG. 8A shows the structure of ASO targeting the BACE1 gene.
  • Figure 8B shows the structure of HDO (all RNA).
  • FIG. 8C shows the structure of HDO (all DNA).
  • FIG. 8D shows the structure of HDO (5MOE wing).
  • FIG. 9 shows acute tolerability scores 30 minutes to 4 hours after administration of mice intracerebroventricularly administered with various nucleic acid agents. Error bars indicate standard error.
  • FIG. 10 shows the motor function of mice intracerebroventricularly administered with various nucleic acid agents 1 hour after administration.
  • FIG. 10A shows the total distance traveled for 5 minutes.
  • FIG. 10B shows maximum movement speed. Error bars indicate standard error.
  • 11 shows the structure of the nucleic acid used in Example 3.
  • FIG. 11A shows the structure of ASO targeting the Malat1 gene.
  • Figure 11B shows the structure of HDO (all RNA).
  • FIG. 11C shows the structure of HDO (all DNA).
  • FIG. 11D shows the structure of HDO (10 MOE wing).
  • FIG. 12 shows acute tolerability scores 30 minutes to 4 hours after administration of mice intracerebroventricularly administered with various nucleic acid agents. Error bars indicate standard error.
  • FIG. 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 for 5 minutes.
  • FIG. 13B shows maximum movement speed. Error bars indicate standard error.
  • 14 shows the structure of the nucleic acid used in Example 4.
  • FIG. 14A shows the structure of HDO (all DNA) containing ASOs targeting the Mapt gene.
  • FIG. 14B shows the structure of HDO (RNA 6MOE wing).
  • FIG. 14C shows the structure of HDO (6MOE wing).
  • FIG. 14D shows the structure of HDO (6OMe wing).
  • FIG. 14A shows the structure of HDO (all DNA) containing ASOs targeting the Mapt gene.
  • FIG. 14B shows the structure of HDO (RNA 6MOE wing).
  • FIG. 14E shows the structure of HDO (6F wing).
  • FIG. 15 shows acute tolerability scores 30 minutes to 4 hours after administration of mice intracerebroventricularly administered with various nucleic acid agents. Error bars indicate standard error.
  • FIG. 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 for 5 minutes. FIG. 16B shows maximum movement speed. Error bars indicate standard error.
  • FIG. 17 shows the motor function of mice intracerebroventricularly administered with various nucleic acid agents 3 hours after administration. Figure 17A shows the total distance traveled for 5 minutes. FIG. 17B shows the maximum movement speed. Error bars indicate standard error. 18 shows the structure of the nucleic acid used in Example 5.
  • FIG. 15 shows acute tolerability scores 30 minutes to 4 hours after administration of mice intracerebroventricularly administered with various nucleic acid agents. Error bars indicate standard error.
  • FIG. 16 shows the motor function of mice intracerebroventricularly administered with various
  • FIG. 18A shows the structure of an ASO targeting the Mapt gene.
  • FIG. 18B shows the structure of HDO (6MOE wing).
  • FIG. 18C shows the structure of HDO (G MOE ).
  • FIG. 18D shows the structure of HDO (G RNA ).
  • Figure 18E shows the structure of HDO (inosine).
  • FIG. 19 shows acute tolerability scores 30 minutes to 4 hours after administration of mice intracerebroventricularly administered with various nucleic acid agents. Error bars indicate standard error.
  • 20 shows the structure of the nucleic acid used in Example 6.
  • FIG. 20A shows the structure of HDO (all DNA) containing ASOs targeting the Mapt gene.
  • FIG. 20B shows the structure of HDO (6MOE-5'&3').
  • FIG. 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').
  • FIG. 20F shows the structure of HDO(10MOE-3′).
  • FIG. 21 shows acute tolerability scores 30 minutes to 4 hours after administration of mice intracerebroventricularly administered with various nucleic acid agents. Error bars indicate standard error.
  • FIG. 22 shows the motor function of mice intracerebroventricularly administered with various nucleic acid agents 1 hour after administration.
  • Figure 22A shows the total distance traveled for 5 minutes.
  • FIG. 22B shows the maximum movement speed. Error bars indicate standard error.
  • FIG. 23 shows the motor function of mice intracerebroventricularly administered with various nucleic acid agents 3 hours after administration.
  • FIG. 23A shows the total distance traveled for 5 minutes.
  • FIG. 23B shows the maximum movement speed. Error bars indicate standard error.
  • FIG. 24 shows Mapt mRNA expression levels in the hippocampus of mice to which various nucleic acid agents were intracerebroventricularly administered. Error bars indicate standard error.
  • 25 shows the structure of the nucleic acid used in Example 7.
  • FIG. 25A shows the structure of HDO (all DNA) containing ASOs 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').
  • FIG. 25G shows the structure of HDO (10MOE-3').
  • FIG. 26 shows acute tolerability scores 30 minutes to 4 hours after administration of mice intracerebroventricularly administered with various nucleic acid agents. Error bars indicate standard error.
  • FIG. 27 shows the motor function of mice intracerebroventricularly administered with various nucleic acid agents 1 hour after administration.
  • Figure 27A shows the total distance traveled for 5 minutes.
  • FIG. 27B shows the maximum movement speed. Error bars indicate standard error.
  • 28 shows the structure of the nucleic acid used in Example 8.
  • FIG. 28A shows the structure of HDO (all DNA) containing ASOs targeting the Mapt gene.
  • FIG. 28B shows the structure of HDO (6MOE wing).
  • FIG. 28C shows the structure of HDO (9MOE wing).
  • FIG. 28D shows the structure of HDO (11MOE wing).
  • FIG. 28E shows the structure of HDO (13MOE wing).
  • FIG. 28F shows the structure of HDO (15 MOE wing).
  • FIG. 29 shows acute tolerability scores 30 minutes to 4 hours after administration of mice intracerebroventricularly administered with various nucleic acid agents. Error bars indicate standard error.
  • FIG. 30 shows the motor function of mice intracerebroventricularly administered with various nucleic acid agents 1 hour after administration.
  • Figure 27A shows the total distance traveled for 5 minutes.
  • FIG. 27B shows the maximum movement speed. Error bars indicate standard error.
  • 31 shows the structure of the nucleic acid used in Example 9.
  • FIG. 31A shows the structure of HDO (all DNA) containing ASOs targeting the Mapt gene.
  • FIG. 31B shows the structure of HDO (A MOE ).
  • FIG. 31C shows the structure of HDO (G MOE ).
  • FIG. 31D shows the structure of HDO ( CMOE ).
  • FIG. 31E shows the structure of HDO( TMOE ).
  • FIG. 32 shows acute tolerability scores 30 minutes to 4 hours after administration of mice intracerebroventricularly administered with various nucleic acid agents. Error bars indicate standard error.
  • FIG. 33 shows the motor function of mice intracerebroventricularly administered with various nucleic acid agents 1 hour after administration.
  • Figure 33A shows the total distance traveled for 5 minutes.
  • FIG. 33B shows maximum movement speed. Error bars indicate standard error.
  • 34 shows the structure of the nucleic acid used in Example 10.
  • FIG. 34A shows the structure of HDO (all DNA) containing ASOs targeting the Mapt gene.
  • FIG. 34B shows the structure of HDO ( CMOE ).
  • Figure 34C shows the structure of HDO (2C MOE -5).
  • FIG. 34D shows the structure of HDO (2C MOE -3).
  • FIG. 34E shows the structure of HDO (3C MOE ).
  • FIG. 35 shows acute tolerability scores 30 minutes to 4 hours after administration of mice intracerebroventricularly administered with various nucleic acid agents. Error bars indicate standard error.
  • FIG. 36 shows the motor function of mice intracerebroventricularly administered with various nucleic acid agents 1 hour after administration.
  • Figure 36A shows the total distance traveled for 5 minutes.
  • FIG. 36B shows maximum movement speed. Error bars indicate standard error.
  • Figure 37 shows the stability of various nucleic acid agents in human cerebrospinal fluid.
  • 37A and B show the structure of the nucleic acid used in Example 11.
  • FIG. Figure 37A shows the structure of an ASO targeting the Mapt gene.
  • Figure 37B shows the structure of HDO (ASO/cRNA).
  • FIG. 37C shows the results of examining stability by electrophoresis after mixing various nucleic acid agents with human cerebrospinal fluid (Human CSF; hCSF) for 10 minutes or 6 hours.
  • FIG. 37D shows the results of quantification of band intensity of HDO duplex in HDO (ASO/cRNA).
  • FIG. 38 shows the stability of various nucleic acid agents in human and rat cerebrospinal fluid.
  • FIG. 38A and 38B show the structure of the nucleic acid used in Example 11.
  • FIG. Figure 38A shows the structure of an HDO containing an ASO targeting the Mapt gene (ASO/cRNA).
  • Figure 38B shows the structure of HDO (ASO/cDNA).
  • FIG. 38C shows the results of examining stability by electrophoresis after mixing various nucleic acid agents with human or rat cerebrospinal fluid for 6 hours.
  • FIG. 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.
  • FIG. 39A and 39B show the structure of the nucleic acid used in Example 12.
  • FIG. 39A shows the structure of HDO (all RNA) containing ASOs targeting the Mapt gene.
  • Figure 39B shows the structure of HDO (all DNA).
  • FIG. 39C shows the results of examining the stability by electrophoresis after mixing various nucleic acid agents with mouse, rat, monkey, and human cerebrospinal fluid for 6 hours.
  • FIG. 40 shows the stability of various nucleic acid agents in mouse, rat, monkey, and human cerebrospinal fluid.
  • 40A and 40B show the structures of the nucleic acids used in Example 12.
  • FIG. 40A shows the structure of HDO (cRNA 6MOE wing) containing an ASO targeting the Mapt gene.
  • FIG. 40B shows the structure of HDO (cDNA 6MOE wing).
  • FIG. 40C shows the results of mixing various nucleic acid agents with cerebrospinal fluid of mice, rats, monkeys and humans for 6 hours and then examining the stability by electrophoresis.
  • 41 shows the structure of the nucleic acid used in Example 13.
  • FIG. 41A shows the structure of HDO (all DNA) containing ASOs targeting the Mapt gene.
  • FIG. 41B shows the structure of HDO(A RNA ).
  • Figure 41C shows the structure of HDO (G RNA ).
  • FIG. 41D shows the structure of HDO(C RNA ).
  • FIG. 41E shows the structure of HDO(U RNA ).
  • FIG. 42 shows the results of examining stability by electrophoresis after mixing various nucleic acid agents with human cerebrospinal fluid for 6 hours.
  • 43 shows the structure of the nucleic acid used in Example 14.
  • FIG. Figure 43A shows the structure of HDO (all DNA) containing ASOs targeting the Mapt gene.
  • FIG. 43B shows the structure of HDO (GA RNA ).
  • FIG. 43C shows the structure of HDO (CU RNA ).
  • FIG. 43D shows the structure of HDO(C RNA ).
  • FIG. 43E shows the structure of HDO(U RNA ).
  • FIG. 44 shows the results of examining stability by electrophoresis after mixing various nucleic acid agents with human cerebrospinal fluid for 1 hour or 6 hours.
  • 45 shows the structure of the nucleic acid used in Example 15.
  • FIG. FIG. 45A shows the structure of HDO (all DNA) containing ASOs targeting the Malat1 gene.
  • FIG. 45B shows the structure of HDO(A RNA ).
  • Figure 45C shows the structure of HDO (G RNA ).
  • FIG. 45D shows the structure of HDO(C RNA ).
  • Figure 45E shows the structure of HDO (U RNA ).
  • FIG. 46 shows the results of examining stability by electrophoresis after mixing various nucleic acid agents with human cerebrospinal fluid for 6 hours.
  • FIG. 47 shows the results of evaluating the central nervous system toxicity of nucleic acid agents in monkeys.
  • 47A-C show the structures of the nucleic acids used in Example 16.
  • FIG. Figure 47A shows the structure of an ASO targeting the Mapt gene.
  • FIG. 47B shows the structure of HDO (RNA-MOE).
  • Figure 47C shows the structure of HDO (DNA-MOE).
  • FIG. 47D shows the procedure for evaluating central nervous system toxicity in monkeys in Example 16.
  • FIG. 47E shows the results of evaluating central nervous system toxicity of various nucleic acid agents in monkeys.
  • 48 shows the structure of the nucleic acid used in Example 17.
  • FIG. Figure 48A shows the structure of HDO (all DNA).
  • FIG. 48B shows the structure of HDO (all MOE).
  • FIG. 48C shows the structure of HDO (bulge1).
  • FIG. 48D shows the structure of HDO (bulge2).
  • FIG. 49 shows Mapt mRNA expression levels and LDH activity in human neuroblastoma-derived cells transfected with various nucleic acid agents.
  • FIG. 49A shows relative Mapt mRNA levels.
  • Figure 49B shows relative LDH release levels in the supernatant. Error bars indicate standard error.
  • 50 shows the structure of the nucleic acid used in Example 18.
  • FIG. 50A shows the structure of HDO(bulge).
  • FIG. 50B shows the structure of HDO (ssHDO).
  • FIG. 50C shows the structure of PEG linker ssHDO.
  • FIG. 50D shows the structure of Bulge plus ssHDO.
  • FIG. 51 shows the results of evaluating motor function in mice to which various nucleic acid agents were intracerebroventricularly administered 1 hour after administration.
  • FIG. 51A shows the total distance traveled for 5 minutes.
  • FIG. 51B shows the maximum movement speed. Error bars indicate standard error.
  • FIG. 51A shows the total distance traveled for 5 minutes.
  • FIG. 51B shows the maximum movement speed. Error bars indicate standard error.
  • FIG. 51A shows the total distance traveled for 5 minutes.
  • FIG. 52 shows the expression levels of Malat1 RNA in the brains of mice to which various nucleic acid agents were intracerebroventricularly administered.
  • FIG. 52A shows results for the left frontal cortex.
  • FIG. 52B shows results for the right frontal cortex. Error bars indicate standard error.
  • FIG. 53 shows the results of incubating various nucleic acid agents in brain tissue homogenate for 7 days and evaluating the efficiency of dissociation of double strands by electrophoresis.
  • FIG. 54 shows acute phase tolerability scores 30 minutes to 4 hours after administration of mice intracerebroventricularly administered with various nucleic acid agents. Error bars indicate standard error.
  • FIG. 55 shows the results of evaluating motor function in mice to which various nucleic acid agents were intracerebroventricularly administered 1 hour after administration.
  • FIG. 55A shows the total distance traveled for 5 minutes.
  • FIG. 55B shows maximum movement speed. Error bars indicate standard error.
  • FIG. 56 shows acute phase tolerability scores 30 minutes to 4 hours after administration of mice intracerebroventricularly administered with various nucleic acid agents. Error bars indicate standard error.
  • FIG. 57 shows the results of evaluating motor function in mice to which various nucleic acid agents were intracerebroventricularly administered 1 hour after administration.
  • Figure 57A shows the total distance traveled for 5 minutes.
  • Figure 57B shows the maximum movement speed. Error bars indicate standard error.
  • FIG. 58 shows the results of measuring the body weight of mice to which various nucleic acid agents were intracerebroventricularly administered. Error bars indicate standard error. Error bars indicate standard error.
  • FIG. 59 shows the results of evaluation of motor function in mice to which various nucleic acid agents were intracerebroventricularly administered one day after administration.
  • FIG. 60 shows the expression level of Mapt mRNA in the right frontal lobe of mice to which various nucleic acid agents were intracerebroventricularly administered. Error bars indicate standard error.
  • FIG. 61 shows the results of incubating various nucleic acid agents in a brain tissue homogenate for 7 days and evaluating the efficiency of dissociation of double strands by electrophoresis.
  • FIG. 62 shows acute phase tolerability scores 30 minutes to 4 hours after administration of mice intracerebroventricularly administered with various nucleic acid agents. Error bars indicate standard error.
  • FIG. 63 shows the results of evaluating motor function in mice to which various nucleic acid agents were intracerebroventricularly administered 1 hour after administration.
  • Figure 63A shows the total distance traveled for 5 minutes.
  • FIG. 63B shows maximum movement speed. Error bars indicate standard error.
  • FIG. 64 shows LDH activity and Bace1 mRNA expression levels in mouse neuroblastoma-derived cells (Neuro2a cell line) introduced with various nucleic acid agents.
  • Figure 64A shows relative LDH release levels in the supernatant.
  • Figure 64B shows relative Bace1 mRNA levels. Error bars indicate standard error.
  • FIG. 65 shows a modified FOB score evaluation method.
  • FIG. 66 shows the results of evaluation of modified FOB scores in monkeys intrathecally administered with various nucleic acid agents.
  • FIG. 67 shows the results of measurement of spontaneous movement time and number of jumps by 3-minute video animation in monkeys to which various nucleic acid agents were intrathecally administered.
  • Double-stranded nucleic acid complex 1-1 A first aspect of the present invention is a double-stranded nucleic acid complex.
  • a double-stranded nucleic acid complex of the present invention comprises a first nucleic acid strand and a second nucleic acid strand, and comprises 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.
  • a "transcript" of a target gene refers to any RNA that is directly targeted by the nucleic acid complex of the present invention and synthesized by RNA polymerase. Specifically, mRNA transcribed from target genes (including mature mRNA, pre-mRNA, and mRNA that has not undergone base modification), non-coding RNA such as miRNA (non-coding RNA, ncRNA), long non-coding RNA RNA (lncRNA), which may include natural antisense RNA.
  • target genes including mature mRNA, pre-mRNA, and mRNA that has not undergone base modification
  • non-coding RNA such as miRNA (non-coding RNA, ncRNA), long non-coding RNA RNA (lncRNA), which may include natural antisense RNA.
  • target gene refers to the function of a transcription product or translation product whose expression level can be suppressed or enhanced by the antisense effect of the double-stranded nucleic acid complex of the present invention. can be inhibited, or steric blocking, splicing switch, RNA editing, exon skipping or exon inclusion can be induced.
  • the type of target gene is not particularly limited as long as it is expressed in vivo.
  • genes derived from organisms into which the double-stranded nucleic acid complex of the present invention is introduced such as genes whose expression is increased in various diseases. is mentioned.
  • scavenger receptor B1 (herein often referred to as "SR-B1”) gene
  • metastasis associated lung adenocarcinoma transcript 1 (herein often referred to as abbreviated as "Malat1”) gene
  • microtubule-associated protein tau in this specification, often abbreviated as "Mapt” gene
  • ⁇ -secretase 1 (beta-secretase 1: in this specification , often referred to as "BACE1” gene
  • DMPK dystrophia myotonica-protein kinase
  • target transcript refers to any RNA that is directly targeted by the nucleic acid complex of the present invention and synthesized by RNA polymerase.
  • transcription products of target genes correspond. Specifically, mRNA transcribed from target genes (including mature mRNA, pre-mRNA, and mRNA that has not undergone base modification), non-coding RNA such as miRNA (non-coding RNA, ncRNA), long non-coding RNA RNA (lncRNA), which may include natural antisense RNA.
  • target gene transcripts examples include SR-B1 mRNA, which is the transcript of the SR-B1 gene, Mapt mRNA, which is the transcript of the Mapt gene, BACE1 mRNA, which is the transcript of the BACE1 gene, and transcript of the Malat1 gene.
