WO2021070959A1 - 修飾ヘテロ核酸 - Google Patents

修飾ヘテロ核酸 Download PDF

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WO2021070959A1
WO2021070959A1 PCT/JP2020/038380 JP2020038380W WO2021070959A1 WO 2021070959 A1 WO2021070959 A1 WO 2021070959A1 JP 2020038380 W JP2020038380 W JP 2020038380W WO 2021070959 A1 WO2021070959 A1 WO 2021070959A1
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Prior art keywords
nucleic acid
modified
sugar
nucleoside
region
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English (en)
French (fr)
Japanese (ja)
Inventor
隆徳 横田
永田　哲也
英紀 古川
能紀 余郷
宮田　健一
章夫 内田
直輝 冨田
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Takeda Pharmaceutical Co Ltd
Tokyo Medical and Dental University NUC
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Takeda Pharmaceutical Co Ltd
Tokyo Medical and Dental University NUC
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Priority to EP20875085.1A priority Critical patent/EP4043565A4/en
Priority to CN202080084535.5A priority patent/CN115103911A/zh
Priority to US17/767,663 priority patent/US20240117346A1/en
Priority to JP2021551735A priority patent/JP7793144B2/ja
Publication of WO2021070959A1 publication Critical patent/WO2021070959A1/ja
Anticipated expiration legal-status Critical
Priority to JP2025130417A priority patent/JP2025156522A/ja
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C12N2310/3341—5-Methylcytosine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/51—Methods for regulating/modulating their activity modulating the chemical stability, e.g. nuclease-resistance

Definitions

  • the present invention relates to a double-stranded nucleic acid complex, a composition containing the double-stranded nucleic acid complex, and the like.
  • nucleic acid drugs In recent years, oligonucleotides have attracted attention in the ongoing development of drugs called nucleic acid drugs, and nucleic acid drugs that utilize the antisense method, especially in view of the high selectivity and low toxicity of the target gene. Development is being actively promoted.
  • the antisense method uses a partial sequence of mRNA or miRNA transcribed from a target gene as the target sense strand, and an oligonucleotide complementary to it (antisense oligonucleotide: often referred to as "ASO (AntiSense Oligonucleotide)" in the present specification. This is a method comprising selectively modifying or inhibiting the expression of a protein encoded by a target gene or the activity of miRNA by introducing the above into a cell.
  • ASO AntiSense Oligonucleotide
  • the present inventors have prepared a double-stranded nucleic acid complex (heteroduplex oligonucleotide (HDO)) in which an antisense oligonucleotide and a complementary strand thereof are annealed.
  • HDO heteroduplex oligonucleotide
  • the mechanism of action of the above double-stranded nucleic acid complex is not limited, but is partially as follows. That is, when introduced into a cell, an RNA region complementary to a part of the antisense oligonucleotide in the complementary strand is cleaved by RNase H to release the antisense oligonucleotide, and then this antisense oligonucleotide is released. For example, it can act to alter the activity or function of the transcript (see, eg, Patent Document 2). This is called the "RNase H-dependent pathway", and it is desirable that the nucleic acid moiety is not modified in order for cleavage by RNase H to occur. On the other hand, if the nucleic acid moiety is not modified, it may be degraded by a nucleic acid-degrading enzyme in vivo and may not exhibit sufficient activity.
  • nucleic acids should be modified in order to suppress the degradation by nucleic acid-degrading enzymes in vivo while maintaining the activity of the double-stranded nucleic acid complex is not sufficient.
  • An object of the present invention is to provide a double-stranded nucleic acid complex capable of maintaining activity and / or suppressing degradation in vivo.
  • the present inventors are a complex of a first nucleic acid chain and a second nucleic acid chain, the first nucleic acid chain is a gapmer, and the second nucleic acid chain complements a part of the first nucleic acid chain.
  • a sugar-unmodified central region also referred to herein as "first exposed region" consisting of one or two consecutive sugar-unmodified ribonucleosides linked by an internucleoside bond. It has been found that the containing complex can maintain activity and / or suppress degradation in vivo.