  • Mapt mRNA which is the transcript of the Mapt gene
  • BACE1 mRNA which is the transcript of the BACE1 gene
  • Malat1 non-coding RNA Malat1 non-coding RNA
  • DMPK mRNA which is a transcription product of the DMPK gene
  • dystrophin mRNA which is a transcription product of the dystrophin gene, or its precursor (pre-mRNA).
  • target transcripts include the exon 23/intron 23 boundary region of Dystrophin pre-mRNA (GenBank Accession Number: NC_000086.7), such as positions 83803482 to 83803566, such as positions 83803512 to 83803536.
  • SEQ ID NO: 7 shows the nucleotide sequence of mouse DMPK mRNA
  • SEQ ID NO: 8 shows the nucleotide sequence of human DMPK mRNA. In each of SEQ ID NOs: 7 to 8, the base sequence of mRNA is replaced with the base sequence of DNA.
  • the nucleotide sequence information of these genes and transcripts can be obtained from known databases such as the NCBI (National Center for Biotechnology Information) database.
  • antisense oligonucleotides or “antisense nucleic acids” are capable of hybridizing (i.e., complementary a) refers to a single-stranded oligonucleotide containing a base sequence and capable of producing an antisense effect on a target transcript.
  • the first nucleic acid strand functions as an ASO, and its target region is 3'UTR, 5'UTR, exon, intron, coding region, translation initiation region, translation termination region, or Any other nucleic acid region may be included.
  • the target region of the target transcript is at least 8 bases long, e.g. It can be 16-22 bases long, or 16-20 bases long.
  • Antisense effect refers to the effect of regulating the expression or editing of a target transcript (eg, RNA sense strand) by hybridizing the ASO to the target transcript.
  • “Modulating the expression or editing of a target transcript” refers to the expression of a target gene or the expression level of a target transcript (herein, “target transcript expression level” is often referred to as “target transcript level”). expression), inhibition of translation, RNA editing, splicing function modification effects (eg, splicing switch, exon inclusion, exon skipping, etc.), or degradation of transcripts.
  • RNA oligonucleotide when introduced into a cell as an ASO, the ASO forms a partial duplex by annealing with mRNA, the transcript of the target gene. This partial double strand serves as a cover to prevent translation by the ribosome, thereby inhibiting the expression of the target protein encoded by the target gene at the translational level (steric blocking).
  • oligonucleotides containing DNA as ASO are introduced into cells, partial DNA-RNA heteroduplexes are formed. Recognition of this heteroduplex structure by RNase H results in degradation of target gene mRNA and inhibition of expression of the protein encoded by the target gene at the expression level.
  • antisense effects can also be produced by targeting introns in pre-mRNAs.
  • antisense effects can also be produced by targeting miRNAs.
  • the functional inhibition of the miRNA can increase the expression of genes whose expression is normally controlled by the miRNA.
  • modulating expression of the target transcript may be reduction in target transcript abundance.
  • the antisense effect is measured, for example, by administering a test nucleic acid compound to a subject (eg, a mouse), and, for example, several days later (eg, 2 to 7 days later), the expression is regulated by the antisense effect provided by the test nucleic acid compound. It can be carried out by measuring the expression level of the target gene or the level (amount) of the target transcript (for example, the amount of mRNA or RNA such as microRNA, the amount of cDNA, the amount of protein, etc.).
  • the measured target gene expression level or target transcript level is 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) If so, it indicates that the test nucleic acid compound is capable of producing an antisense effect (eg, reduction in target transcript abundance).
  • a negative control e.g., vehicle administration
  • the number, type and position of non-natural nucleotides in the nucleic acid chain can affect the antisense effect provided by the nucleic acid complex.
  • the selection of modification may differ depending on the sequence of the target gene, etc., but a person skilled in the art should refer to the literature related to the antisense method (for example, WO 2007/143315, WO 2008/043753, and WO 2008/049085).
  • the preferred embodiment can be determined by reference.
  • the measured value thus obtained is not significantly lower than the measured value of the nucleic acid complex before modification (e.g., When the measured value obtained after modification is 70% or more, 80% or more, or 90% or more of the measured value of the nucleic acid complex before modification), the relevant modification can be evaluated.
  • target gene translation product refers to any polypeptide or protein synthesized by translation of the target transcript or target gene transcript that is the direct target of the nucleic acid complex of the present invention. say.
  • aptamer refers to a nucleic acid molecule that specifically binds to a specific target molecule intracellularly, on the cell membrane, or extracellularly, for example, on the cell membrane or extracellularly. Aptamers can be produced by methods known in the art, for example, in vitro selection using the SELEX (systematic evolution of ligands by exponential enrichment) method.
  • the term “decoy” refers to a nucleic acid having a binding site sequence of a transcription factor (for example, NF-kB) or a similar sequence.
  • a transcription factor for example, NF-kB
  • a substance that suppresses the action represses transcription if it is a transcription activator, or promotes transcription if it is a transcription repressor.
  • a decoy nucleic acid can be easily designed based on the information of the binding sequence of the target transcription factor.
  • bait refers to a nucleic acid molecule that specifically binds to a specific target molecule in cells and modifies the function of the target molecule.
  • a target that interacts with a bait is also called a "prey”.
  • nucleic acid or “nucleic acid molecule” may refer to a monomeric nucleotide or nucleoside, or may refer to an oligonucleotide composed of more than one monomer, or if a polymer means a polynucleotide.
  • Natural nucleic acid refers to a nucleic acid that occurs in nature. Natural nucleic acids include natural nucleosides, natural nucleotides, and the like, which will be described later.
  • Non-natural nucleic acid or “artificial nucleic acid” refers to any nucleic acid other than a naturally occurring nucleic acid. Non-natural nucleic acids or artificial nucleic acids include non-natural nucleosides, non-natural nucleotides, and the like, which will be described later.
  • nucleic acid strand or simply “strand” means two or more nucleosides linked by internucleoside linkages, and may be, for example, oligonucleotides or polynucleotides. Nucleic acid strands can be produced full length or partial strands by chemical synthetic methods, for example, using automated synthesizers, or by enzymatic processes using polymerases, ligases, or restriction reactions. A nucleic acid strand may comprise naturally occurring and/or non-naturally occurring nucleotides.
  • Nucleoside generally refers to a molecule consisting of a combination of a base and a sugar.
  • the sugar moiety of a nucleoside is usually, but not limited to, composed of pentofuranosyl sugars, specific examples of which include ribose and deoxyribose.
  • the base portion (nucleobase) of a nucleoside is usually a heterocyclic base portion. Non-limiting examples include adenine, cytosine, guanine, thymine, or uracil, as well as other modified nucleobases (modified bases).
  • Nucleotide refers to a molecule in which a phosphate group is covalently bonded to the sugar moiety of the nucleoside. Nucleotides containing a pentofuranosyl sugar typically have a phosphate group attached to the hydroxyl group at the 2', 3', or 5' position of the sugar.
  • Oligonucleotide refers to a linear oligomer formed by covalently linking several to several tens of hydroxyl groups and phosphate groups of sugar moieties between adjacent nucleotides.
  • polynucleotide refers to a linear polymer formed by linking several tens or more, preferably several hundred or more nucleotides by covalent bonds, which is larger than that of oligonucleotides.
  • phosphate groups are commonly considered to form internucleoside linkages.
  • Natural nucleoside refers to a nucleoside that exists in nature. Examples thereof include ribonucleosides composed of ribose and a base such as adenine, cytosine, guanine, or uracil, and deoxyribonucleosides composed 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 as "DNA nucleosides" and "RNA nucleosides", respectively.
  • naturally occurring nucleotide refers to a naturally occurring nucleotide in which a phosphate group is covalently bonded to the sugar moiety of the natural nucleoside.
  • ribonucleotides known as structural units of RNA, in which a phosphate group is bound to a ribonucleoside
  • deoxyribonucleotides known as a structural unit of DNA, in which a phosphate group is bound to a deoxyribonucleoside.
  • non-natural nucleotide refers to any nucleotide other than natural nucleotides, including modified nucleotides and nucleotide mimetics.
  • modified nucleotide means a nucleotide having any one or more of modified sugar moieties, modified internucleoside linkages, and modified nucleobases.
  • nucleotide mimetics include structures that are used to replace nucleosides and linkages at one or more positions of an oligomeric compound.
  • Peptide Nucleic Acid is a nucleotide mimetic with a backbone of N-(2-aminoethyl)glycine instead of sugar linked by amide bonds.
  • Nucleic acid strands comprising unnatural oligonucleotides, as used herein, are often characterized by, for example, enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity. has the desired properties of Therefore, it is preferred over natural nucleotides.
  • non-natural nucleoside refers to any nucleoside other than natural nucleosides. Examples include modified nucleosides and nucleoside mimetics. As used herein, “modified nucleoside” means a nucleoside having a modified sugar moiety and/or modified nucleobase.
  • mimetics refers to functional groups that replace sugars, nucleobases, and/or internucleoside linkages. In general, mimetics are used in place of the sugar or sugar-internucleoside linkage combination so that the nucleobase is maintained for hybridization to a target of choice.
  • nucleoside mimetic refers to substitution of a sugar at one or more positions of an oligomeric compound, substitution of a sugar and a base, or binding between monomer subunits constituting an oligomeric compound. Contains the structure used to replace.
  • oligomeric compound is meant a polymer of linked monomeric subunits capable of hybridizing to at least a region of a nucleic acid molecule.
  • Nucleoside mimetics include, for example, morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclic or tricyclic sugar mimetics, eg, nucleoside mimetics having non-furanose sugar units.
  • Modified sugar refers to a sugar that has a substitution and/or any change from the natural sugar moiety (i.e., sugar moieties found in DNA (2'-H) or RNA (2'-OH));
  • a “sugar modification” refers to a substitution and/or any change from a naturally occurring sugar moiety.
  • Nucleic acid strands may optionally contain one or more modified nucleosides, including modified sugars.
  • a “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 nucleic acid chains.
  • nucleosides include chemically modified ribofuranose ring moieties.
  • chemically modified ribofuranose rings include, but are not limited to, addition of substituents (including 5' and 2' substituents), bridging of non-geminal ring atoms to form bicyclic nucleic acids (bridged nucleic acids, BNA ), S, N(R), or C(R1)(R2) of the ribosyl ring oxygen atom (R, R1 and R2 are each independently H, C1 - C12 alkyl, or a protecting group ), and combinations thereof.
  • sugar modified nucleosides include, but are not limited to, 5'-vinyl, 5'-methyl (R or S), 5'-allyl (R or S), 4'-S, 2'-F ( 2'-fluoro group), 2'- OCH3 (2'-O-Me group or 2'-O-methyl group), 2'-O-[2-(N-methylcarbamoyl)ethyl] (2'- O-MCE group), and nucleosides containing 2'-O-methoxyethyl (2'-O- MOE or 2-O( CH2 ) 2OCH3 ) substituents.
  • a "2'-modified sugar” means a furanosyl sugar modified at the 2' position. Nucleosides containing 2'-modified sugars are sometimes referred to as "2'-modified nucleosides" or "2'-sugar modified nucleosides.”
  • Bicyclic nucleoside refers to a modified nucleoside containing a bicyclic sugar moiety. Nucleic acids containing bicyclic sugar moieties are commonly referred to as bridged nucleic acid (BNA). Nucleosides containing a bicyclic sugar moiety are sometimes referred to as “bridged nucleosides,” “bridged non-natural nucleosides,” or “BNA nucleosides.” Some examples of crosslinked nucleic acids are shown in FIG.
  • 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 of skill in the art.
  • One subgroup of bicyclic sugar containing nucleic acids (BNA) or BNA nucleosides are 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 R 3 is a hydrogen atom, an alkyl group, an alkenyl group, a cyclo Represents an alkyl group, an aryl group,
  • R 1 and R 2 are typically is a hydrogen atom, which may be the same or different, and is also a hydroxyl group-protecting group for nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl a sulfonyl group, a silyl group, a phosphate group, a phosphate group protected by a protecting group for nucleic acid synthesis, or P(R 4 )R 5 [wherein R 4 and R 5 are 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, 1
  • Non-limiting examples of such BNA nucleosides include methyleneoxy(4'- CH2 -O-2') BNA nucleosides (LNA nucleosides, also known as 2',4'-BNA nucleosides) (e.g.
  • amine BNA nucleosides as 2'-Amino-LNA nucleosides
  • 2'-O,4'-C-spirocyclopropylene bridged nucleosides also known as scpBNA nucleosides
  • other BNA nucleosides known to those skilled in the art.
  • a "cationic nucleoside” refers to, at a certain pH (e.g., human physiological pH (about 7.4), pH of a delivery site (e.g., organelle, cell, tissue, organ, organism, etc.)) A modified nucleoside that exists as a cationic form as compared to a neutral form (such as the neutral form of a ribonucleoside).
  • Cationic nucleosides may contain one or more cationic modifying groups at any position of the nucleoside.
  • the cationic nucleosides are 2'-Amino-LNA nucleosides (e.g.
  • Bicyclic nucleosides with a methyleneoxy (4'- CH2 -O-2') bridge are sometimes referred to as LNA nucleosides.
  • modified internucleoside linkage refers to an internucleoside linkage that has a substitution or any change from a naturally occurring internucleoside linkage (ie, a phosphodiester linkage).
  • Modified internucleoside linkages include internucleoside linkages containing a phosphorus atom and internucleoside linkages without a phosphorus atom.
  • Representative phosphorus-containing internucleoside linkages include phosphodiester linkages, phosphorothioate linkages, phosphorodithioate linkages, phosphotriester linkages (methylphosphotriester and ethylphosphotriester linkages described in US Pat. No.
  • alkyl Phosphonate linkages e.g., methylphosphonate linkages described in U.S. Pat. Nos. 5,264,423 and 5,286,717, methoxypropylphosphonate linkages described in WO 2015/168172
  • alkylthiophosphonate linkages e.g., methylthiophosphonate linkages, boranophosphate linkages, cyclic guanidine moieties
  • Internucleoside linkages containing e.g., the substructure represented by the following formula (I):
  • an internucleoside linkage comprising a guanidine moiety substituted with 1 to 4 C 1-6 alkyl groups e.g., a tetramethylguanidine (TMG) moiety
  • TMG tetramethylguanidine
  • a phosphorothioate linkage refers to an internucleoside linkage in which a sulfur atom replaces the non-bridging oxygen atom of a phosphodiester linkage.
  • Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known.
  • Modified internucleoside linkages are preferably linkages that are more resistant to nucleases than naturally occurring internucleoside linkages.
  • the internucleoside linkage When the internucleoside linkage has a chiral center, the internucleoside linkage may be chirally controlled. "Chirally controlled” is intended to exist in a single diastereomer about a chiral center, eg, a chiral bound phosphorus. Chirally controlled internucleoside linkages can be completely chirally pure or highly chiral pure, e.g. It may have a chiral purity of %de, 99.9% de, or higher.
  • chiral purity refers to the proportion of one diastereomer in a mixture of diastereomers, expressed as diastereomeric excess (% de), (diastereomer of interest minus other diastereomer defined as (stereomers)/(total diastereomers) ⁇ 100 (%).
  • internucleoside linkages may be phosphorothioate linkages chiral controlled to the Rp or Sp configuration, guanidine moieties substituted with 1-4 C 1-6 alkyl groups (e.g., tetramethylguanidine (TMG) moieties; e.g. Alexander A. Lomzov et al., Biochem Biophys Res Commun., 2019, 513(4), 807-811) and/or an internucleoside linkage comprising a cyclic guanidine moiety.
  • TMG tetramethylguanidine
  • nucleobase or “base” used herein is a base component (heterocyclic moiety) that constitutes a nucleic acid, and adenine, guanine, cytosine, thymine, and uracil are mainly known.
  • nucleobase or “base” includes both modified and unmodified nucleobases (bases) unless otherwise specified.
  • the purine base can be either modified or unmodified purine base.
  • the pyrimidine base may be a modified or unmodified pyrimidine base.
  • Modified nucleobase or “modified base” means any nucleobase other than adenine, cytosine, guanine, thymine, or uracil.
  • Unmodified nucleobases or “unmodified bases” (natural nucleobases) include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and means uracil (U).
  • modified nucleobases include hypoxanthine, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine or N4-methylcytosine; N6-methyladenine or 8-bromoadenine; thymine; and N2-methylguanine or 8-bromoguanine, but are not limited to these.
  • the modified nucleobase is preferably 5-methylcytosine.
  • the term “complementary” means that the nucleobases can form so-called Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen bases) through hydrogen bonding. It means a relationship that can form an equality).
  • the antisense oligonucleotide region in the first nucleic acid strand does not necessarily have to be completely complementary to at least part of the target transcript (for example, the transcript of the target gene). Complementarity of at least 70%, preferably at least 80%, even more preferably at least 90% (eg, 95%, 96%, 97%, 98%, or 99% or more) is acceptable.
  • the antisense oligonucleotide region in the first nucleic acid strand is complementary in base sequence (typically when the base sequence is complementary to the base sequence of at least a portion of the target transcript), It can hybridize to a target transcript.
  • the region of complementarity in the second nucleic acid strand need not necessarily be completely complementary to at least a portion of the first nucleic acid strand, but has a base sequence of at least 70%, preferably at least 80%, and even More preferably, at least 90% (eg, 95%, 96%, 97%, 98%, or 99% or more) complementarity is acceptable.
  • the complementary regions in the second nucleic acid strand can anneal when they are complementary in base sequence to at least a portion of the first nucleic acid strand.
  • Complementarity of base sequences can be determined by using the BLAST program or the like.
  • a person skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which two strands can anneal or hybridize, taking into consideration the degree of complementarity between the strands.
  • a person skilled in the art can easily design an antisense nucleic acid complementary to the target transcript, for example, based on information on the base sequence of the target gene.
  • Hybridization conditions may be various stringent conditions such as low stringent conditions and high stringent conditions.
  • Low stringent conditions may be relatively low temperature and high salt conditions, eg, 30° C., 2 ⁇ SSC, 0.1% SDS.
  • Highly stringent conditions may be relatively high temperature and low salt conditions, eg, 65° C., 0.1 ⁇ SSC, 0.1% SDS.
  • Hybridization stringency can be adjusted by varying conditions such as temperature and salt concentration.
  • 1 ⁇ SSC contains 150 mM sodium chloride and 15 mM sodium citrate.
  • toxicity includes, for example, death, pain, tremor, convulsion, movement disorder, cognitive impairment, consciousness disturbance, general malaise, fatigue, nausea, vomiting, dizziness, numbness, lightheadedness, etc. An action that causes unfavorable objective or subjective symptoms or functional abnormalities in a specimen. Toxicity can be toxicity in any organ. The toxicity may be neurotoxic.
  • neurotoxicity refers to an action that causes damage to nerve tissue, including central nerve tissue and peripheral nerve tissue, and interferes with normal activity of the nervous system.
  • Neurotoxicity includes death, respiratory abnormalities, cardiovascular abnormalities, headache, nausea or vomiting, unresponsiveness or hyporesponsiveness, altered consciousness, psychiatric disturbances, personality changes, hallucinations, delusions, cognitive impairment, postural abnormalities, involuntary movements, and tremors. , convulsions, hyperactive motor dysfunction, paralysis, sensory disturbances or autonomic dysfunction.
  • the neurotoxicity may be acute neurotoxicity.
  • Acute neurotoxicity can be neurotoxicity that occurs within 1, 3, 6, 9, 12, 24 or 48 hours of administration. Toxicity can be evaluated, for example, by acute phase tolerability score, adverse event rate, mortality rate, etc., as described in the Examples below.
  • central neurotoxicity refers to an action that causes damage to at least the central nervous system among nerve tissues and interferes with normal activity of the nervous system.
  • the term "subject” refers to a subject to which the double-stranded nucleic acid complex or pharmaceutical composition of the present invention is applied.
  • Subjects include organs, tissues, and cells as well as individuals.
  • any animal including humans can be applicable.
  • various livestock, poultry, pets, experimental animals, and the like can be mentioned.
  • a subject may be an individual in need of decreased expression of a target transcript or an individual in need of treatment or prevention of a disease.
  • the double-stranded nucleic acid complex of the present invention comprises a first nucleic acid strand and a second nucleic acid strand. Specific configurations of each nucleic acid strand are shown below.
  • the first nucleic acid strand can hybridize to at least a portion of the target gene or its transcript and has an antisense effect on the target gene or its transcript.
  • the second nucleic acid strand comprises a base sequence complementary to the first nucleic acid strand and contains one or more 2′-modified nucleosides.
  • the number of 2'-modified nucleosides contained in the second nucleic acid strand is at least one, and is equal to or less than the number of all nucleosides constituting the second nucleic acid strand (that is, the base length of the second nucleic acid strand).
  • the specific number of 2′-modified nucleosides contained in the second nucleic acid strand is, for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 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 It may be 1 or less, 4 or less, 3 or less, or 2 or less.
  • the number of 2'-modified nucleosides in the second nucleic acid strand is 1 to 30, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 to 21, 1 to 20. , 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10 , 1-9, 1-8, 1-7, or 1-6.
  • it may be 1, 2, 3, 4, 5, or 6.
  • 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% of the nucleosides in the second nucleic acid strand % 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, and/or 100% or less, 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55 % or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less are 2'-modified nucleosides be. For example, 10%-90%, 20%-80%, 30%-70%,
  • the nucleosides other than the 2'-modified nucleosides contained in the second nucleic acid strand may be natural nucleosides, non-natural nucleosides such as crosslinked nucleosides, or any combination thereof.
  • the number of nucleosides other than the 2'-modified nucleosides contained in the second nucleic acid strand is not limited, but is, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 1 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, 1 to 8, or 1 to 6, such as 1 to 5, such as 1, 2, 3, 4, or 5.
  • the second nucleic acid strand contains one 2'-modified nucleoside. In another embodiment, all of the nucleosides making up the second nucleic acid strand are 2'-modified nucleosides. For example, in the second nucleic acid strand, all 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 comprises one or more but less than all 2'-modified nucleosides.
  • the second nucleic acid strand "containing less than all" of 2'-modified nucleosides means that the second nucleic acid strand contains at least one arbitrary nucleoside other than a 2'-modified nucleoside.
  • the second nucleic acid strand comprises one or more contiguous 2'-modified nucleosides located at the 5' terminus.
  • “comprising multiple consecutive 2'-modified nucleosides” means comprising multiple 2'-modified nucleosides linked by any internucleoside linkage.
  • the second nucleic acid strand has one located at the 5' end, or consecutive 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, Alternatively, it may contain 2-3, eg, 2, 3, or 4, 2'-modified nucleosides.
  • the second nucleic acid strand comprises one or more consecutive 2'-modified nucleosides located at the 3' terminus.
  • the second nucleic acid strand has one located at the 3′ end, or consecutive 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, Alternatively, it may contain 2-3, eg, 2, 3, or 4, 2'-modified nucleosides.
  • 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.
  • the second nucleic acid strand contains 2'-modified nucleosides at positions other than the 5' and 3' ends.
  • the second nucleic acid strand "containing a 2'-modified nucleoside at a position other than the 5' end and the 3' end" means that one or more consecutive 2' -modified nucleosides and 2'-modified nucleosides at positions other than one or more consecutive 2'-modified nucleosides located at the 3'-end.
  • the second nucleic acid strand has 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 12, 1 to 10, 1 9, 1-8, 1-7, 1-6, 1-5, 1-4, or 1-3, such as 1 or 2, 2'-modified nucleosides.
  • the second nucleic acid strand contains 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 (eg, in the region consisting of the base sequence complementary to the first nucleic acid strand) can be deoxyribonucleosides.
  • the 2'-modified nucleosides are 2'-O-methoxyethyl modified nucleosides and/or 2'-O-methyl modified nucleosides.
  • the 2'-O-methoxyethyl modified nucleoside has the following formula (III): is indicated by 2'-O-methyl modified nucleosides may also be represented by the following formula (IV): is indicated by
  • the 2'-modified nucleosides are 2'-O-methyl modified nucleosides.
  • the 2'-modified nucleoside is a 2'-O-methoxyethyl modified nucleoside.
  • all of the nucleosides in the first nucleic acid strand may be non-natural nucleosides or modified nucleosides.
  • all of the nucleosides in the first nucleic acid strand may be 2'-modified nucleosides.
  • all of the nucleosides may be 2'-O-methoxyethyl modified nucleosides.
  • the first nucleic acid strand can contain at least 4, at least 5, at least 6, or at least 7 contiguous nucleosides that are recognized by RNase H when hybridized to the target transcript. Generally, any region containing consecutive nucleosides of 4 to 20 bases, 5 to 16 bases, or 6 to 12 bases may be used. As a nucleoside recognized by RNase H, for example, natural deoxyribonucleoside can be used. Suitable nucleosides containing modified deoxyribonucleosides and other bases are well known in the art. It is also known that nucleosides having a hydroxy group at the 2'-position, such as ribonucleosides, are unsuitable as the nucleosides.
  • the suitability of nucleosides for use in this region containing "at least 4 contiguous nucleosides" can readily be determined.
  • the first nucleic acid strand may comprise at least 4 contiguous deoxyribonucleosides.
  • the nucleosides of the first nucleic acid strand comprise or consist of deoxyribonucleosides, e.g., 70% or more, 80% or more, 90% or more, or 95% or more of the nucleosides of the first nucleic acid strand are deoxyribonucleosides. be.
  • the first nucleic acid strand may be a gapmer.
  • the term "gapmer” basically refers to a central region (DNA gap region) and wing regions located directly at its 5' and 3' ends (5' wing region and 3' wing region, respectively). It refers to a single-stranded nucleic acid consisting of The length of the DNA gap region is 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, or 7 to 14 bases. long, or 8-12 bases long.
  • the central region in the gapmer contains at least 3 or at least 4 contiguous deoxyribonucleosides and the wing regions contain at least one non-natural nucleoside.
  • non-natural nucleosides contained in the wing regions usually have higher binding strength to RNA than natural nucleosides and have high resistance to nucleolytic enzymes (nucleases and the like).
  • the non-naturally occurring nucleosides making up the wing regions comprise or consist of bridging nucleosides
  • said gapmers are specifically referred to as "BNA/DNA gapmers”.
  • the number of bridging nucleosides contained in the 5' wing region and the 3' wing region is at least 1, and may be, for example, 2 or 3.
  • the bridging nucleosides contained in the 5' wing region and the 3' wing region may be present continuously or non-contiguously within the 5' wing region and the 3' wing region.
  • Bridging nucleosides can further include modified nucleobases (eg, 5-methylcytosine).
  • modified nucleobases eg, 5-methylcytosine
  • the bridging nucleosides are LNA nucleosides
  • said gapmers are referred to as "LNA/DNA gapmers”.
  • the non-natural nucleosides making up the 5' and 3' wing regions comprise or consist of peptide nucleic acids
  • said gapmers are specifically referred to as "peptide nucleic acid gapmers”.
  • the unnatural nucleosides making up the 5' and 3' wing regions comprise or consist of morpholino nucleic acids
  • said gapmers are specifically referred to as "morpholino nucleic acid gapmers”.
  • 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.
  • the 5′ wing region and/or the 3′ wing region may contain at least one non-natural nucleoside, and may further contain natural nucleosides.
  • the 5' and 3' wing regions are, for example, non-natural nucleosides linked by modified internucleoside linkages such as phosphorothioate linkages, bridged nucleosides such as LNA nucleosides, and 2' -It may be a modified nucleoside.
  • the central region contains at least 3 or at least 4 consecutive deoxyribonucleosides, and the wing regions (5' wing region and 3' wing region) contain at least one unnatural nucleoside.
  • the central region can be functionally defined as the region that can be recognized by RNase H1.
  • RNase H1 means that when the gapmer binds to the target RNA, the sequence in the target RNA paired with the gapmer can be cleaved by RNase H1.
  • the boundary positions can be determined with the region that can be recognized by RNase H1 in the gapmer as the central region and the region that is not recognized by RNase H1 as the wing regions (5' wing region and 3' wing region).
  • nucleosides adjacent to the central region in the 5' wing region and 3' wing region are defined as non-natural nucleosides
  • nucleosides adjacent to the 5' wing region or 3' wing region in the central region are defined as natural nucleosides. .
  • the first nucleic acid strand constituting the gapmer is, in order from the 5′ end, a bridging nucleoside of 2 to 7 bases or 3 to 5 bases (for example, 2 or 3 bases), 4 to 15 bases or 8 to 12 bases. It may be composed of ribonucleosides or deoxyribonucleosides having a base length (eg, 8 or 10 base lengths) and bridging nucleosides having a length of 2 to 7 bases or 3 to 5 bases (eg, 2 or 3 base lengths).
  • a nucleic acid strand having a wing region only on either the 5'-end side or the 3'-end side is called a "hemigapmer" in the art. shall be included.
  • the second nucleic acid strand contains 2'-modified nucleosides in a region consisting of a base sequence complementary to the 5' wing region and/or 3' wing region of the first nucleic acid strand.
  • the second nucleic acid strand may have 2'-modified nucleosides for 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.
  • 2'-modified nucleosides may be, for example, 2'-O-methoxyethyl modified nucleosides or 2'-O-methyl modified nucleosides.
  • the nucleoside containing a purine base in the region consisting of the base sequence complementary to the central region of the first nucleic acid strand may be a ribonucleoside.
  • all nucleosides containing purine bases can be ribonucleosides in the region consisting of the nucleotide sequence complementary to the central region of the first nucleic acid strand.
  • the nucleosides containing pyrimidine bases in the region consisting of the base sequence complementary to the central region of the first nucleic acid strand may be deoxyribonucleosides.
  • all nucleosides containing pyrimidine bases in the region consisting of the nucleotide sequence complementary to the central region of the first nucleic acid strand can be deoxyribonucleosides.
  • all nucleosides containing purine bases are ribonucleosides and all nucleosides containing pyrimidine bases in the region consisting of a nucleotide sequence complementary to the central region of the first nucleic acid strand.
  • the second nucleic acid strand has 2′-modified nucleosides (e.g., 2′- O-methoxyethyl-modified nucleoside or 2'-O-methyl-modified nucleoside), and in the second nucleic acid strand, all the purine bases in the region consisting of the base sequence complementary to the central region of the first nucleic acid strand.
  • the nucleosides are ribonucleosides and all nucleosides containing a pyrimidine base can be deoxyribonucleosides.
  • nucleoside in the first nucleic acid strand may be a 2'-modified nucleoside.
  • nucleosides in the first nucleic acid strand may be 2'-modified nucleosides.
  • At least one guanosine nucleoside in the 3' wing region and/or 5' wing region of the first nucleic acid strand is a 2'-modified nucleoside.
  • nucleosides in the 3' wing region and/or 5' wing region of the first nucleic acid strand may be a further embodiment, (i) all guanosine nucleosides in the 3' wing region and/or 5' wing region of the first nucleic acid strand, (ii) the nucleosides 5' adjacent to said guanosine nucleosides, (iii) said Even if 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 is a 2'-modified nucleoside good.
  • 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.
  • nucleosides in a region consisting of base sequences complementary to the 5' wing region and 3' wing region of the first nucleic acid strand are 2'-modified nucleosides, and All nucleosides in the region consisting of complementary nucleotide sequences can be deoxyribonucleosides.
  • At least one of the nucleosides containing a pyrimidine base in the second nucleic acid strand may be a 2'-modified nucleoside and/or deoxyribonucleoside.
  • the second nucleic acid strand does not contain natural ribonucleosides containing pyrimidine bases, for example all of the nucleosides containing pyrimidine bases in the second nucleic acid strand may be 2'-modified nucleosides and/or deoxyribonucleosides. .
  • the second nucleic acid strand may contain modified nucleosides other than 2'-modified nucleosides in addition to 2'-modified nucleosides.
  • R represents a hydrogen atom or a methyl group.
  • the crosslinked non-natural nucleoside represented by formula (V) above is a 2'-O,4'-C-spirocyclopropylene crosslinked nucleic acid, and is mainly referred to as "scpBNA" in this specification.
  • R may be either a hydrogen atom or a methyl group.
  • R may be either a hydrogen atom or a methyl group.
  • a methyl group can also be written as AmNA[N-Me].
  • the first nucleic acid strand may be a mixmer.
  • the term "mixmer” refers to a nucleic acid chain that contains alternating natural and unnatural nucleosides of periodic or random segment length and is free of four or more contiguous deoxyribonucleosides and ribonucleosides. .
  • Mixmers in which the unnatural nucleoside is a bridged nucleoside and the natural nucleoside is a deoxyribonucleoside are particularly referred to as "BNA/DNA mixmers".
  • the bridged nucleoside may be a bridged non-natural nucleoside represented by formula (V) or formula (VI) above.
  • Mixmers in which the non-natural nucleoside is a peptide nucleic acid and the natural nucleoside is a deoxyribonucleoside are particularly referred to as "peptide nucleic acid/DNA mixmers".
  • Mixmers in which the unnatural nucleoside is a morpholinonucleic acid and the natural nucleoside is a deoxyribonucleoside are particularly referred to as "morpholinonucleic acid/DNA mixmers”.
  • Mixmers are not limited to containing only two nucleosides.
  • Mixmers can include any number of types of nucleosides, whether natural or modified nucleosides or nucleoside mimetics.
  • bridging nucleoside e.g., LNA nucleoside or a bridging non-natural nucleoside represented by formula (V) or formula (VI) above.
  • Bridging nucleosides may further include modified nucleobases (eg, 5-methylcytosine).
  • the second nucleic acid strand comprises at least 4 contiguous ribonucleosides complementary to the at least 4 contiguous nucleosides (e.g., deoxyribonucleosides) in the central region of the first nucleic acid strand. good too. This is so that the second nucleic acid strand forms a partial DNA-RNA heteroduplex with the first nucleic acid strand and is recognized and cleaved by RNaseH. At least four consecutive ribonucleosides in the second nucleic acid strand are preferably linked by naturally occurring internucleoside linkages, ie phosphodiester linkages.
  • the second nucleic acid strand may further comprise at least 2 consecutive deoxyribonucleosides in addition to the at least 4 consecutive ribonucleosides described above.
  • the at least two contiguous deoxyribonucleosides are complementary to the first nucleic acid strand and may be included in a region complementary to the central region of the first nucleic acid strand.
  • the at least two consecutive deoxyribonucleosides may be positioned on either the 5' side or the 3' side of the at least four consecutive ribonucleosides, and may be positioned on both the 5' and 3' sides.
  • the at least two consecutive deoxyribonucleosides may be 2, 3, 4, 5, 6 or more consecutive deoxyribonucleosides.
  • Modified nucleosides may include modified sugars and/or modified nucleobases.
  • Modified sugars may be 2'-modified sugars (eg, sugars containing a 2'-O-methyl group).
  • a modified nucleobase can also be 5-methylcytosine.
  • the second nucleic acid strand is, in order from the 5' end, a modified nucleoside (e.g., a modified nucleoside containing a 2'-modified sugar) having a length of 2 to 7 bases or a length of 3 to 5 bases (for example, 2 or 3 bases), 4 to ribonucleosides or deoxyribonucleosides (optionally linked by modified internucleoside linkages) 15 bases long or 8-12 bases long (eg 8 or 10 bases long) and 2-7 bases long or 3-5 bases long ( (eg, 2 or 3 bases long) modified nucleosides (eg, modified nucleosides containing 2'-modified sugars).
  • the first nucleic acid strand may be a gapmer.
  • the first nucleic acid strand and/or the second nucleic acid strand may contain nucleoside mimics or nucleotide mimics in whole or in part.
  • Nucleotide mimetics may be peptide nucleic acids and/or morpholino nucleic acids.
  • the second nucleic acid strand may contain non-complementary bases and/or insertions and/or deletions of one or more bases relative to the first nucleic acid strand.
  • the number of non-complementary bases in the second nucleic acid strand is not limited, but for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to It may be 4, 1-3, 1-2, or 1 or 2.
  • a sequence consisting of non-complementary bases may form a bulge structure, which will be described later.
  • the number of bases in the insertion sequence in the second nucleic acid strand is not limited, but is, for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to It may be 4, 1-3, 1-2, or 1 or 2.
  • the insertion sequence may be a sequence that forms a bulge structure as described below.
  • the length of consecutive bases to be deleted in the second nucleic acid strand is not limited, but for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 It may be ⁇ 4, 1-3, 1-2, or 1 or 2.
  • the second nucleic acid strand may contain a bulge structure described later at the deletion position.
  • the base lengths of the first nucleic acid strand and the second nucleic acid strand are not particularly limited, but are at least 8 bases long, at least 9 bases long, at least 10 bases long, at least 11 bases long, at least 12 bases long, at least 13 bases long, at least It may be 14 bases long, or at least 15 bases long.
  • the base lengths of the first nucleic acid strand and the second nucleic acid strand are 35 base lengths or less, 30 base lengths or less, 25 base lengths or less, 24 base lengths or less, 23 base lengths or less, 22 base lengths or less, and 21 base lengths 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 nucleic acid strand and the second nucleic acid strand may have the same length or different lengths (eg, one may be 1 to 3 bases shorter or longer).
  • the length of the second nucleic acid strand is shorter than the length of the first nucleic acid strand.
  • the position at which the second nucleic acid strand can bind to the first nucleic acid strand does not matter. For example, it may bind to any of the 5'-side region, the central region, or the 3'-side region in the first nucleic acid strand.
  • the second nucleic acid strand may be at least 8 bases long.
  • the double-stranded structure formed by the first nucleic acid strand and the second nucleic acid strand may contain a bulge.
  • the choice of length can be determined by the balance between the strength of the antisense effect and the specificity of the nucleic acid strand for its target, among other factors such as cost, synthetic yield, and the like.
  • the second nucleic acid strand may contain at least one overhang region located on one or both of its 5' and 3' ends.
  • the "overhang region” is a region adjacent to a region complementary to the first nucleic acid strand in the second nucleic acid strand, and when the first nucleic acid strand and the second nucleic acid strand 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.
  • the overhang region in the second nucleic acid strand may be located on the 5'-terminal side or the 3'-terminal side of the complementary region.
  • the overhang regions in the second nucleic acid strand may be located on the 5'-end side and the 3'-end side of the complementary region.
  • the length of the overhang region is not limited, but may be 1 to 20 bases long, for example, 2 to 15 bases long, 2 to 12 bases long, 2 to 10 bases long, 2 to 8 bases long, 2 to It may be 6 bases long, 2-5 bases long, 2-4 bases long, or 2-3 bases long.