  • the present inventors have found that the function of the complex can be further improved by including a protective region containing a modified or unmodified pyrimidine base in the second nucleic acid chain, and have completed the present invention. It was.
  • the first nucleic acid chain can (1) hybridize to at least a part of the target transcript.
  • the central region which has (2) antisense effect on the target transcript and (3) contains at least four contiguous deoxyribonucleosides, and the 5'wing and 3'wing regions containing unnatural nucleosides, respectively.
  • the second nucleic acid chain is a sugar-unmodified central region consisting of one or two or three consecutive sugar-unmodified ribonucleosides linked by an internucleoside bond, which is complementary to a part of the first nucleic acid chain.
  • the nucleic acid complex containing at least one (first exposed region) and the first nucleic acid strand being annealed to the second nucleic acid strand.
  • the second nucleic acid chain is Linked by modified or unmodified nucleoside linkages,
  • [5] The description according to any one of [1] to [4], wherein the sugar-unmodified central region (first exposed region) consists of three consecutive sugar-unmodified ribonucleosides linked by an internucleoside bond.
  • Nucleic acid complex [6] The nucleic acid complex according to any one of [1] to [5], wherein the second nucleic acid chain contains only one sugar-unmodified central region (first exposed region). [7] The nucleic acid complex according to any one of [1] to [6], wherein the sugar-unmodified ribonucleoside in the sugar-unmodified central region (first exposed region) is a natural ribonucleoside. [8] The nucleic acid complex according to any one of [1] to [7], wherein the sugar-unmodified central region (first exposed region) contains at least one modified nucleoside-linked bond.
  • nucleic acid complex according to [11], wherein the second nucleic acid chain does not contain a sugar-unmodified region other than the sugar-unmodified terminal region and the sugar-unmodified central region (first exposed region).
  • Nucleic acid complex [15] The nucleic acid complex according to any one of [1] to [14], wherein the sugar-unmodified central region (first exposed region) is located 3'side from the center of the second nucleic acid chain.
  • the present invention further includes the following embodiments.
  • a nucleic acid complex containing a first nucleic acid chain and a second nucleic acid chain.
  • the first nucleic acid chain can (1) hybridize to at least a part of the target transcript.
  • the central region which has (2) antisense effect on the target transcript and (3) contains at least four contiguous deoxyribonucleosides, and the 5'wing and 3'wing regions containing unnatural nucleosides, respectively. , Gapmers contained on the 5'end and 3'ends of the central region, respectively.
  • the second nucleic acid strand comprises at least one first exposed region and at least one defense region.
  • the first exposed region consists of one or two or three consecutive unmodified ribonucleosides linked by internucleoside linkages that are complementary to a portion of the first nucleic acid chain.
  • the defense region is from one or two or more (a) deoxyribonucleosides, (b) sugar-modified nucleosides and / or (c) nucleosides having a modified nucleoside bond on the 3'side linked by an internucleoside bond.
  • the first nucleic acid strand is the nucleic acid complex annealed to the second nucleic acid strand.
  • [3A] The nucleic acid complex according to [1A] or [2A], wherein the first exposed region consists of three consecutive sugar-unmodified ribonucleosides linked by internucleoside linkages.
  • [4A] The nucleic acid complex according to any one of [1A] to [3A], wherein the first exposed region contains a nucleoside containing a modified or unmodified purine base.
  • [5A] The nucleic acid complex according to any one of [1A] to [4A], wherein the second nucleic acid strand contains only one first exposed region.
  • [6A] The nucleic acid complex according to any one of [1A] to [4A], wherein the second nucleic acid chain contains at least two first exposed regions.
  • [7A] The nucleic acid complex according to any one of [1A] to [6A], wherein the second nucleic acid strand contains at least two defense regions.
  • [8A] The nucleic acid complex according to any one of [1A] to [7A], wherein the sugar-unmodified ribonucleoside in the first exposed region is a natural ribonucleoside.
  • [9A] A second exposed region consisting of four or more consecutive unmodified ribonucleosides linked by nucleoside linkage, which is complementary to a part of the first nucleic acid chain, is further included in the second exposed region.
  • At least one of the defense regions contains (a) a deoxyribonucleoside containing a modified or unmodified pyrimidine base, (b) a sugar-modified nucleoside and / or (c) a nucleoside having a modified nucleoside interlinkage on the 3'side.
  • the nucleic acid complex according to any one of [1A] to [9A] which comprises.
  • All of the defense regions include (a) deoxyribonucleosides containing modified or unmodified pyrimidine bases, (b) sugar-modified nucleosides and / or (c) nucleosides with modified nucleoside linkages on the 3'side. , [10A].
  • At least one of the defense regions comprises one or two or more modified or unmodified pyrimidine bases linked by nucleoside interlinks (a) deoxyribonucleosides, (b) sugar-modified nucleosides and / Alternatively, (c) the nucleic acid complex according to any one of [1A] to [11A], which comprises a nucleoside having a modified nucleoside interlinkage on the 3'side.
  • nucleic acid complex according to any one of [1A] to [12A] or [13A-1] to [13A-2], which is a nucleoside having an interconjugation and / or a nucleoside interlinking containing a cyclic guanidine moiety.
  • nucleoside having a modified nucleoside interlink on the 3'side in the defense region is a nucleoside having a phosphorothioate bond on the 3'side, [1A] to [12A] or [13A-1] to [13A].
  • the nucleic acid complex according to any one of.
  • the sugar-modified nucleoside in the defense region is a 2'-O-methyl-modified nucleoside, and / or a nucleoside having a modified nucleoside-linked bond on the 3'side is a nucleoside having a phosphorothioate bond on the 3'side.
  • the nucleic acid complex according to.
  • At least one defense region of the second nucleic acid strand is linked to the first exposed region and / or the second exposed region by nucleoside binding at the 5'end and 3'end, [1A].
  • nucleic acid complex Described in any of [1A] to [20A] or [13A-1] to [13A-4], which comprises an internucleoside bond containing a guanidine moiety substituted with a group and / or a nucleoside bond containing a cyclic guanidine moiety.
  • Nucleic acid complex [22A]
  • the defense region of the second nucleic acid chain contains a guanidine moiety substituted with at least one Rp or Sp configuration chiral-controlled phosphorothioate bond and 1 to 4 C 1 to 6 alkyl groups.
  • nucleic acid complex according to any one of [1A] to [21A] or [13A-1] to [13A-4], which comprises an internucleoside bond and / or a nucleoside interlink containing a cyclic guanidine moiety.
  • nucleic acid complex according to [21A] or [22A] wherein the nucleoside-to-nucleoside bond containing the cyclic guanidine moiety is a nucleoside-to-nucleoside bond represented by a partial structure represented by the following formula (II).
  • the internucleoside bond containing a guanidine moiety substituted with the above 1 to 4 alkyl groups of C 1 to 6 is an internucleoside bond represented by a partial structure represented by the following formula (III). , [21A] to [23A]. [25A] Either [17A] or [19A] to [24A], wherein the second nucleic acid chain does not contain a sugar-unmodified region other than the first exposed region, the second exposed region, and the sugar-unmodified terminal region. Nucleic acid complex described in Crab.
  • [26A] The description according to any one of [17A] to [25A], wherein at least one first exposed region, second exposed region and / or defense region of the second nucleic acid strand contains one or more mismatched bases.
  • [27A] The nucleic acid complex according to any one of [17A] to [25A], wherein the first exposed region, the second exposed region and / or the defense region of the second nucleic acid chain does not contain a mismatched base.
  • the second nucleic acid strand is bound to a functional moiety having a function selected from a labeling function, a purification function, and a delivery function to a target, [1A] to [27A] or [13A-1]. ] To [13A-4].
  • [29A] A pharmaceutical composition containing the nucleic acid complex according to any one of [1A] to [28A] or [13A-1] to [13A-4] as an active ingredient.