  • the type of nucleosides constituting the overhang region is not limited. For example, it may be composed of natural nucleosides (eg deoxyribonucleosides) or non-natural nucleosides (eg bridged nucleosides such as LNA nucleosides). Also, all or part of the internucleoside linkages in the overhang region may be modified internucleoside linkages.
  • Modified internucleoside linkages may be, for example, phosphorothioate linkages.
  • the overhang regions are preferably protein-binding, lipophilic and/or nuclease-resistant and may be composed of, for example, deoxyribonucleosides or LNA nucleosides linked by phosphorothioate linkages.
  • the nucleotide sequence of the overhang region may be a sequence unrelated to the nucleotide sequence of the target gene.
  • At least 1, at least 2 (eg, 2), at least 3, or at least 4 nucleosides from the end (5' end, 3' end, or both ends) of the second nucleic acid strand are non-natural nucleosides (modified nucleoside).
  • Modified nucleosides may include modified sugars and/or modified nucleobases.
  • Modified sugars may be 2'-modified sugars (eg, sugars containing a 2'-O-methyl group).
  • a modified nucleobase can also be 5-methylcytosine.
  • the second nucleic acid strand is 1 to 8, 1 to 7, 1 to 6, 1 ⁇ 5, 1-4, or 1-3 (e.g., 1-2, or 1) non-complementary bases, 1-8, 1-7, 1-6, 1-5 1 to 4, or 1 to 3 (eg, 1 to 2, or 1) deleted bases, and/or 1 to 20 (eg, 1 to 15, 1 to 12, 1 ⁇ 10, 1-8, 1-6, 1-4, 1-3, or 1) inserted bases.
  • a sequence region consisting of inserted bases may form a bulge structure.
  • the two nucleic acid strands contain at least one bulge structure consisting of a non-complementary base sequence to the first nucleic acid strand.
  • the term “bulge structure” refers to a portion of a double-stranded nucleic acid in which part of the nucleic acid of any 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 is 1 to 50 bases long, 1 to 40 bases long, 1 to 30 bases long, 1 to 20 bases long, 1 to 15 bases long, preferably 1 to 10 bases long.
  • the bulge structure comprises sugar-unmodified nucleosides. In a further embodiment, all nucleosides in the bulge structure are sugar unmodified nucleosides.
  • all nucleosides other than the bulge structure may be 2'-modified nucleosides.
  • the internucleoside linkages in the first nucleic acid strand and the second nucleic acid strand may be naturally occurring internucleoside linkages and/or modified internucleoside linkages. At least 1, at least 2, or at least 3 internucleoside linkages from the end (5′ end, 3′ end, or both ends) of the first nucleic acid strand and/or the second nucleic acid strand are modified internucleoside linkages, but are not limited to A bond is preferred.
  • two internucleoside bonds from the end of a nucleic acid chain mean the internucleoside bond closest to the end of the nucleic acid chain and the internucleoside bond adjacent thereto and located on the opposite side of the end. Modified internucleoside linkages in terminal regions of nucleic acid strands are preferred because they can reduce or inhibit unwanted degradation of the nucleic acid strand.
  • all or part of the internucleoside linkages of the first nucleic acid strand and/or the second nucleic acid strand may be modified internucleoside linkages.
  • the first nucleic acid strand and/or the second nucleic acid strand each has 1, 2, 3, 4, 5, 6, 7, 8, 9 modified internucleoside linkages. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, It may contain 26, 27, 28, 29, 30, 35, 40, 45, 50 or more.
  • the first nucleic acid strand and/or the second nucleic acid strand each have 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%.
  • the modified internucleoside linkage may be a phosphorothioate linkage or a boranophosphate linkage.
  • all or part of the internucleoside linkages in the first nucleic acid strand may be phosphorothioate linkages.
  • the first nucleic acid strand and/or the second nucleic acid strand each have 1, 2, 3, 4, 5, 6, 7, 8 chirally controlled internucleoside linkages. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, It may contain 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more.
  • the first nucleic acid strand and/or the second nucleic acid strand each have at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% chirally controlled internucleoside linkages. %, at least 60%, at least 70%, at least 80%, at least 90%, or more.
  • the first nucleic acid strand and/or the second nucleic acid strand each have 1, 2, 3, 4 non-negatively charged internucleoside linkages (preferably neutral internucleoside linkages), 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.
  • the first nucleic acid strand and/or the second nucleic acid strand each have at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50% non-negatively charged internucleoside linkages. , at least 60%, at least 70%, at least 80%, at least 90%, or more.
  • At least 1, at least 2, or at least 3 internucleoside linkages from the 5' end of the second nucleic acid strand may be modified internucleoside linkages. At least 1, at least 2, or at least 3 internucleoside linkages from the 3′ end of the second nucleic acid strand are replaced with modified internucleoside linkages, such as phosphorothioate linkages, 1-4 C 1-6 alkyl groups may be an internucleoside linkage comprising a modified guanidine moiety (eg, a TMG moiety) and/or an internucleoside linkage comprising a cyclic guanidine moiety. Modified internucleoside linkages may be chirally controlled to the Rp or Sp configuration.
  • At least one (eg, three) internucleoside linkage from the 3' end of the second nucleic acid strand may be a modified internucleoside linkage such as a highly RNase-resistant phosphorothioate linkage.
  • the double-stranded nucleic acid complex has an improved gene suppressing activity, which is preferable.
  • the modified internucleoside linkages of the first nucleic acid strand and/or the second nucleic acid strand are controlled at a pH (e.g., human physiological pH (about 7.4), a delivery site (e.g., an organelle, cell, tissue, organ).
  • a pH e.g., human physiological pH (about 7.4)
  • a delivery site e.g., an organelle, cell, tissue, organ.
  • the modified internucleoside linkage is in an anionic form (e.g., -OP(O)(O - )-O- (anionic form of natural phosphate linkage), -OP(O) (S ⁇ )—O— (anionic form of phosphorothioate linkages, etc.)), including non-negatively charged (neutral or cationic, respectively) internucleoside linkages that exist as neutral or cationic forms.
  • the modified internucleoside linkages of the first and/or second nucleic acid strand comprise neutral internucleoside linkages.
  • the modified internucleoside linkages of the first and/or second nucleic acid strand comprise cationic internucleoside linkages.
  • a non-negatively charged internucleoside linkage e.g., a neutral internucleoside linkage
  • the non-negatively charged internucleoside linkage is, for example, a methylphosphonate linkage as described in U.S. Patent Nos.
  • the non-negatively charged internucleoside linkage comprises a triazole or alkyne moiety.
  • the non-negatively charged internucleoside linkage comprises a cyclic guanidine moiety and/or a guanidine moiety (preferably a TMG moiety) substituted with 1-4 C 1-6 alkyl groups.
  • a modified internucleoside linkage comprising a cyclic guanidine moiety has a substructure represented by Formula (I).
  • the guanidine moiety substituted with 1-4 C 1-6 alkyl groups has a substructure represented by Formula (II).
  • neutral internucleoside linkages comprising cyclic guanidine moieties and/or guanidine moieties substituted with 1-4 C 1-6 alkyl groups are chirally controlled.
  • the disclosure relates to a composition
  • a composition comprising an oligonucleotide comprising at least one neutral internucleoside linkage and at least one phosphorothioate internucleoside linkage.
  • neutral internucleotide linkages have properties compared to equivalent nucleic acids that do not contain neutral internucleotide linkages. and/or have improved activity, e.g., improved delivery, improved resistance to exonucleases and endonucleases, improved cellular uptake, improved endosomal escape, and/or improved nuclear uptake, etc.
  • the second nucleic acid strand may be bound to a ligand (also referred to herein as a binder).
  • Ligands include small molecules (small molecule ligands), medium molecules (medium molecule ligands), macromolecules (macromolecular ligands), peptides (peptide ligands), lipids (lipid ligands), and aptamers (e.g., nucleic acid aptamers).
  • peptide refers to an amino acid polymer having one or more peptide bonds.
  • a “peptide” is not limited by the number of amino acid residues it contains.
  • peptide includes 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 peptides.
  • Peptide ligands may bind to molecules present on the cell surface or inside cells, or in body fluids.
  • the peptide may be an antibody or active fragment thereof.
  • antibodies include monoclonal antibodies, polyclonal antibodies, recombinant antibodies such as chimeric antibodies and humanized antibodies, Fab, F(ab') 2 , Fab', VHH and the like.
  • active fragments of antibodies include scFv (single chain fragment of variable region: single chain antibody), diabody, triabody, tetrabody and the like.
  • Lipids include tocopherols, cholesterol, fatty acids, phospholipids and their analogues; folic acid, vitamin C, vitamin B1, vitamin B2; estradiol, androstane and their analogues; steroids and their analogues; SRBI or LRP1/2 ligands; FK-506, and cyclosporin; lipids described in PCT/JP2019/012077, PCT/JP2019/010392 and PCT/JP2020/035117, but are not limited thereto.
  • Lipids also include tocopherol or analogues thereof and/or cholesterol or analogues thereof , substituted or unsubstituted C 1-30 alkyl groups, substituted or unsubstituted C 2-30 It may be a 30 alkenyl group or a substituted or unsubstituted C 1-30 alkoxy group.
  • tocopherol is a methylated derivative of tocorol, a fat-soluble vitamin (vitamin E) with a cyclic structure called chroman. Tocorol has a strong antioxidant action, and therefore, in vivo, as an antioxidant substance, it has the function of quenching free radicals generated by metabolism and protecting cells from injury.
  • tocopherol Several different forms of tocopherol are known, consisting of ⁇ -tocopherol, ⁇ -tocopherol, ⁇ -tocopherol, and ⁇ -tocopherol, based on the position of the methyl group that binds to the chroman.
  • a tocopherol herein can be any tocopherol.
  • Analogues of tocopherol include various unsaturated analogues of tocopherol, such as ⁇ -tocotrienol, ⁇ -tocotrienol, ⁇ -tocotrienol and ⁇ -tocotrienol.
  • the tocopherol is ⁇ -tocopherol.
  • cholesterol is a type of sterol, also known as steroidal alcohol, and is particularly abundant in animals. Cholesterol plays an important role in metabolic processes in vivo, and in animal cells, it is also a major constituent of the cell membrane system together with phospholipids. Analogs of cholesterol also refer to various cholesterol metabolites and analogs, which are alcohols having a sterol backbone, including, but not limited to, cholestanol, lanosterol, cerebrosterol, dehydrocholesterol, and coprostanol. etc.
  • analog refers to compounds having the same or similar basic skeleton and having similar structures and properties. Analogs include, for example, biosynthetic intermediates, metabolites, compounds with substituents, and the like. A person skilled in the art can determine whether a compound is an analog of another compound based on common general technical knowledge.
  • Cholesterol analogs refer to various cholesterol metabolites and analogs, etc., which are alcohols with a sterol backbone, including, but not limited to, cholestanol, lanosterol, cerebrosterol, dehydrocholesterol, and coprostanol. include.
  • the second nucleic acid strand may be bound with tocopherol or cholesterol or analogs thereof.
  • the second nucleic acid strand bound to cholesterol or its analogue may have a group represented by the following general formula (VII).
  • R c represents an optionally substituted alkylene group having 4 to 18 carbon atoms, preferably 5 to 16 carbon atoms (wherein the substituent is a halogen atom or a hydroxy an alkyl group having 1 to 3 carbon atoms which may be substituted with a group such as a hydroxymethyl group, and non-adjacent carbon atoms of the alkylene group may be substituted with oxygen atoms).
  • Rc is, 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- may be
  • 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.
  • a ligand such as cholesterol or an analogue thereof may be bound to the 5' end, 3' end, or both ends of the second nucleic acid strand.
  • a ligand such as cholesterol or an analogue thereof may also be attached to an internal nucleotide of the second nucleic acid strand.
  • the second nucleic acid strand contains multiple cholesterols or analogues thereof, they may be the same or different. For example, this applies to the case where the second nucleic acid strand has one cholesterol bound to the 5′ end and one other cholesterol analogue bound to the 3′ end. With respect to the binding position, the cholesterol or analogue thereof may be bound at multiple positions and/or at one position as a group on the second nucleic acid strand. Cholesterol or analogs thereof may be linked to the 5' end and the 3' end of the second nucleic acid strand, respectively.
  • the binding between the second nucleic acid strand and the ligand may be direct binding or indirect binding mediated by another substance.
  • the second nucleic acid strand When the second nucleic acid strand directly binds to the ligand, it may be bound to the second nucleic acid strand via, for example, covalent bond, ionic bond, hydrogen bond, or the like.
  • a covalent bond is preferred in view of the fact that a more stable bond can be obtained.
  • the second nucleic acid strand is not bound to a ligand.
  • "not bound to a ligand” means that a ligand such as tocopherol or cholesterol is not bound.
  • the double-stranded nucleic acid complex of the invention is unliganded, ie neither the first nor the second nucleic acid strand is bound with a ligand.
  • linker When the second nucleic acid strand and the ligand are indirectly bound, they may be bound via a linking group (herein often referred to as "linker").
  • a linker can be either a cleavable linker or an uncleavable linker.
  • “Cleavable linker” means a linker that can be cleaved under physiological conditions, for example, intracellularly or in an animal body (eg, human body). Cleavable linkers are selectively cleaved by endogenous enzymes such as nucleases. Cleavable linkers include, but are not limited to, amides, esters, phosphodiester esters or both, phosphate esters, carbamates, and disulfide bonds, as well as natural DNA linkers. As an example, cholesterol or an analogue thereof may be linked via a disulfide bond.
  • Non-cleavable linker is meant a linker that is not cleaved under physiological conditions, eg, in cells or in animals (eg, in humans).
  • Non-cleavable linkers include, but are not limited to, phosphorothioate linkages, and linkers composed of modified or unmodified deoxyribonucleosides or modified or unmodified ribonucleosides linked by phosphorothioate linkages.
  • the linker is a nucleic acid such as DNA or an oligonucleotide
  • the chain length is not particularly limited, but usually it may be 2 to 20 bases, 3 to 10 bases, or 4 to 6 bases.
  • a specific example of the linker is a linker represented by formula (VIII) below.
  • L 2 is a substituted or unsubstituted C 1 -C 12 alkylene group (e.g., propylene, hexylene, dodecylene), a substituted or unsubstituted C 3 -C 8 cycloalkylene group, (e.g., cyclohexylene), -( CH2 ) 2- O-( CH2 ) 2- O-( CH2 ) 2- O-( CH2 ) 3- , -( CH2 ) 2- O-(CH 2 ) 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 ) 3- , L3 represents -NH- or a bond, L4 is substituted or unsubstituted C1 - C12 alkylene groups (e.g.
  • C3-8 cycloalkylene groups e.g. cyclohexylene
  • the linker represented by formula (VIII) is such that L 2 is an unsubstituted C 3 -C 6 alkylene group (eg, propylene, hexylene), —(CH 2 ) 2 —O—( CH2 ) 2- O-( CH2 ) 2- O-( CH2 ) 3- or -( CH2 ) 2- O-(CH2) 2- O-( CH2 ) 2- O- (CH 2 ) 2 -O-( CH2 ) 3- , L3 is -NH-, and L4 and L5 are a bond.
  • C 3 -C 6 alkylene group eg, propylene, hexylene
  • L3 is -NH-
  • L4 and L5 are a bond.
  • linker is a linker represented by the following general formula (IX).
  • n 0 or 1;
  • the first nucleic acid strand and/or the second nucleic acid strand may further contain at least one functional moiety bound to the polynucleotides constituting the nucleic acid strand.
  • a “functional portion” refers to a portion that imparts a desired function to a double-stranded nucleic acid complex and/or a nucleic acid strand to which the functional portion is bound. Desired functions include, for example, labeling functions, purification functions, and the like. Examples of moieties that provide a labeling function include compounds such as fluorescent proteins and luciferase.
  • moieties that impart a purification function include compounds such as biotin, avidin, His-tag peptides, GST-tag peptides and FLAG-tag peptides.
  • a functional moiety is added to the second nucleic acid strand.
  • a molecule having an activity of delivering a double-stranded nucleic acid complex in certain embodiments to a target site is bound.
  • moieties that provide targeted delivery functions include lipids, antibodies, aptamers, ligands for specific receptors, and the like.
  • the first nucleic acid strand and/or the second nucleic acid strand (preferably the second nucleic acid strand) is associated with a functional moiety.
  • the binding between the second nucleic acid strand and the functional moiety may be direct binding or indirect binding via another substance. It is preferable that the second nucleic acid strand and the functional moiety are directly bonded by, for example, a hydrogen bond, and a covalent bond is more preferable from the viewpoint of obtaining a more stable bond.
  • the first nucleic acid strand and the second nucleic acid strand may be linked via a linker.
  • the first nucleic acid strand and the second nucleic acid strand can be linked via a linker to form a single strand.
  • the double-stranded nucleic acid complex in this case can be called hinge nucleic acid, single-stranded HDO, ssHDO, or the like.
  • the functional region has the same configuration as that of the double-stranded nucleic acid complex even in that case, such a single-stranded nucleic acid is also included in the present specification as an embodiment of the double-stranded nucleic acid complex of the present invention. do.
  • the linker joins the 5' end of the first nucleic acid strand and the 3' end of the second nucleic acid strand. In another embodiment, a linker joins the 3' end of the first nucleic acid strand and the 5' end of said second nucleic acid strand. In a further embodiment, the linker joins the 5' end of the first nucleic acid strand and the 3' end of the second nucleic acid strand, and connects the 3' end of the first nucleic acid strand and the 5' end of said second nucleic acid strand. Join. In this case, the double-stranded nucleic acid complex has a circular structure.
  • the linker can be any polymer.
  • linkers that connect the second nucleic acid strand and the ligand as described above can also be used.
  • it can be composed of, for example, natural nucleotides and nucleosides such as DNA and RNA, or non-natural nucleotides and nucleosides such as peptide nucleic acids and morpholino nucleic acids.
  • it may be composed of a polyether such as polyethylene glycol.
  • the chain length of the linker is at least 1 base, such as 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 It can have a chain length of 10 bases or 4-6 bases. A chain length of 4 bases is preferred.
  • the position of the linker can be either on the 5' side or the 3' side of the first 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.
  • Linkers may be either cleavable or uncleavable.
  • first nucleic acid strand and the second nucleic acid strand are linked via a linker, and the second nucleic acid strand has at least a bulge structure consisting of a non-complementary base sequence to the first nucleic acid strand.
  • the antisense effect of the first nucleic acid strand on the target transcript can be measured by a method known in the art. For example, after introducing a double-stranded nucleic acid complex into a cell or the like, measurement may be performed using a known technique such as Northern blotting, quantitative PCR, or Western blotting. By measuring the expression level of target genes or the level of target transcripts (e.g., the amount of mRNA or RNA such as microRNA, the amount of cDNA, the amount of protein, etc.) in specific tissues, double-stranded nucleic acid complexes can be detected at those sites. It can be determined whether target gene expression is repressed by the body. For exon skipping, for example, the effect can be determined by comparing the product produced by exon skipping with the product produced without exon skipping.
  • a known technique such as Northern blotting, quantitative PCR, or Western blotting.
  • the double-stranded nucleic acid complex of the present invention is not limited to the above exemplary embodiments.