  • [30A] A nucleic acid complex containing a first nucleic acid chain and a second nucleic acid chain.
  • the first nucleic acid chain can (1) hybridize to at least a part of the target transcript.
  • the central region which has (2) antisense effect on the target transcript and (3) contains at least four contiguous deoxyribonucleosides, and the 5'wing and 3'wing regions containing unnatural nucleosides, respectively. , Gapmers contained on the 5'end and 3'ends of the central region, respectively.
  • the second nucleic acid strand comprises at least one second exposed region and at least one defense region.
  • the second exposed region consists of four or more contiguous sugar-unmodified ribonucleosides linked by nucleoside linkages that are complementary to a portion of the first nucleic acid chain.
  • the defense region is from one or two or more (a) deoxyribonucleosides, (b) sugar-modified nucleosides and / or (c) nucleosides having a modified nucleoside bond on the 3'side linked by an internucleoside bond.
  • the first nucleic acid strand is the nucleic acid complex annealed to the second nucleic acid strand.
  • [31A] The nucleic acid complex according to [30A], wherein the first nucleic acid chain has a length of 13 to 22 bases.
  • [32A] The nucleic acid complex according to [30A] or [31A], wherein the second nucleic acid chain contains only one second exposed region.
  • [33A] The nucleic acid complex according to any one of [30A] to [32A], wherein the second nucleic acid chain contains at least two second exposed regions.
  • [34A] Further comprises a first exposed region consisting of one or two consecutive unmodified ribonucleosides linked by internucleoside linkages complementary to a portion of the first nucleic acid chain [34A].
  • This specification includes the disclosure of Japanese Patent Application No. 2019-188042, which is the basis of the priority of the present application.
  • the present invention provides a double-stranded nucleic acid complex having a novel structure.
  • the nucleic acid complex of the present invention may be highly active and / or suppressed in vivo degradation.
  • FIG. 1A and 1B are schematic views showing an example in which the second nucleic acid chain contains a lipid in a specific embodiment of the nucleic acid complex according to the present invention.
  • 2A to 2C are schematic views showing an example in which the second nucleic acid chain contains a lipid and includes a complementary region and an overhang region in a specific embodiment of the nucleic acid complex according to the present invention.
  • FIG. 3 is a diagram showing an example of a general mechanism of the antisense method.
  • FIG. 4 is a diagram showing the structures of various crosslinked nucleic acids.
  • FIG. 5 is a diagram showing the structure of various natural or non-natural nucleotides.
  • FIG. 6 is a schematic diagram of the structure of the nucleic acid used in Example 1. Chol indicates cholesterol.
  • Figure 7 shows ASO alone, ASO and Chol-cRNA (mMalat1) double-stranded complex (Chol-HDO), and ASO and Chol-cRNA (mMalat1) full OMe double-stranded complex (Chol-HDO with full OMe).
  • the results of electrophoresis of cRNA) after cleavage by RNase H and / or RNase A are shown.
  • “+” indicates the result of reacting 10 ⁇ M of nucleic acid with 10 ⁇ L of Ribonuclease H
  • “++” indicates the result of reacting 20 ⁇ M of nucleic acid with 5 ⁇ L of Ribonuclease H with 10 U.
  • FIG. 8 is a graph showing the concentration of the undecomposed band based on the electrophoresis result of FIG. 7 in terms of the relative intensity level (%) with respect to the untreated.
  • FIG. 9 shows the positions of three consecutive sugar-unmodified ribonucleosides for a double-stranded nucleic acid agent containing three consecutive sugar-unmodified ribonucleosides complementary to a part of the gap region of the first nucleic acid chain. It is a graph showing the concentration of undecomposed bands in relative intensity level (%) as in FIG. 8 after performing a cleavage test with RNase H and / or RNase A.
  • FIG. 9 shows the concentration of undecomposed band based on the electrophoresis result of FIG. 7 in terms of the relative intensity level (%) with respect to the untreated.
  • FIG. 9 shows the positions of three consecutive sugar-unmodified ribonucleosides for a double-stranded nucleic acid agent containing three consecutive sugar-unmodified rib
  • FIG. 10 shows the relative RNA expression level when the expression level of Malat1 / Actb ( ⁇ -actin) in PBS treatment is 1 when cells are treated with various double-stranded nucleic acid complexes.
  • FIG. 11 is a schematic diagram of the structure of the nucleic acid used in Example 3. Chol indicates cholesterol.
  • FIG. 12 is a schematic diagram of the structure of the nucleic acid used in Example 3. Chol indicates cholesterol.
  • FIG. 13 shows the position of the sugar-unmodified ribonucleoside being changed for a double-stranded nucleic acid agent containing one or two consecutive sugar-unmodified ribonucleosides complementary to a part of the gap region of the first nucleic acid chain.
  • FIG. 14 a double-stranded nucleic acid agent containing one or two consecutive RNAs complementary to a part of the gap region of the first nucleic acid strand is cleaved with RNase A and then undegraded in the same manner as in FIG. It is a graph which showed the density
  • FIG. 15 is a schematic diagram showing the structure of the nucleic acid used in Example 4. Chol indicates cholesterol.
  • FIG. 15A shows the double-stranded nucleic acid complex Chol-HDO (Default) containing ASO (mDMPK) and Chol-cRNA (Default).
  • FIG. 15A shows the double-stranded nucleic acid complex Chol-HDO (Default) containing ASO (mDMPK) and Chol-cRNA (Default).
  • FIG. 15B shows the double-stranded nucleic acid complex Chol-HDO (CUOMe) containing ASO (mDMPK) and Chol-cRNA (CUOMe).
  • Figure 16 shows the gastrocnemius (GC) and triceps brachii (TB) with double-stranded nucleic acid complexes Chol-HDO (Default) and Chol-cRNA (CUOMe), and PBS for negative control.
  • GC gastrocnemius
  • TB double-stranded nucleic acid complexes Chol-HDO (Default) and Chol-cRNA (CUOMe)
  • PBS for negative control.
  • Tibialis anterior (TA) triceps femoris
  • Back quadriceps femoris
  • Quantadriceps myocardium
  • the relative RNA expression level when the Dmpk / Actb ( ⁇ -actin) expression level is 1.
  • FIG. 17 is a diagram showing the effect of double-stranded nucleic acid complexes Chol-HDO (Default) and Chol-cRNA (CUOMe), and PBS for negative control on the expression-suppressing effect of the Dmpk gene in the kidney and liver.
  • the relative RNA expression level when the Dmpk / Actb ( ⁇ -actin) expression level is 1.
  • FIG. 18 is a schematic diagram showing the structure of the nucleic acid used in Example 5. Chol indicates cholesterol.
  • FIG. 18A shows the double-stranded nucleic acid complex Chol-HDO (Default) containing ASO (hSOD1) and Chol-cRNA (Default).
  • FIG. 18B shows the double-stranded nucleic acid complex Chol-HDO (CUOMe) containing ASO (hSOD1) and Chol-cRNA (CUOMe).
  • FIG. 19 shows the double-stranded nucleic acid complexes Chol-HDO (Default) and Chol-cRNA (CU OMe), as well as the intrinsic back muscle (Back), quadriceps femoris (Quadriceps), and myocardium (Heart) by PBS for negative control.
  • the diaphragm (Diaphragm) show the effect of suppressing the expression of the SOD1 gene.
  • the relative RNA expression level when the SOD1 / Actb ( ⁇ -actin) expression level is 1.
  • FIG. 20 is a schematic diagram showing the structure of the nucleic acid used in Example 6. Chol indicates cholesterol.
  • FIG. 20A shows the double-stranded nucleic acid complex Chol-HDO (Default) containing ASO (Malat1) and Chol-cRNA (Default).
  • FIG. 20B shows the double-stranded nucleic acid complex Chol-HDO (CT DNA) containing ASO (Malat1) and Chol-cRNA (CT DNA).