  • the double-stranded nucleic acid complex of the present invention can be produced by a person skilled in the art by appropriately selecting a known method. Although not limited, it usually starts with designing and manufacturing each of the first nucleic acid strand and the second nucleic acid strand that constitute the double-stranded nucleic acid complex.
  • the first nucleic acid strand is designed based on information on the nucleotide sequence of the target transcript (for example, the nucleotide sequence of the target gene), and the second nucleic acid strand is designed as its complementary strand.
  • each nucleic acid chain may be synthesized using, for example, a commercially available automatic nucleic acid synthesizer from GE Healthcare, Thermo Fisher Scientific, Beckman Coulter, and the like. After that, the obtained oligonucleotide can be purified using a reverse phase column or the like.
  • the first nucleic acid strand may be produced according to the above method.
  • the functional moiety-bound second nucleic acid strand can be produced by performing the above-described synthesis and purification using a nucleic acid species to which a functional moiety has been bound in advance.
  • a nucleic acid species with pre-bound cholesterol or an analog thereof may be used to produce a second nucleic acid strand by performing the synthesis and purification described above.
  • cholesterol or an analogue thereof may be bound to the second nucleic acid strand produced by carrying out the synthesis and purification described above by a known method.
  • 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.
  • the nucleic acids are mixed in a suitable buffer solution, denatured at about 90° C.-98° C. for several minutes (eg, 5 minutes), and then the nucleic acids are annealed at about 30° C.-70° C. for about 1-8 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. Nucleic acid chains can also be obtained by ordering from various manufacturers (eg, Gene Design Co., Ltd.) specifying the nucleotide sequence, modification site and type.
  • the double-stranded nucleic acid complex of the present invention has at least one of the following effects: an effect of suppressing or enhancing the expression level of a transcription product or translation product of a target gene, a target Those intended for the action of inhibiting the function of gene transcription or translation products, the action of regulating RNA splicing, and the action of inhibiting the binding of target genes to proteins, such as exon skipping good.
  • the double-stranded nucleic acid complex of the present invention may have at least one of the following actions: exon skipping, exon inclusion, steric blocking, and RNA expression enhancement.
  • the double-stranded nucleic acid complex of the present invention exerts the above action in specific tissues such as brain, spinal cord, kidney, liver, lung, intestinal tract, spleen, adrenal gland, eye, retina, skin, peripheral nerves such as brain.
  • the brain may be any of the cerebrum, diencephalon, brainstem, and cerebellum, and may be, for example, any one or more of the cerebrum (cerebral cortex, etc.), brainstem, cerebellum, hippocampus, and striatum.
  • the double-stranded nucleic acid complex of the present invention may be one for exerting the above-mentioned action on muscle tissue including cardiac muscle and skeletal muscle.
  • the double-stranded nucleic acid complex of the present invention is for disease or prevention.
  • the disease may be skeletal muscle dysfunction or cardiac dysfunction.
  • diseases include muscular dystrophy (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 ganglia syndrome (CBD), Pick's disease, frontotemporal dementia, neuronal inclusions disease, spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), Parkinson's disease, Huntington's disease, hereditary spinocerebellar ataxia (SCA), multiple system atrophy, hereditary spastic paraplegia, multiple Severe sclerosis, cerebral infarction,
  • toxicity such as central nervous system toxicity of the double-stranded nucleic acid complex can be reduced or eliminated without impairing the efficacy of the double-stranded nucleic acid complex. That is, compared with conventional double-stranded nucleic acid complexes, the central nervous system toxicity is reduced without impairing the antisense effect on the target gene.
  • 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 nervous system toxicity and can be administered intrathecally or intracerebroventricularly without side effects.
  • Each component that can be included in the pharmaceutical composition of the present invention is specifically described below.
  • the pharmaceutical composition of the present invention includes at least the double-stranded nucleic acid complex according to the first aspect as an active ingredient.
  • the pharmaceutical composition of the present invention may contain two or more types of double-stranded nucleic acid complexes.
  • the amount (content) of the double-stranded nucleic acid complex contained in the pharmaceutical composition depends on the type of the double-stranded nucleic acid complex, the delivery site, the dosage form of the pharmaceutical composition, the dosage of the pharmaceutical composition, and the carrier described later. depends on the type of Therefore, it is sufficient to determine them as appropriate in consideration of each condition.
  • a single dose of the pharmaceutical composition is adjusted to include an effective amount of the double-stranded nucleic acid complex.
  • Effective amount means an amount necessary for the double-stranded nucleic acid complex to exhibit its function as an active ingredient, and an amount that imparts little or no harmful side effects to the living body to which it is applied. Say.
  • Subject information is various individual information of a living body to which the pharmaceutical composition is applied. For example, if the subject is a human, the information includes age, weight, sex, dietary habits, health condition, disease progression and severity, drug sensitivity, presence or absence of concomitant drugs, and the like.
  • Carrier A pharmaceutical composition of the present invention can comprise a pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” refers to additives commonly used in the field of formulation technology. For example, solvents, vegetable oils, bases, emulsifiers, suspending agents, surfactants, pH adjusters, stabilizers, flavorants, fragrances, excipients, vehicles, preservatives, binders, diluents, Tonicity agents, soothing agents, bulking agents, disintegrating agents, buffering agents, coating agents, lubricants, coloring agents, sweetening agents, thickening agents, flavoring agents, solubilizing agents, and other additives.
  • the solvent may be, for example, water or other pharmaceutically acceptable aqueous solution, or a pharmaceutically acceptable organic solvent.
  • Aqueous solutions include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, phosphate buffers, and sodium acetate buffers.
  • auxiliary agents include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, and the like.
  • the carrier is used to avoid or suppress in vivo degradation of the double-stranded nucleic acid complex, which is an active ingredient, by enzymes, etc., facilitate formulation and administration methods, and maintain the dosage form and efficacy. and can be used as needed.
  • the dosage form of the pharmaceutical composition of the present invention is such that the active ingredient, the double-stranded nucleic acid complex according to the first aspect, is delivered to the target site without being inactivated by degradation or the like, and is effective in vivo.
  • the form can exhibit the pharmacological effect of the component (antisense effect on target gene expression).
  • Specific dosage forms differ depending on the administration method and/or prescription conditions. Since administration methods can be broadly classified into parenteral administration and oral administration, dosage forms suitable for each administration method may be used.
  • the preferred dosage form is a liquid formulation that can be administered directly to the target site or systemically administered via the circulatory system.
  • liquid formulations include injections. Injections are appropriately combined with the aforementioned excipients, elixirs, emulsifiers, suspending agents, surfactants, stabilizers, pH adjusters, etc., and mixed in a unit dosage form generally accepted for pharmaceutical practice. It can be formulated by Other formulations include ointments, plasters, cataplasms, transdermal formulations, lotions, inhalants, aerosols, eye drops, and suppositories.
  • preferred dosage forms may be solids or liquids, such as tablets, capsules, drops, lozenges, pills, granules, powders, powders, and liquids for internal use. , emulsions, syrups, pellets, sublingual agents, peptizers, buccal preparations, pastes, suspensions, elixirs, coatings, ointments, plasters, cataplasms, oral Skins, lotions, inhalants, aerosols, eye drops, injections, and suppositories.
  • a solid formulation it is optionally made into a dosage form with a coating known in the art, such as a sugar-coated tablet, a gelatin-coated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, and a multilayer tablet. be able to.
  • a coating known in the art, such as a sugar-coated tablet, a gelatin-coated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet, and a multilayer tablet.
  • each dosage form described above are not particularly limited as long as they are within the range of dosage forms known in the art for each dosage form.
  • the method for producing the pharmaceutical composition of the present invention it may be formulated according to a conventional method in the technical field.
  • the double-stranded nucleic acid complex of the present invention has excellent solubility in water, Japanese Pharmacopoeia Dissolution Test 2nd Fluid, or Japanese Pharmacopoeia Disintegration Test 2nd Fluid, drug half-life, brain penetration, metabolic stability, CYP inhibition) and low toxicity (e.g., acute toxicity, chronic toxicity, genotoxicity, reproductive toxicity, cardiotoxicity, drug interaction, carcinogenicity, phototoxicity) It is superior as a drug in terms of, etc.) and has excellent properties as a drug, such as less side effects (for example, suppression of sedation and avoidance of lamellar necrosis).
  • parenteral administration there is no particular limitation herein on the preferred dosage form of the pharmaceutical composition.
  • 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 continuous subcutaneous implanted administration), intradermal administration, tracheal/bronchial administration, rectal administration, and by blood transfusion.
  • Intrathecal administration may be, for example, an occipital fovea puncture or a lumbar puncture.
  • the dosage or ingestion is, for example, 0.00001 mg/kg/day to 10000 mg/kg/day, or 0.001 mg/kg/day of the included double-stranded nucleic acid complex. It should be between 100mg/kg/day.
  • the pharmaceutical composition may be single-dose or multi-dose. In the case of multiple administrations, it can be administered daily or at appropriate time intervals (eg, at intervals of 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month), for example, 2 to 20 doses.
  • a single dose of the double-stranded nucleic acid complex is, 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, 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, 30mg/kg or more, 40mg/kg or more, 50mg/kg or more, 75mg/kg or more, 100mg/kg or more, 150mg/kg or more, 200mg/kg or more, 300mg/kg or more, 400mg/kg or more, or 500 mg/kg or more, for example, any amount within the range of 0.001 mg/kg to 500 mg/kg (e.g., 0.001 mg/kg,
  • the double-stranded nucleic acid complex of the present invention may be administered at a dose of 0.01 to 10 mg/kg (eg, about 6.25 mg/kg) twice a week for 4 times.
  • the double-stranded nucleic acid complex is administered at a dose of 0.05 to 30 mg/kg (eg, about 25 mg/kg) at a frequency of 1 to 2 times a week, 2 to 4 times, for example, twice a week. good too.
  • Employment of such a dosing regimen can reduce toxicity (eg, avoid platelet depletion) and reduce burden on the subject compared to single administration of higher doses.
  • the pharmaceutical composition has an additive inhibitory effect in cells even after repeated administration.
  • the pharmaceutical composition of this aspect is administered intracerebroventricularly or intrathecally.
  • 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, or 5 mg or more is administered to monkeys or humans.
  • 1 mg to 20 mg may be administered, and in the case of mice, 1 ⁇ g or more may be administered.
  • the pharmaceutical composition of this aspect is administered intravenously or subcutaneously.
  • the diseases to which the pharmaceutical composition is applied are not limited.
  • the antisense effect of the double-stranded nucleic acid complex of the present invention can suppress or enhance the expression level of the transcription product or translation product, inhibit the function of the transcription product or translation product, or steric blocking or splicing switch. , RNA editing, exon skipping or exon inclusion-inducible genes may be of interest. Specific examples of diseases conform to the description in “1-5. Use of double-stranded nucleic acid complex”.
  • the pharmaceutical composition of this aspect can be used to treat central nervous system disease in a subject.
  • the central nervous system disease to which the pharmaceutical composition of this aspect is applied is not particularly limited, but examples thereof include brain tumor, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, and the like. be done.
  • 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 by intracerebroventricular or intrathecal administration.
  • treatment of neurological diseases such as Alzheimer's disease requires administration of high doses of nucleic acid agents, and there is a risk of side effects.
  • the pharmaceutical composition of the present invention can significantly reduce such side effects. becomes possible.
  • a method for treating and/or preventing a disease such as a central nervous system disease comprises administering the double-stranded nucleic acid complex or pharmaceutical composition described above to a subject.
  • double-stranded nucleic acid complex of the present invention in the manufacture of a medicament for treating and/or preventing diseases.
  • Example 1 MOE modification of HDO targeting Mapt gene>
  • a heteroduplex oligonucleotide comprising 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 (hereinafter referred to as "HDO”) is administered intracerebroventricularly, and the toxicity-reducing effect based on MOE modification is verified by in vivo experiments.
  • ASO antisense nucleic acid
  • HDO second nucleic acid strand having a base sequence complementary to the first nucleic acid strand
  • ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid that targets mouse microtubule-associated protein tau (Mapt) mRNA, and has a base sequence complementary to a portion of Mapt mRNA. , 3 LNA nucleosides at the 5' end, 3 LNA nucleosides at the 3' end, and 10 DNA nucleosides therebetween linked by phosphorothioate bonds.
  • HDO (all RNA), HDO (all DNA), HDO (6MOE wing), and HDO (all MOE) used in this example all contain the above ASO as the first nucleic acid strand, and the second nucleic acid strand It has a sequence complementary to one nucleic acid strand.
  • 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 (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 wing) (c(6MOE wing)) consists of three 2'-O-MOE-RNA nucleosides at the 5' end and three 2'-O-MOE- It has a structure in which RNA nucleosides and 10 DNA nucleosides between them 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.
  • the 2'-O-MOE-RNA nucleoside used in the examples of the present specification has the following formula (III): is a non-natural nucleoside represented by
  • the first and second nucleic acid strands were mixed in equimolar amounts and the solution was heated to 95°C for 5 minutes and then to 37°C. The mixture was cooled and held for 1 hour to anneal the nucleic acid strands and prepare a double-stranded nucleic acid complex. Annealed nucleic acids were stored at 4°C or on ice. All oligonucleotides were custom synthesized by Gene Design (Osaka, Japan).
  • Each category contains two behavioral endpoints.
  • Each behavioral evaluation item is evaluated on a 5-point scale of 0 to 5 (Fig. 3, score 0 to 5), with 0 for normal and higher scores for higher toxicity.
  • score 0 to 5 For each category, the score for the higher of the two behavioral measures is taken as the score for that category.
  • the acute phase tolerability score (0-20 points) is the sum of the five category scores.
  • Quantitative RT-PCR was performed using the resulting cDNA as a template to measure the expression levels of Mapt mRNA and Actb mRNA (internal standard genes). Quantitative RT-PCR was performed by TaqMan (Roche Applied Science). Primers used in quantitative RT-PCR were products designed and manufactured by Thermo Fisher Scientific (formerly Life Technologies Corp). 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 for 40 cycles.
  • the ratio of the expression level of Mapt mRNA to the expression level of Actb mRNA was calculated, and the value normalized to the value of the PBS administration group was taken as the relative Mapt mRNA level.
  • FIG. 5 shows the results of evaluation of central nervous system toxicity in mice to which various nucleic acid agents were intracerebroventricularly administered. At 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours after administration of the nucleic acid agent, ASO, Compared to the HDO(all RNA) and HDO(all DNA) administration groups, the acute tolerability score was remarkably decreased. These results indicated that HDO containing 2'-O-MOE-RNA nucleosides significantly reduced central nervous system toxicity.
  • Figure 6 shows the results of evaluating motor function in mice intracerebroventricularly administered with various nucleic acid agents 1 hour after administration.
  • total movement distance Fig. 6A
  • maximum movement velocity Figure 6B
  • Fig. 7 shows Mapt mRNA expression levels in the hippocampus 7 days after intracerebroventricular administration of various nucleic acid agents.
  • the gene suppression effect was lower than in the ASO, HDO (all RNA), and HDO (all DNA) administration groups.
  • the HDO (6MOE wing) administration group showed gene suppression effects equivalent to those of the ASO, HDO (all RNA), and HDO (all DNA) administration groups.
  • Example 2 MOE modification of HDO targeting BACE1 gene> (the purpose) MOE modification for central neurotoxicity observed when intracerebroventricularly administered HDO containing a first nucleic acid strand consisting of an ASO targeting the BACE1 gene and a second nucleic acid strand having a nucleotide sequence complementary to the first nucleic acid strand To verify the toxicity reduction effect based on the in vivo experiment.
  • Table 2 and FIG. 8 show the base sequences and chemical modifications of the first and second nucleic acid strands constituting the ASO and HDO used in this example.
  • ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid that targets mouse ⁇ -secretase 1 (BACE1) mRNA, and has a base complementary to a part of BACE1 mRNA. It has a structure in which 2 LNA nucleosides at the 5' end, 3 LNA nucleosides at the 3' end, and 8 DNA nucleosides therebetween are linked by phosphorothioate bonds.
  • HDO (all RNA), HDO (all DNA), and HDO (5MOE wing) used in this example all contain the above ASO as the first nucleic acid strand, and the second nucleic acid strand is complementary to the first nucleic acid strand. array.
  • 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 (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(5MOE wing)) of HDO(5MOE wing) consists of three 2'-O-MOE-RNA nucleosides at the 5' end and two 2'-O-MOE- It has a structure in which RNA nucleosides and eight DNA nucleosides between them are linked by phosphodiester bonds.
  • HDO all RNA
  • HDO all DNA
  • HDO all DNA
  • HDO all DNA
  • HDO 5MOE wing
  • FIG. 9 shows the results of evaluation of central nervous system toxicity in mice to which various nucleic acid agents were intracerebroventricularly administered. At 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours after administration of the nucleic acid agent, in the HDO (5MOE wing) administration group, ASO, HDO (all RNA), and The acute tolerability score was significantly decreased compared to the HDO (all DNA) administration group. These results indicated that HDO containing 2'-O-MOE-RNA nucleosides significantly reduced central nervous system toxicity.
  • Fig. 10 shows the results of evaluating motor function in mice intracerebroventricularly administered with various nucleic acid agents 1 hour after administration.
  • the HDO(5MOE wing) administration group significantly improved the total moving distance (Fig. 10A) and the maximum moving speed (Fig. 10B) compared to the ASO, HDO(all RNA), and HDO(all DNA) administration groups.
  • Fig. 10A the total moving distance
  • Fig. 10B maximum moving speed
  • HDO containing 2'-O-MOE-RNA nucleosides had a very small inhibitory effect on motor function and a very low toxicity.
  • Example 3 MOE modification of HDO targeting Malat1 gene> (the purpose) MOE modification for central neurotoxicity observed when intracerebroventricularly administered HDO containing a first nucleic acid strand consisting of an ASO targeting the Malat1 gene and a second nucleic acid strand having a nucleotide sequence complementary to the first nucleic acid strand To verify the toxicity reduction effect based on the in vivo experiment.
  • Table 3 and FIG. 11 show the base sequences and chemical modifications of the first and second nucleic acid strands that constitute the ASO and HDO used in this example.
  • the ASO used in this example is a 2'-O-MOE-RNA/DNA gapmer-type anti-RNA that targets metastasis associated lung adenocarcinoma transcript 1 (Malat1) non-coding RNA in mice. It is a sense nucleic acid, has a base sequence complementary to a part of Malat1 ncRNA, has five 2'-O-MOE-RNA nucleosides at the 5' end and five 2'-O-MOE at the 3' end - RNA nucleosides and 10 DNA nucleosides between them linked by phosphorothioate bonds.
  • Malat1 metastasis associated lung adenocarcinoma transcript 1
  • 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 is complementary to the first nucleic acid strand. array.
  • 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 (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(10MOE wing)) of HDO(10MOE wing) consists of five 2'-O-MOE-RNA nucleosides at the 5' end and five 2'-O-MOE- It has a structure in which RNA nucleosides and 10 DNA nucleosides between them are linked by phosphodiester bonds.
  • HDO all RNA
  • HDO all DNA
  • HDO (10MOE wing) listed in Table 3 the same methods as in Example 1 were used to prepare nucleic acids, perform in vivo experiments, and evaluate central nervous system toxicity. Motor function evaluation was performed. However, in this example, the dose of the nucleic acid agent per mouse was 13.86 nmol/mouse.
  • FIG. 12 shows the results of evaluating central nervous system toxicity in mice to which various nucleic acid agents were intracerebroventricularly administered.
  • ASO and HDO all DNA
  • the acute phase tolerability score decreased significantly compared to the group.
  • the HDO (5MOE wing) administration group had a higher acute phase tolerability score than the HDO (all RNA) administration group. was low.
  • Fig. 13 shows the results of evaluating the motor function of mice to which various nucleic acid agents were intracerebroventricularly administered 1 hour after administration.
  • the HDO (10 MOE wing) administration group significantly improved the total moving distance (Fig. 13A) and the maximum moving speed (Fig. 13B) compared to the ASO, HDO (all RNA), and HDO (all DNA) administration groups. .
  • Example 4 Comparison of MOE modification, 2'OMe modification, and 2'F modification> (the purpose) MOE modification for central neurotoxicity observed when intracerebroventricularly administered 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 , 2'OMe modification, and 2'F modification are compared by in vivo experiments.
  • Table 4 and FIG. 14 show the base sequences and chemical modifications of the first and second nucleic acid strands that constitute the ASO and HDO used in this example.
  • the ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid that targets Mapt mRNA, has a nucleotide sequence complementary to a part of Mapt mRNA, and has three LNAs at the 5' end. It has a structure in which a nucleoside, 3 LNA nucleosides at the 3' end, and 10 DNA nucleosides therebetween are linked by phosphorothioate bonds.
  • the second nucleic acid strand has a sequence complementary to the first nucleic acid strand.
  • 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) consists of three 2'-O-MOE-RNA nucleosides at the 5' end and three 2'-O- It has a structure in which MOE-RNA nucleosides and 10 RNA nucleosides between them are linked by phosphodiester bonds.
  • the second nucleic acid strand of HDO(6MOE wing) (c(6MOE wing)) consists of three 2'-O-MOE-RNA nucleosides at the 5' end and three 2'-O-MOE- It has a structure in which RNA nucleosides and 10 DNA nucleosides between them are linked by phosphodiester bonds.
  • the second nucleic acid strand (c(6OMe wing)) of HDO(6OMe wing) consists of three 2'-O-Me-RNA nucleosides at the 5' end and three 2'-O-Me- It has a structure in which RNA nucleosides and 10 DNA nucleosides between them are linked by phosphodiester bonds.
  • the second nucleic acid strand (c(6F wing)) of HDO(6F wing) consists of three 2'F-RNA nucleosides at the 5' end, three 2'F-RNA nucleosides at the 3' end, and their It has a structure in which 10 DNA nucleosides in between are linked by phosphodiester bonds.
  • RNA 6MOE wing RNA 6MOE wing
  • HDO (6MOE wing) HDO (6OMe wing)
  • HDO (6F wing HDO (6F wing) listed in Table 4, preparation of nucleic acids, in vivo experiments, and Central nervous system toxicity evaluation and motor function evaluation were performed. However, in this example, the dose of the nucleic acid agent per mouse was 19 nmol/mouse.
  • FIG. 15 shows the results of evaluating central nervous system toxicity in mice to which various nucleic acid agents were intracerebroventricularly administered.
  • HDO RNA 6MOE wing
  • HDO (6MOE wing) RNA 6MOE wing
  • 6MOE wing HDO Acute phase tolerability scores were significantly reduced compared to the (all DNA), HDO (6OMe wing), and HDO (6F wing) administration groups.
  • the HDO (6MOE wing) administration group showed a lower acute tolerability score than the HDO (RNA 6MOE wing) administration group. From these results, HDOs containing 2'-O-MOE-RNA nucleosides can achieve lower CNS toxicity than HDOs containing 2'-O-Me-RNA and 2'F-RNA nucleosides. was shown.
  • Figures 16 and 17 show the results of evaluating motor function in mice intracerebroventricularly administered with various nucleic acid agents 1 hour and 3 hours after administration.
  • the total distance traveled Fig. 16A, Fig. 17A
  • maximum movement speed Fig. 16B, Fig. 17B
  • HDOs containing 2'-O-MOE-RNA nucleosides are more effective in suppressing motor function than HDOs containing 2'-O-Me-RNA and 2'F-RNA nucleosides. It was shown to be extremely small and of extremely low toxicity.
  • Example 5 Substitution of guanosine nucleoside> (the purpose) Regarding central nervous toxicity observed when intracerebroventricularly administered 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, the second To investigate the toxicity-reducing effect of guanosine nucleoside substitution in nucleic acid chains by in vivo experiments.
  • Table 5 and FIG. 18 show the base sequences and chemical modifications of the first and second nucleic acid strands that constitute the ASO and HDO used in this example.
  • ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid that targets Mapt mRNA, has a nucleotide sequence complementary to a part of Mapt mRNA, and has three LNAs at the 5' end. It has a structure in which a nucleoside, 3 LNA nucleosides at the 3' end, and 10 DNA nucleosides therebetween are linked by phosphorothioate bonds.
  • HDO (6MOE wing), HDO (G MOE ), HDO (G RNA ), and HDO (inosine) used in this example all contain the above ASO as the first nucleic acid strand, and the second nucleic acid strand is the first It has a sequence complementary to a nucleic acid strand.
  • the second nucleic acid strand (c(6MOE wing)) of HDO(6MOE wing) consists of three 2'-O-MOE-RNA nucleosides at the 5' end and three 2' It has a structure in which -O-MOE-RNA nucleosides and 10 DNA nucleosides therebetween are linked by phosphodiester bonds.
  • a DNA nucleoside containing a guanine base is replaced with a 2′-O-MOE-RNA nucleoside in c(6MOE wing).
  • a DNA nucleoside containing a guanine base is replaced with an RNA nucleoside in c(6MOE wing).
  • guanine bases contained in DNA nucleosides are substituted with inosine bases in c(6MOE wing).
  • HDO 6MOE wing
  • HDO G MOE
  • HDO G RNA
  • HDO inosine
  • FIG. 19 shows the results of evaluating central nervous system toxicity in mice to which various nucleic acid agents were intracerebroventricularly administered. At 30 minutes, 1 hour, 2 hours, 3 hours, and 4 hours after administration of the nucleic acid agent, the HDO (G MOE ) and HDO (G RNA ) administration groups 6MOE wing) showed an acute phase tolerability score equivalent to or lower than the administration group.
  • Example 6 Various MOE modifications in HDO targeting the Mapt gene> (the purpose) Various studies have been conducted on the central nervous system toxicity observed when intracerebroventricularly administered HDO containing a first nucleic acid strand consisting of an ASO targeting the Mapt gene and a second nucleic acid strand having a nucleotide sequence complementary to the first nucleic acid strand. We will verify the toxicity-reducing effect based on MOE modification by in vivo experiments.
  • Table 6 and FIG. 20 show the base sequences and chemical modifications of the first and second nucleic acid strands constituting the HDO used in this example.
  • the ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid that targets Mapt mRNA, has a nucleotide sequence complementary to a part of Mapt mRNA, and has three LNAs at the 5' end. It has a structure in which a nucleoside, 3 LNA nucleosides at the 3' end, and 10 DNA nucleosides therebetween are linked by phosphorothioate bonds.
  • HDO (all DNA), HDO (6MOE-5'&3'), HDO (6MOE-5'), HDO (6MOE-3'), HDO (10MOE-5'), and HDO (10MOE) used in this example -3') 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.
  • 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')) consists of three 2'-O-MOE-RNA nucleosides at the 5' end, three 2'-O-MOE-RNA nucleosides and 10 DNA nucleosides between them linked by phosphodiester bonds.
  • the second nucleic acid strand of HDO(6MOE-5') (c(6MOE-5')) consists of 6 2'-O-MOE-RNA nucleosides at the 5' end and 10 DNA nucleosides at the 3' end. have a structure in which are linked by phosphodiester bonds.
  • the second nucleic acid strand (c(6MOE-3')) of HDO(6MOE-3') consists of 10 DNA nucleosides at the 5' end and 6 2'-O-MOE-RNA nucleosides at the 3' end. have a structure in which are linked by phosphodiester bonds.
  • the second nucleic acid strand (c(10MOE-5')) of HDO(10MOE-5') consists of 10 2'-O-MOE-RNA nucleosides at the 5' end and 6 DNA nucleosides at the 3' end. have a structure in which are linked by phosphodiester bonds.
  • the second nucleic acid strand (c(10MOE-3')) of HDO(10MOE-3') consists of 6 DNA nucleosides at the 5' end and 10 2'-O-MOE-RNA nucleosides at the 3' end. have a structure in which are linked by phosphodiester bonds.
  • HDO(all DNA), HDO(6MOE-5'&3'), HDO(6MOE-5'), HDO(6MOE-3'), HDO(10MOE-5'), and HDO(10MOE) listed in Table 6 -3′) was subjected to nucleic acid preparation, in vivo experiments, central nervous system toxicity evaluation, motor function evaluation, and gene suppression effect evaluation in the same manner as in Example 1.
  • the dose of the nucleic acid agent per mouse was 18.86 nmol/mouse.
  • FIG. 21 shows the results of evaluation of central nervous system toxicity in mice to which various nucleic acid agents were intracerebroventricularly administered.
  • HDO(6MOE-5'&3'), HDO(6MOE-5'), HDO(6MOE-3'), HDO(10MOE-5'), and HDO(10MOE-3') significantly reduced acute tolerability scores compared to HDO(all DNA) bottom.
  • the acute tolerability score in the HDO (10MOE-5') and HDO (10MOE-3') administration groups was the lowest in the HDO administration group.
  • Figures 22 and 23 show the results of evaluating motor function in mice intracerebroventricularly administered with various nucleic acid agents 1 hour and 3 hours after administration.
  • HDO(all DNA) significantly improved the total moving distance (Figs. 22A and 23A) and the maximum moving speed (Figs. 22B and 23B) compared to the administration group.
  • the improvement effect in the HDO (10MOE-5') and HDO (10MOE-3') administration groups was greatest in the HDO administration group.
  • Three hours after administration the improvement effect in the HDO (6MOE-5'&3') administration group was the greatest in the HDO administration group.
  • Fig. 24 shows Mapt mRNA expression levels in the hippocampus 7 days after intracerebroventricular administration of various nucleic acid agents.
  • Administration of HDO(all DNA), HDO(6MOE-5'&3'), HDO(6MOE-5'), HDO(6MOE-3'), HDO(10MOE-5'), and HDO(10MOE-3') The group showed a significant reduction in Mapt mRNA expression levels compared to negative controls that received PBS alone.
  • Example 7 Various MOE modifications in HDO targeting the BACE1 gene> (the purpose) Various studies have been conducted on the central nervous system toxicity observed when intracerebroventricularly administered HDO containing a first nucleic acid strand consisting of an ASO targeting the BACE1 gene and a second nucleic acid strand having a nucleotide sequence complementary to the first nucleic acid strand. We will verify the toxicity-reducing effect based on MOE modification by in vivo experiments.
  • Table 7 and FIG. 25 show the base sequences and chemical modifications of the first and second nucleic acid strands that constitute the HDO used in this example.
  • ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid that targets BACE1 mRNA, has a nucleotide sequence complementary to a part of BACE1 mRNA, and has two LNAs at the 5' end. It has a structure in which a nucleoside, 3 LNA nucleosides at the 3' end, and 8 DNA nucleosides therebetween are linked by phosphorothioate bonds.
  • HDO (all DNA), HDO (5MOE-5'), HDO (5MOE-3'), HDO (8MOE-5'), HDO (8MOE-3'), HDO (10MOE-5') used in this example ), and HDO(10MOE-3′) both 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.
  • 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(5MOE-5')) of HDO(5MOE-5') consists of five 2'-O-MOE-RNA nucleosides at the 5' end and eight DNA nucleosides at the 3' end. have a structure in which are linked by phosphodiester bonds.
  • the second nucleic acid strand (c(5MOE-3')) of HDO(5MOE-3') consists of 8 DNA nucleosides at the 5' end and 5 2'-O-MOE-RNA nucleosides at the 3' end. have a structure in which are linked by phosphodiester bonds.
  • the second nucleic acid strand (c(8MOE-5')) of HDO(8MOE-5') consists of 8 2'-O-MOE-RNA nucleosides at the 5' end and 5 DNA nucleosides at the 3' end. have a structure in which are linked by phosphodiester bonds.
  • the second nucleic acid strand (c(8MOE-3')) of HDO(8MOE-3') consists of 5 DNA nucleosides at the 5' end and 8 2'-O-MOE-RNA nucleosides at the 3' end. have a structure in which are linked by phosphodiester bonds.
  • the second nucleic acid strand (c(10MOE-5')) of HDO(10MOE-5') consists of 10 2'-O-MOE-RNA nucleosides at the 5' end and 3 DNA nucleosides at the 3' end. have a structure in which are linked by phosphodiester bonds.
  • the second nucleic acid strand (c(10MOE-3')) of HDO(10MOE-3') consists of 3 DNA nucleosides at the 5' end and 10 2'-O-MOE-RNA nucleosides at the 3' end. have a structure in which are linked by phosphodiester bonds.
  • FIG. 26 shows the results of evaluating central nervous system toxicity in mice to which various nucleic acid agents were intracerebroventricularly administered.
  • HDO(5MOE-5'), HDO(5MOE-3'), HDO( 8MOE-5'), HDO(8MOE-3'), HDO(10MOE-5'), and HDO(10MOE-3') showed better acute phase tolerance than HDO(all DNA). Tolerability score decreased significantly.
  • Fig. 27 shows the results of evaluating the motor function of mice to which various nucleic acid agents were intracerebroventricularly administered 1 hour after administration.
  • HDO(5MOE-5'), HDO(5MOE-3'), HDO(8MOE-5'), HDO(8MOE-3'), HDO(10MOE-5'), and HDO(10MOE-3') Compared with the HDO(all DNA) administration group, the total distance traveled (Fig. 27A) and the maximum movement speed (Fig. 27B) were significantly improved in the administration group.
  • Example 8 Various MOE modifications in HDO targeting the Mapt gene> (the purpose) Various studies have been conducted on the central nervous system toxicity observed when intracerebroventricularly administered HDO containing a first nucleic acid strand consisting of an ASO targeting the Mapt gene and a second nucleic acid strand having a nucleotide sequence complementary to the first nucleic acid strand. We will verify the toxicity-reducing effect based on MOE modification by in vivo experiments.
  • Table 8 and FIG. 28 show the base sequences and chemical modifications of the first and second nucleic acid strands that constitute the HDO used in this example.
  • the ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid that targets Mapt mRNA, has a nucleotide sequence complementary to a part of Mapt mRNA, and has three LNAs at the 5' end. It has a structure in which a nucleoside, 3 LNA nucleosides at the 3' end, and 10 DNA nucleosides therebetween are linked by phosphorothioate bonds.
  • the second nucleic acid strand has a sequence complementary to the first nucleic acid strand.
  • 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 wing) (c(6MOE wing)) consists of three 2'-O-MOE-RNA nucleosides at the 5' end and three 2'-O-MOE- It has a structure in which RNA nucleosides and 10 DNA nucleosides between them are linked by phosphodiester bonds.
  • the second nucleic acid strand (c(9MOE wing)) of HDO(9MOE wing) consists of five 2'-O-MOE-RNA nucleosides at the 5' end and four 2'-O-MOE- It has a structure in which RNA nucleosides and seven DNA nucleosides between them are linked by phosphodiester bonds.
  • the second nucleic acid strand (c(11MOE wing)) of HDO(11MOE wing) consists of six 2'-O-MOE-RNA nucleosides at the 5' end and five 2'-O-MOE- It has a structure in which RNA nucleosides and five DNA nucleosides between them are linked by phosphodiester bonds.
  • the second nucleic acid strand (c(13MOE wing)) of HDO(13MOE wing) consists of seven 2'-O-MOE-RNA nucleosides at the 5' end and six 2'-O-MOE- It has a structure in which an RNA nucleoside and three DNA nucleosides therebetween are linked by phosphodiester bonds.
  • the second nucleic acid strand (c(15MOE wing)) of HDO(15MOE wing) consists of eight 2'-O-MOE-RNA nucleosides at the 5' end and seven 2'-O-MOE- It has a structure in which RNA nucleosides and one DNA nucleoside between them are linked by phosphodiester bonds.
  • HDO all DNA
  • HDO (6 MOE wing) HDO (9 MOE wing)
  • HDO 11 MOE wing
  • HDO 13 MOE wing
  • HDO 15 MOE wing
  • the same Preparation of nucleic acids, in vivo experiments, evaluation of central nervous system toxicity and motor function, and evaluation of gene suppression effects were carried out according to the method.
  • the dose of the nucleic acid agent per mouse was 18.86 nmol/mouse.
  • FIG. 29 shows the results of evaluating central nervous system toxicity in mice to which various nucleic acid agents were intracerebroventricularly administered.
  • HDO (6MOE wing), HDO (9MOE wing), HDO (11MOE wing), HDO (11MOE wing), In the HDO (13MOE wing) and HDO (15MOE wing) administration groups, the acute phase tolerability score significantly decreased compared to the HDO (all DNA) administration group.
  • Fig. 30 shows the results of evaluating the motor function of mice to which various nucleic acid agents were intracerebroventricularly administered 1 hour after administration.
  • HDO (6 MOE wing), HDO (9 MOE wing), HDO (11 MOE wing), HDO (13 MOE wing), and HDO (15 MOE wing) treatment groups compared with HDO (all DNA) treatment group Distance (Fig. 30A) and maximum movement speed (Fig. 30B) improved significantly.
  • Example 9 Comparison of base species in MOE modification> (the purpose) Regarding central nervous toxicity observed when intracerebroventricularly administered 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, the second In vivo experiments are used to compare the toxicity-reducing effects of different types of nucleosides (nucleosides containing an adenine base, a guanine base, a cytosine base, or a thymine base) into which MOE modifications are introduced in the nucleic acid chain.
  • nucleosides nucleosides containing an adenine base, a guanine base, a cytosine base, or a thymine base
  • Table 9 and FIG. 31 show the base sequences and chemical modifications of the first and second nucleic acid strands that constitute the ASO and HDO used in this example.
  • ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid that targets Mapt mRNA, has a nucleotide sequence complementary to a part of Mapt mRNA, and has three LNAs at the 5' end. It has a structure in which a nucleoside, 3 LNA nucleosides at the 3' end, and 10 DNA nucleosides therebetween are linked by phosphorothioate bonds.
  • HDO (all DNA), HDO (A MOE ), HDO (G MOE ), HDO (C MOE ), and HDO (T MOE ) used in this example all contain the ASO as the first nucleic acid strand
  • the second nucleic acid strand has a sequence complementary to the first nucleic acid strand.
  • 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 (a(A MOE )) of HDO(A MOE ) has a structure in which all DNA nucleosides containing adenine bases are replaced with 2′-O-MOE-RNA nucleosides.
  • HDO( CMOE ) have guanine bases
  • HDO( CMOE ) have cytosine bases
  • HDO( TMOE ) have structures in which DNA nucleosides containing thymine bases are all substituted with 2′-O-MOE-RNA nucleosides.
  • FIG. 32 shows the results of evaluating central nervous system toxicity in mice to which various nucleic acid agents were intracerebroventricularly administered.
  • HDO A MOE
  • HDO C MOE
  • HDO T MOE
  • the acute phase tolerability score decreased significantly compared to the HDO (all DNA ) administration group
  • Tolerability score decreased slightly.
  • FIG. 33 shows the results of evaluating motor function in mice to which various nucleic acid agents were intracerebroventricularly administered 1 hour and 3 hours after administration.
  • the total distance traveled ( Figure 33A) and the maximum travel speed ( Figure 33B) was markedly improved, and the HDO(G MOE )-administered group had slightly improved total moving distance (FIG. 33A) and maximum moving speed (FIG. 33B) compared to the HDO(all DNA)-administered group.
  • Example 10 Comparison of adjacent nucleic acids in MOE modification> (the purpose) Regarding central nervous toxicity observed when intracerebroventricularly administered 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, the second Toxicity-reducing effects by introducing MOE modifications to nucleosides containing cytosine bases and nucleosides adjacent to the 5' and/or 3' sides thereof in nucleic acid chains are compared by in vivo experiments.
  • Table 10 and FIG. 34 show the base sequences and chemical modifications of the first and second nucleic acid strands constituting the ASO and HDO used in this example.
  • ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid that targets Mapt mRNA, has a nucleotide sequence complementary to a part of Mapt mRNA, and has three LNAs at the 5' end. It has a structure in which a nucleoside, 3 LNA nucleosides at the 3' end, and 10 DNA nucleosides therebetween are linked by phosphorothioate bonds.
  • HDO(all DNA), HDO(C MOE ), HDO(2C MOE ⁇ 5), HDO(2C MOE ⁇ 3) and HDO(3C MOE ) used in this example are all and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand.
  • 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( CMOE )) of HDO( CMOE ) has a structure in which all DNA nucleosides containing cytosine bases are replaced with 2'-O-MOE-RNA nucleosides.
  • the second nucleic acid strand (c(2C MOE -5)) of HDO(2C MOE -5) converts a DNA nucleoside containing a cytosine base and its 5'-adjacent DNA nucleoside into 2'-O-MOE-RNA nucleosides.
  • the second nucleic acid strand (c(2C MOE -3)) of HDO(2C MOE -3) replaces all DNA nucleosides containing cytosine bases and its 3' adjacent DNA nucleosides with 2'-O-MOE-RNA nucleosides. It has a structure that The second nucleic acid strand (c(3C MOE )) of HDO(3C MOE ) converts a DNA nucleoside containing a cytosine base and its 5′ and 3′ flanking DNA nucleosides into 2′-O-MOE-RNA nucleosides. It has a permuted structure.
  • HDO (all DNA), HDO (C MOE ), HDO (2C MOE -5), HDO (2C MOE -3) and HDO (3C MOE ) listed in Table 10 were subjected to nucleic acid preparation in the same manner as in Example 1. preparation, in vivo experiments, central nervous system toxicity evaluation and motor function evaluation. However, in this example, the dose of the nucleic acid agent per mouse was 19 nmol/mouse.
  • FIG. 35 shows the results of evaluating central nervous system toxicity in mice to which various nucleic acid agents were intracerebroventricularly administered.
  • FIG. 36 shows the results of evaluating motor function in mice to which various nucleic acid agents were intracerebroventricularly administered 1 hour and 3 hours after administration.
  • the total distance traveled (Fig. 36A) and maximum locomotion velocity (Fig. 36B) were significantly improved.
  • Fig. 36A the total distance traveled
  • Fig. 36B maximum locomotion velocity
  • HDO stability in cerebrospinal fluid (the purpose) 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 was incubated in human or rat cerebrospinal fluid, and its stability was evaluated. think about.
  • the ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid that targets Mapt mRNA, has a nucleotide sequence complementary to a part of Mapt mRNA, and has three LNAs at the 5' end. It has a structure in which a nucleoside, 3 LNA nucleosides at the 3' end, and 10 DNA nucleosides therebetween are linked by phosphorothioate bonds.
  • Both HDO (ASO/cRNA) and HDO (ASO/cDNA) used in this example 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.
  • the second nucleic acid strand (cRNA) of HDO has three phosphorothioate bonds from the 5' end, three phosphorothioate bonds from the 3' end, and three phosphorothioate bonds between them. It has a structure linked by nine phosphodiester bonds.
  • the second nucleic acid strand (cDNA) of HDO (ASO/cDNA) consists of three phosphorothioate bonds from the 5' end, three phosphorothioate bonds from the 3' end, and nine phosphodiester bonds between them. It has a bond-linked structure.
  • a 16% acrylamide gel (1 x TBE) was prepared and produced. 6 ⁇ L of the above sample was loaded onto the gel and run at 100 V for 80 minutes. As a control, ASO alone and cRNA alone were electrophoresed simultaneously. After that, a solution was prepared by diluting GelRed ( ⁇ 10000) aqueous solution (Biotium) with 1 ⁇ TBE to a concentration of 1/10000. The gel was permeated with the solution for 10 minutes. Gels were then imaged with a ChemiDoc Touch imaging system (BioRad).
  • FIG. 37C shows electrophoresis results after incubating HDO (ASO/cRNA) in human cerebrospinal fluid (Human CSF) for 10 minutes and 6 hours.
  • FIG. 37D shows the results of quantifying the band intensity of the HDO double-stranded band in the electrophoresis results of FIG. 37C.
  • HDO(ASO/cRNA) was mostly degraded after 10 minutes of incubation in human cerebrospinal fluid and completely degraded after 6 hours.
  • FIG. 38C shows the results of electrophoresis after incubating HDO (ASO/cRNA) or HDO (ASO/cDNA) in human cerebrospinal fluid or rat cerebrospinal fluid for 6 hours.
  • HDO ASO/cRNA
  • HDO ASO/cDNA
  • Example 12 HDO stability in cerebrospinal fluid> (the purpose) An HDO comprising 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 was incubated in mouse, rat, monkey, or human cerebrospinal fluid. , to consider its stability.
  • the ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid that targets Mapt mRNA, has a nucleotide sequence complementary to a part of Mapt mRNA, and has three LNAs at the 5' end. It has a structure in which a nucleoside, 3 LNA nucleosides at the 3' end, and 10 DNA nucleosides therebetween are linked by phosphorothioate bonds.
  • HDO (all RNA), HDO (all DNA), HDO (cRNA 6MOE wing), and HDO (cDNA 6MOE 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.
  • 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 of HDO(cRNA 6MOE wing) (cRNA(6MOE wing)) consists of three 2'-O-MOE-RNA nucleosides at the 5' end and three 2'-O-MOE at the 3' end. - RNA nucleosides and 10 RNA nucleosides between them are linked by phosphodiester bonds.
  • the second nucleic acid strand of HDO (cDNA 6MOE wing) (cDNA (6MOE wing)) consists of three 2'-O-MOE-RNA nucleosides at the 5' end and three 2'-O-MOE at the 3' end. - RNA nucleosides and 10 DNA nucleosides between them linked by phosphodiester bonds.
  • HDO all RNA
  • HDO all DNA
  • HDO cRNA 6MOE wing
  • HDO cDNA 6MOE wing
  • FIG. 39C shows electrophoresis results after incubation of HDO(all RNA) and HDO(all DNA) in mouse, rat, monkey and human cerebrospinal fluid for 6 hours.
  • HDO (all RNA) was stable and not degraded in mouse and rat cerebrospinal fluids, while it was degraded and unstable in monkey and human cerebrospinal fluids.
  • HDO(all DNA) was stable without degradation in the cerebrospinal fluids of mice, rats, monkeys and humans.
  • FIG. 40C shows the results of electrophoresis after incubating HDO (cRNA 6MOE wing) and HDO (cDNA 6MOE wing) in mouse, rat, monkey, and human cerebrospinal fluid for 6 hours.
  • HDO cRNA 6MOE wing
  • HDO cDNA 6MOE wing
  • FIG. 40C shows the results of electrophoresis after incubating HDO (cRNA 6MOE wing) and HDO (cDNA 6MOE wing) in mouse, rat, monkey, and human cerebrospinal fluid for 6 hours.
  • HDO cRNA 6MOE wing
  • HDO cDNA 6MOE wing
  • HDO stability in cerebrospinal fluid (the purpose) In an HDO comprising 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, part of the DNA nucleosides in the second nucleic acid strand are replaced with RNA nucleosides.
  • ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid that targets Mapt mRNA, has a nucleotide sequence complementary to a part of Mapt mRNA, and has three LNAs at the 5' end. It has a structure in which a nucleoside, 3 LNA nucleosides at the 3' end, and 10 DNA nucleosides therebetween are linked by phosphorothioate bonds.
  • HDO(all DNA), HDO(A RNA ), HDO(G RNA ), HDO(C RNA ), and HDO(U RNA ) used in this example all contain the above ASO as the first nucleic acid strand
  • the second nucleic acid strand has a sequence complementary to the first nucleic acid strand.
  • 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(A RNA )) of HDO(A RNA ) has a structure in which all DNA nucleosides containing adenine bases in c(all DNA) are replaced with RNA nucleosides.
  • the second nucleic acid strand (c(G RNA )) of HDO(G RNA ) has a structure in which all DNA nucleosides containing guanine bases in c(all DNA) are replaced with RNA nucleosides.
  • the second nucleic acid strand (c(C RNA )) of HDO(C RNA ) has a structure in which all DNA nucleosides containing cytosine bases in c(all DNA) are replaced with RNA nucleosides.
  • the second nucleic acid strand (c(U RNA )) of HDO(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.
  • HDO all DNA
  • HDO A RNA
  • HDO G RNA
  • HDO C RNA
  • HDO U RNA
  • Figure 42 shows the results of electrophoresis after incubating HDO (all DNA), HDO (A RNA ), HDO (G RNA ), HDO (C RNA ), and HDO (U RNA ) in human cerebrospinal fluid for 6 hours. show. HDO(all DNA), HDO(A RNA ), and HDO(G RNA ) were stable and not degraded in human cerebrospinal fluid. On the other hand, HDO(C RNA ) and HDO(U RNA ) were degraded and unstable in human cerebrospinal fluid.
  • Example 14 HDO stability in cerebrospinal fluid> (the purpose) In an HDO comprising 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, part of the DNA nucleosides in the second nucleic acid strand are replaced with RNA nucleosides. The stability in cerebrospinal fluid when replaced with
  • ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid that targets Mapt mRNA, has a nucleotide sequence complementary to a part of Mapt mRNA, and has three LNAs at the 5' end. It has a structure in which a nucleoside, 3 LNA nucleosides at the 3' end, and 10 DNA nucleosides therebetween are linked by phosphorothioate bonds.
  • HDO (all DNA), HDO (GA RNA ), HDO (CU RNA ), HDO (C RNA ), and HDO (U RNA ) used in this example all contain the ASO as the first nucleic acid strand
  • the second nucleic acid strand has a sequence complementary to the first nucleic acid strand.
  • 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(GA RNA )) of HDO(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.
  • all DNA nucleosides containing cytosine bases in c(all DNA) are replaced with RNA nucleosides, and all DNA nucleosides containing thymine bases are replaced with uracil bases. It has a structure in which RNA nucleosides containing are substituted.
  • the second nucleic acid strand (c(C RNA )) of HDO(C RNA ) has a structure in which all DNA nucleosides containing cytosine bases in c(all DNA) are replaced with RNA nucleosides.
  • the second nucleic acid strand (c(U RNA )) of HDO(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.
  • HDO all DNA
  • HDO GA RNA
  • CU RNA HDO
  • C RNA HDO
  • U RNA U RNA
  • Figure 44 shows the electrical activity of HDO(all DNA), HDO(GA RNA ), HDO(CU RNA ), HDO(C RNA ), and HDO(U RNA ) after incubation in human cerebrospinal fluid for 1 hour and 6 hours. The results of electrophoresis are shown. HDO(all DNA) and HDO(GA RNA ) were stable and not degraded in human cerebrospinal fluid. On the other hand, HDO(CU RNA ), HDO(C RNA ), and HDO(U RNA ) were degraded and unstable in human cerebrospinal fluid.
  • Example 15 HDO stability in cerebrospinal fluid> (the purpose) In an HDO comprising 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, part of the DNA nucleosides in the second nucleic acid strand are replaced with RNA nucleosides. The stability in cerebrospinal fluid when replaced with
  • the ASO used in this example is a 2'-O-MOE-RNA/DNA gapmer-type antisense nucleic acid targeting Malat1 ncRNA, and has a base sequence complementary to a part of Malat1 ncRNA.
  • the 5 2'-O-MOE-RNA nucleosides at the 'end, the 5 2'-O-MOE-RNA nucleosides at the 3' end, and the 10 DNA nucleosides in between were linked with phosphorothioate linkages. have a structure.
  • HDO(all DNA), HDO(A RNA ), HDO(G RNA ), HDO(C RNA ), and HDO(U RNA ) used in this example all contain the above ASO as the first nucleic acid strand
  • the second nucleic acid strand has a sequence complementary to the first nucleic acid strand.
  • 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(A RNA )) of HDO(A RNA ) has a structure in which all DNA nucleosides containing adenine bases in c(all DNA) are replaced with RNA nucleosides.
  • the second nucleic acid strand (c(G RNA )) of HDO(G RNA ) has a structure in which all DNA nucleosides containing guanine bases in c(all DNA) are replaced with RNA nucleosides.
  • the second nucleic acid strand (c(C RNA )) of HDO(C RNA ) has a structure in which all DNA nucleosides containing cytosine bases in c(all DNA) are replaced with RNA nucleosides.
  • the second nucleic acid strand (c(U RNA )) of HDO(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.
  • HDO all DNA
  • HDO A RNA
  • HDO G RNA
  • HDO C RNA
  • HDO U RNA
  • Figure 46 shows the results of electrophoresis after incubating HDO (all DNA), HDO (A RNA ), HDO (G RNA ), HDO (C RNA ), and HDO (U RNA ) in human cerebrospinal fluid for 6 hours. show. HDO(all DNA), HDO(A RNA ), and HDO(G RNA ) were stable and not degraded in human cerebrospinal fluid. On the other hand, HDO(C RNA ) and HDO(U RNA ) were degraded and unstable in human cerebrospinal fluid.
  • Nucleic acids were prepared in the same manner as in Example 1 for ASO, HDO (RNA-MOE), and HDO (DNA-MOE) listed in Table 16.
  • HDO having a bulge structure> (the purpose) An HDO comprising 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. To verify the toxicity-reducing effect and gene-suppressing effect by introducing a bulge structure into the second nucleic acid strand by in vitro experiments.
  • the ASO used in this example is an LNA/DNA gapmer-type antisense nucleic acid that targets Mapt mRNA, has a nucleotide sequence complementary to a part of Mapt mRNA, and has three LNAs at the 5' end. It has a structure in which a nucleoside, 3 LNA nucleosides at the 3' end, and 10 DNA nucleosides therebetween are linked by phosphorothioate bonds.
  • HDO (all DNA), HDO (all MOE), HDO (bulge1), and HDO (bulge2) used in this example all contain the ASO as the first nucleic acid strand, and the second nucleic acid strand is the first nucleic acid. It contains a sequence complementary to the strand.
  • 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.
  • 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.
  • a bulge structure has a structure in which two DNA nucleosides are linked by a phosphodiester bond.
  • the second nucleic acid strand (c(bulge2)) of HDO(bulge2) includes 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.
  • the region has a single base deletion relative to the first nucleic acid strand, and a bulge structure is located at the site of the deletion.
  • 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.
  • Nucleic acids were prepared in the same manner as in Example 1 for HDO (all DNA), HDO (all MOE), HDO (bulge1), and HDO (bulge2) listed in Table 17.
  • the ratio of the Mapt mRNA expression level to the Actb mRNA (internal standard gene) expression level was calculated, and the value normalized to the value of the PBS administration group was taken as the relative Mapt mRNA level.
  • LDH activity in the cell supernatant was measured as evaluation of neuronal cytotoxicity.
  • LDH activity was measured using Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories) according to the attached protocol.
  • a value normalized to the LDH activity in the PBS-administered group was defined as the relative LDH release level.
  • FIG. 49A shows the results of evaluation of target gene inhibitory effects of various nucleic acid agents in human neuroblastoma-derived cells (BE(2)-M17 cell line).
  • the suppressive effect was significantly attenuated compared to HDO (all DNA).
  • the HDO(bulge1) and HDO(bulge2) administration groups exhibited a strong gene-silencing effect equivalent to that of HDO(all DNA).
  • Fig. 49B shows the results of evaluating the LDH activity in the supernatant as cytotoxicity when various nucleic acid agents were introduced into cells.
  • the HDO (all DNA) administration group showed an increase in LDH activity
  • the HDO (all MOE), HDO (bulge1), and HDO (bulge2) groups showed LDH activity equal to or lower than that of the PBS administration group, resulting in cytotoxicity. decreased significantly.
  • HDO body1
  • HDO body2
  • Example 18 Single-stranded HDO> (the purpose) Regarding central nervous toxicity observed when intracerebroventricularly administered 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, the second Introduction of a bulge structure into the nucleic acid strand, linking the first and second nucleic acid strands with a linker, and toxicity-reducing effects and gene-suppressing effects of both are verified by in vivo experiments.
  • HDO (bulge), ssHDO, PEG linker ssHDO, and bulge plus ssHDO used in this example all contain a first nucleic acid strand and a second nucleic acid strand.
  • the first nucleic acid strands of HDO(bulge), ssHDO, PEG linker ssHDO, and Bulge plus ssHDO are all LNA/DNA gapmer-type antisense nucleic acids, having a base sequence complementary to a portion of Mapt mRNA, It has a structure in which 3 LNA nucleosides at the 5' end, 3 LNA nucleosides at the 3' end, and 10 DNA nucleosides therebetween are linked by phosphorothioate bonds.
  • the first and second nucleic acid strands of HDO are not linked by a linker, but in ssHDO, PEG linker ssHDO, and Bulge plus ssHDO, the 3' end of the second nucleic acid strand is 5 ' is bound to the end via a linker.
  • the second nucleic acid strand (c(bulge)) of HDO(bulge) includes 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.
  • the second nucleic acid strand of ssHDO has a sequence complementary to the first nucleic acid strand and contains three 2'-O-MOE-RNA nucleosides, ten DNA nucleosides, and three 2'-O-MOE -Have a structure in which RNA nucleosides are sequentially linked by phosphodiester bonds from the 5' end.
  • the linker that connects the first and second nucleic acid strands in ssHDO consists of three DNA nucleosides linked by phosphodiester bonds.
  • the configuration of the second nucleic acid strand of PEG linker ssHDO is the same as that of the second nucleic acid strand of ssHDO described above.
  • the linker that connects the first nucleic acid strand and the second nucleic acid strand consists of PEG (polyethylene glycol).
  • the second nucleic acid strand of Bulge plus ssHDO includes 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 Bulge plus ssHDO consists of three DNA nucleosides linked by phosphodiester bonds.
  • HDO(bulge) ssHDO, PEG linker ssHDO, and Bulge plus ssHDO were intracerebroventricularly administered to mice, and compared with HDO(all DNA)-administered mice used in Example 1, 30 minutes to 4 minutes after administration. There is a marked decrease in post-hour acute tolerability scores.
  • mice intracerebroventricularly administered HDO(bulge), ssHDO, PEG linker ssHDO, and Bulge plus ssHDO compared with mice etc. administered with HDO (all DNA) used in Example 1, 1 hour after administration Subsequent motor functions, such as total distance traveled and maximum speed of travel, are significantly improved.
  • ⁇ Example 19 Examination of nucleic acid species to be introduced into the gap region of the second nucleic acid strand> (the purpose) Regarding the central nervous system toxicity observed when HDO targeting the Malat1 gene is intracerebroventricularly administered, a region consisting of a nucleotide sequence complementary to the central region (gap region) of the first nucleic acid strand in the second nucleic acid strand (hereinafter referred to as the second nucleic acid strand) The influence of the type of nucleic acid to be introduced into the "gap region” (also referred to as a "gap region" in two nucleic acid strands) is examined.
  • HDO in which all nucleosides are RNA nucleosides HDO in which all nucleosides are DNA nucleosides, and RNA nucleosides are arranged at the positions of adenine base and guanine base, and cytosine
  • the toxicity of HDOs with DNA nucleosides placed at base and thymine base positions is compared by in vivo experiments.
  • Table 19 shows the base sequences and chemical modifications of the first and second nucleic acid strands constituting the ASOs and HDOs used in this example.
  • HDO (all DNA), HDO (cRNA 10MOE), HDO (cDNA 10MOE), and HDO (agRNA 10MOE) used in this example contain the common ASO as the first nucleic acid strand, and the second nucleic acid strand It has a sequence complementary to a nucleic acid strand.
  • 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 has two regions consisting of base sequences complementary to the wing regions of the first nucleic acid strand (hereinafter also referred to as "wing regions" in the second nucleic acid strand).
  • the second nucleic acid strand c (DNA 10MOE) of HDO (cDNA 10MOE) is composed of '-O-MOE-RNA nucleosides and the gap region is composed of RNA nucleosides;
  • the second nucleic acid strand c (agRNA 10MOE) of HDO (agRNA 10MOE) is composed of RNA nucleosides, and the gap region is composed of DNA nucleosides;
  • the wing region is composed of 2'-O-MOE-RNA nucleosides.
  • nucleosides with adenine and guanine bases are RNA nucleosides
  • nucleosides with cytosine and thymine bases are DNA nucleosides.
  • nucleic acid molecules shown in Table 19 preparation of nucleic acids, in vivo experiments, evaluation of central nervous system toxicity, and evaluation of motor function were performed in the same manner as in Example 1. However, in this example, the dose of the nucleic acid agent per mouse was 14 nmol/mouse.
  • FIG. 51 shows the results of evaluating motor function in mice to which various nucleic acid agents were intracerebroventricularly administered 1 hour after administration.
  • HDO all DNA
  • HDO cRNA 10MOE
  • HDO cDNA 10MOE
  • HDO agRNA 10MOE
  • the total distance traveled Fig. 51A
  • the average speed of movement Fig. 51B
  • the total moving distance and average moving speed were improved compared to the HDO (all DNA) administration group.
  • FIG. 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)-administered group showed a marked effect of suppressing the Malat1 RNA expression level compared to the ASO-administered group and other HDO-administered groups. From this result, HDO containing the second nucleic acid strand in which the positions of the adenine base and the guanine base in the gap region are RNA nucleosides, and the positions of the cytosine base and the thymine base are DNA nucleosides, all nucleosides in the gap region are DNA nucleosides. It was shown that an excellent gene expression-suppressing effect can be achieved compared to HDO containing a second nucleic acid strand that is an RNA nucleoside or an RNA nucleoside.
  • Example 20 Evaluation of double strand dissociation efficiency in brain tissue> (the purpose)
  • the first nucleic acid strand is dissociated from the second nucleic acid strand in the target tissue and can exert an antisense effect on the target gene or its transcript. Therefore, HDOs containing the first and second nucleic acid strands are incubated in mouse brain tissue homogenate to examine the efficiency of duplex dissociation.
  • a solution was prepared by diluting GelRed ( ⁇ 10000) aqueous solution (Biotium) with 1 ⁇ TBE to a concentration of 1/10000. The gel was permeated with the solution for 10 minutes. The gel was then imaged with a ChemiDoc Touch imaging system (BioRad).
  • Figure 53 shows electrophoresis results after incubation of ASO, HDO (cRNA 10MOE), HDO (cDNA 10MOE) and HDO (agRNA 10MOE) in mouse brain tissue homogenate for 7 days.
  • HDO agRNA 10MOE
  • An HDO comprising a second nucleic acid strand in which the positions of adenine and guanine bases in the gap region are RNA nucleosides and the positions of cytosine and thymine bases are DNA nucleosides, all nucleosides in the gap region are either DNA nucleosides or RNA nucleosides.
  • the double-strand dissociation efficiency was extremely high in brain tissue, which strongly supported the result of marked suppression of target gene expression in Example 19 above.
  • Example 21 Examination of nucleic acid modification to be introduced into the wing region of the second nucleic acid strand> (the purpose) Regarding the central neurotoxicity observed when HDO targeting the Malat1 gene is administered intracerebroventricularly, the effect of introduction of MOE modified nucleic acid or natural nucleic acid (RNA or DNA) in the wing region and gap region of the second nucleic acid strand is investigated in vivo. Compare by experiment.
  • Table 20 shows the base sequences and chemical modifications of the first and second nucleic acid strands constituting the HDO used in this example.
  • the second nucleic acid strand has a sequence complementary to the first nucleic acid strand.
  • the second nucleic acid strand of HDO (all DNA) has a structure in which DNA nucleosides are linked by phosphodiester bonds.
  • the wing region is composed of 2'-O-MOE-RNA nucleosides;
  • the gap region is composed of RNA nucleoside structures;
  • the second nucleic acid strand of HDO (gapMOE RNA) , the region corresponding to the wing region is composed of RNA nucleosides, and the region corresponding to the gap region is composed of 2'-O-MOE-RNA nucleoside structures;
  • the wing region is composed of 2'-O-MOE-RNA nucleosides, and the gap structure is composed of DNA nucleoside structures;
  • the gap region is composed of 2'-O-MOE-RNA nucleoside structures;
  • the second nucleic acid strand of HDO (agRNA 10MOE) is composed of 2'-O-MOE-RNA nucleosides in the wing region
  • nucleosides with adenine and guanine bases are RNA nucleosides, and nucleosides with adenine and gu
  • nucleic acid molecules shown in Table 21 preparation of nucleic acids, in vivo experiments, evaluation of central nervous system toxicity, and evaluation of motor function were performed in the same manner as in Example 1. However, in this example, the dose of the nucleic acid agent per mouse was 28 nmol/mouse.
  • FIG. 54 shows the results of evaluation of central nervous system toxicity in mice to which various nucleic acid agents were intracerebroventricularly administered.
  • the HDO (cRNA MOEwing), HDO (gapMOE RNA), HDO (cDNA MOEwing), HDO (gapDNA MOE) and HDO (agRNA 10MOE) administration groups had better acute phase tolerance than the HDO (all DNA) administration group. Tolerability score decreased significantly.
  • the HDO (gapMOE RNA), HDO (cDNA MOEwing), HDO (gapDNA MOE), and HDO (agRNA 10MOE) administration groups had higher acute phase tolerability scores than the HDO (cDNA MOEwing) administration group. decreased further.
  • FIG. 55 shows the results of evaluating motor function in mice to which various nucleic acid agents were intracerebroventricularly administered 1 hour after administration.
  • the total distance traveled was (Fig. 55A) and mean locomotion speed (Fig. 55B) were significantly improved.
  • the HDO (gapMOE RNA), HDO (cDNA MOEwing), HDO (gapDNA MOE) and HDO (agRNA 10MOE) administration groups compared with the HDO (cDNA MOEwing) administration group, total moving distance and average moving speed improved.
  • Example 22 Examination of nucleic acid species to be introduced into the gap region of the second nucleic acid strand: comparison of acute/delayed neurotoxicity> (the purpose) After intracerebroventricular administration of HDO containing a first nucleic acid strand consisting of an ASO targeting the Mapt gene and a MOE wing-modified second nucleic acid strand having a nucleotide sequence complementary to the first nucleic acid strand Acute neurotoxicity within 1 day and delayed neurotoxicity after 1 day after administration will be compared by in vivo experiments.
  • Table 21 shows the base sequences and chemical modifications of the first and second nucleic acid strands constituting the ASOs and HDOs used in this example.
  • HDO (all DNA), HDO (cRNA 6MOE), HDO (cDNA 6MOE), and HDO (agRNA 6MOE) used in this example contain the ASO as a common first nucleic acid strand, and the second nucleic acid strand It has a sequence complementary to one nucleic acid strand.
  • 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) consists of three 2'-O-MOE-RNA nucleosides at the 5' end, three 2'-O-MOE-RNA nucleosides at the 3' end, and has a structure in which 10 RNA nucleosides are linked by phosphodiester bonds.
  • the second nucleic acid strand of HDO (cDNA 6MOE) consists of three 2'-O-MOE-RNA nucleosides at the 5' end, three 2'-O-MOE-RNA nucleosides at the 3' end, and has a structure in which 10 DNA nucleosides are linked by phosphodiester bonds.
  • the second nucleic acid strand of HDO (agRNA 6MOE) consists of three 2'-O-Me-RNA nucleosides at the 5' end, three 2'-O-Me-RNA nucleosides at the 3' end, and In the gap region, nucleosides having adenine and guanine bases are RNA nucleosides, and nucleosides having cytosine and thymine bases are DNA nucleosides.
  • HDO all DNA
  • HDO cRNA 6MOE
  • HDO cDNA 6MOE
  • HDO agRNA 6MOE
  • preparation of nucleic acids in vivo experiments, and In addition, central nervous system toxicity evaluation and motor function evaluation were performed.
  • the dose of the nucleic acid agent per mouse was 28 nmol/mouse.
  • FIG. 56 shows the results of evaluating acute central nervous system toxicity within 1 day of administration in mice to which various nucleic acid agents were intracerebroventricularly administered.
  • HDO cRNA 6MOE
  • HDO cDNA 6MOE
  • HDO agRNA 6MOE
  • the acute phase tolerability score was significantly decreased in the ASO and HDO (all DNA) administration groups.
  • the HDO (agRNA 6MOE) administration group showed a lower acute phase tolerability score than the HDO (cRNA 6MOE) and HDO (cDNA 6MOE) administration groups.
  • FIG. 57 shows the results of evaluating motor function in mice to which various nucleic acid agents were intracerebroventricularly administered 1 hour after administration. Total distance traveled (Fig. 57A) and maximum speed Figure 57B) improved.
  • HDO agRNA 6MOE
  • cDNA 6MOE a nucleoside having adenine base and a guanine base
  • a nucleoside having a cytosine base and a thymine base is a DNA nucleoside in the gap region of the second nucleic acid strand.
  • FIG. 58 shows the results of evaluation of body weight loss in mice to which various nucleic acid agents were intracerebroventricularly administered as delayed central nervous system toxicity after 1 day after administration. Body weight was measured on days 0, 7, 14, and 21 after administration.
  • HDO (cRNA 6MOE), HDO (cDNA 6MOE), and HDO (agRNA 6MOE) administration groups showed reduced weight loss effects compared to ASO and HDO (all DNA) administration groups.
  • FIG. 59 shows the results of evaluation of motor function in mice intracerebroventricularly administered with various nucleic acid agents as delayed central nervous system toxicity after one day of administration.
  • HDO (cRNA 6MOE), HDO (cDNA 6MOE), and HDO (agRNA 6MOE) groups were compared with ASO and HDO (all DNA) groups at any time point 7, 14, or 21 days after administration. As a result, the reduction effect on maximum movement speed has been reduced.
  • FIG. 60 shows Mapt mRNA expression levels in the right frontal lobe 21 days after intracerebroventricular administration of various nucleic acid agents.
  • the HDO (agRNA 6MOE)-administered group showed a marked effect of suppressing the Mapt mRNA expression level compared to the HDO (cRNA 6MOE) and HDO (cDNA 6MOE)-administered groups.
  • the nucleoside having an adenine base and a guanine base in the gap region of the second nucleic acid strand is an RNA nucleoside
  • the nucleoside having a cytosine base and a thymine base is a DNA nucleoside
  • the second nucleic acid strand is a DNA nucleoside or It was shown that a superior effect of suppressing gene expression can be achieved compared to HDOs composed of RNA nucleosides.
  • FIG. 61 shows HDO (cRNA 6MOE), HDO (cDNA 6MOE), and HDO (agRNA 6MOE) using methods similar to Example 20 for evaluating the duplex dissociation efficiency of nucleic acid agents in brain tissue homogenates. shows the results of electrophoresis after incubation for 7 days in mouse brain tissue. HDO (agRNA 6MOE) showed higher duplex dissociation efficiency in brain tissue homogenates compared to HDO (cRNA 6MOE) and HDO (cDNA 6MOE).
  • the second nucleic acid strand is a DNA nucleoside or Compared to HDO consisting of RNA nucleosides, the double-strand dissociation efficiency was extremely high in the brain tissue, which strongly supported the result that the expression of the target gene was remarkably suppressed in the above result 3.
  • Example 23 Introduction of MOE modification into second nucleic acid strand of HDO containing all MOE-modified ASOs> (the purpose)
  • an HDO comprising, as the first nucleic acid strand, an ASO in which all nucleosides are MOE-modified nucleosides (hereinafter referred to as "all-MOE-modified ASO") capable of regulating splicing by targeting the Mecp2 gene. target.
  • all-MOE-modified ASO capable of regulating splicing by targeting the Mecp2 gene.
  • Table 22 shows the base sequences and chemical modifications of the first and second nucleic acid strands constituting the ASOs and HDOs used in this example.
  • ASO used in this example is a non-gapmer type antisense nucleic acid that targets MeCP2 (methyl-CpG binding protein 2) pre-mRNA, and has a base sequence complementary to a part of MECP2 pre-mRNA. and all nucleosides are 2'-O-MOE-RNA nucleosides linked by phosphorothioate linkages.
  • HDO (all DNA) and HDO (cRDNA MOE) used in this example contain the above ASO as a common first nucleic acid strand, and the second nucleic acid strand has a sequence complementary to the first nucleic acid strand.
  • 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) consists of three 2'-O-MOE-RNA nucleosides at the 5' end, three 2'-O-MOE-RNA nucleosides at the 3' end, and In the gap region, nucleosides having adenine and guanine bases are RNA nucleosides, and nucleosides having cytosine and thymine bases are DNA nucleosides.
  • ASO, HDO (all DNA), and HDO (cRDNA MOE) listed in Table 22 were subjected to nucleic acid preparation, in vivo experiments, central nervous system toxicity evaluation, and motor function evaluation in the same manner as in Example 1. .
  • the dose of the nucleic acid agent per mouse was 28 nmol/mouse.
  • FIG. 62 shows the results of evaluating acute central nervous system toxicity in mice to which various nucleic acid agents were intracerebroventricularly administered.
  • ASO administration in the HDO (all DNA) and HDO (cRDNA MOE) administration groups The acute phase tolerability score decreased significantly compared to the group.
  • the HDO(cRDNA MOE)-administered group showed a lower acute-phase tolerability score than the HDO(all DNA)-administered group.
  • the wing region of the second nucleic acid strand is composed of 2'-O-MOE-RNA nucleosides, and in the gap region, nucleosides having adenine bases and guanine bases are RNA nucleosides, and have cytosine bases and thymine bases. It was shown that HDOs in which the nucleoside is a DNA nucleoside can achieve lower central nervous system toxicity than other HDOs.
  • FIG. 63 shows the results of evaluating motor function in mice to which various nucleic acid agents were intracerebroventricularly administered 1 hour after administration. In the HDO (all DNA) and HDO (cRDNA MOE) administration groups, the total moving distance (Fig. 63A) and the maximum moving speed (Fig.
  • the wing region of the second nucleic acid strand is composed of 2'-O-MOE-RNA nucleosides, and in the gap region, nucleosides having adenine bases and guanine bases are RNA nucleosides, and have cytosine bases and thymine bases. It was shown that HDO, in which the nucleoside is a DNA nucleoside, has a very small effect of suppressing motor function and a very low toxicity compared with other HDOs.
  • Example 24 Single-chain HDO> (the purpose) The neurocytotoxicity of single-stranded HDO (ssHDO), in which the first nucleic acid strand consisting of ASO targeting the Bace1 gene and the second nucleic acid strand having a complementary base sequence to the first nucleic acid strand are linked by a linker, was investigated in It is verified by in vitro experiments.
  • ssHDO single-stranded HDO
  • HDO prepared in (1) was introduced into human neuroblastoma-derived cells (Neuro2a cell line) using the lipofection method (lipofectamine2000). 48 hours after introduction of HDO, RNA was extracted from the cells using the IsogenI kit (Genedesign 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 standard genes). Quantitative RT-PCR was performed by TaqMan (Roche Applied Science).
  • Primers used in quantitative RT-PCR were products 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 (one cycle). The ratio of the Bace1 mRNA expression level to the Actb mRNA (internal standard gene) expression level was calculated, and the value normalized to the value of the PBS-administered group was defined as the relative Bace1 mRNA level.
  • LDH activity in the cell supernatant was measured as evaluation of neuronal cytotoxicity.
  • LDH activity was measured using Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories) according to the attached protocol.
  • a value normalized to the LDH activity in the PBS-administered group was defined as the relative LDH release level.
  • FIG. 64B shows the results of evaluating target gene inhibitory effects of various nucleic acid agents in mouse neuroblastoma-derived cells (Neuro2a cell line).
  • ssHDO-administered group a strong gene-silencing effect equivalent to that of HDO (all DNA) was obtained.
  • FIG. 64A shows the results of evaluating LDH activity in the supernatant as cytotoxicity when various nucleic acid agents were introduced into cells.
  • Cells treated with ssHDO at 5 nM and 25 nM exhibited a suppressed increase in LDH activity and markedly decreased cytotoxicity compared to cells treated with HDO (all DNA) at 5 nM and 25 nM, respectively.
  • ssHDO containing 2'-O-MOE-RNA nucleosides significantly reduced cytotoxicity.
  • Example 25 Evaluation of central nervous system toxicity in monkeys> (the purpose) HDO containing MOE modification is administered intrathecally to monkeys to assess central neurotoxicity.
  • HDO all DNA
  • HDO DNA MOE
  • HDO RNA MOE
  • HDO DNA RNA MOE
  • FIG. 66 shows the results of evaluating acute central nervous system toxicity in monkeys intrathecally administered with various nucleic acid agents.
  • Administration of HDO (all DNA), HDO (DNA MOE), HDO (RNA MOE), and HDO (DNA RNA MOE) at any of 1 hour, 2 hours, and 3 hours after administration of the nucleic acid agent The modified FOB score significantly decreased in the group compared with the ASO-treated group.
  • the modified FOB score decreased in the HDO (DNA MOE) and HDO (DNA RNA MOE) administration groups compared to the HDO (all DNA) administration group.
  • FIG. 67 shows the results of measuring the spontaneous exercise time and the number of jumps using a 3-minute video animation.
  • HDO (all DNA), HDO (DNA MOE), HDO (RNA MOE), and HDO (DNA RNA MOE) administration groups suppressed the effect of reducing the spontaneous exercise time or the number of jumps compared to the ASO administration group. was done. Furthermore, in the HDO (DNA MOE) and HDO (DNA RNA MOE) administration groups, compared with the HDO (all DNA) administration group, the effect of reducing spontaneous exercise time or the number of jumps was suppressed. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

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