  • FIG. 21 shows the effect of double-stranded nucleic acid complexes Chol-HDO (Default) and Chol-cRNA (CU OMe), and PBS for negative control on the expression suppression effect of the Malat1 gene in the cervical spinal cord (Cervical spinal cord). It is a figure.
  • FIG. 22A shows the sequence, chemical modification and structure of the oligonucleotide used in Example 7.
  • FIG. 22B shows the sequence, chemical modification and structure of the oligonucleotide used in Example 7.
  • FIG. 23 shows a double-stranded nucleic acid complex containing a first nucleic acid strand containing an antisense oligonucleotide and a complementary strand (second nucleic acid strand) containing a modified ribonucleoside as mouse serum for a time (0 hr, 24 hr). This is the result of mixing and examining the stability by electrophoresis.
  • FIG. 22A shows the sequence, chemical modification and structure of the oligonucleotide used in Example 7.
  • FIG. 22B shows the sequence, chemical modification and structure of the oligonucleotide used in Example 7.
  • FIG. 23 shows a double-stranded nucleic acid complex containing a first nucleic acid strand containing an antisense oligonu
  • FIG. 24 is a graph showing the concentration of the double-stranded band after 24 hours based on the electrophoresis result of FIG. 23 in terms of the relative intensity level (%) with respect to the double-stranded band after 0 hr.
  • FIG. 25 shows a double-stranded nucleic acid complex containing a first nucleic acid strand containing an antisense oligonucleotide and a complementary strand (second nucleic acid strand) containing a modified ribonucleoside as human serum for a time (0 hr, 2 hr). This is the result of mixing and examining the stability by electrophoresis.
  • FIG. 26 is a graph showing the concentration of the double-stranded band after 2 hr based on the electrophoresis result of FIG. 25 in terms of the relative intensity level (%) with respect to the double-stranded band after 0 hr.
  • the present invention relates to a nucleic acid complex comprising a first nucleic acid strand and a second nucleic acid strand, wherein the first nucleic acid strand is annealed to the second nucleic acid strand.
  • the first nucleic acid strand can (1) hybridize to at least a part of the target transcript, (2) have an antisense effect on the target transcript, and (3) is a gapmer.
  • the nucleic acid complex of the present invention and the nucleic acid chains constituting the nucleic acid complex will be described in detail below.
  • the first nucleic acid strand may be a nucleotide strand containing or consisting of an antisense oligonucleotide region against the target transcript.
  • An "antisense oligonucleotide” or “antisense nucleic acid” comprises a (ie, complementary) base sequence capable of hybridizing to at least a portion of a target transcript (mainly a transcript of a target gene). Refers to a single-stranded oligonucleotide that can provide an antisense effect on a target transcript.
  • the antisense oligonucleotide region in the first nucleic acid strand can provide an antisense effect on the target transcript.
  • the target region of the target transcript is at least 8 bases long, eg, 10-35 bases, 12-25 bases, 13-20 bases, 14-19 bases, or 15-18 bases, or 13-22. It can be base length, 16 to 22 base length, or 16 to 20 base length.
  • the "antisense effect” is a high of a target transcript (RNA sense strand) and a strand (eg, a DNA strand) that is complementary to a partial sequence of the transcript and is designed to cause the antisense effect. It means the regulation of expression of the target transcript resulting from hybridization. Regulation of target transcript expression suppresses or reduces the expression of the target gene or the level (expression level) of the target transcript, or, in certain cases, translational inhibition or splicing function modification effects, such as exon skipping. , Or, including degradation of transcripts (see Figure 3).
  • an oligonucleotide containing RNA when introduced into a cell as an antisense oligonucleotide (ASO), the ASO binds to the transcript (mRNA) of the target gene, forming a partial double strand. Will be done. This partial double strand acts as a cover to prevent translation by the ribosome, thus inhibiting the expression of the protein encoded by the target gene at the translational level ( Figure 3, x-marked outside the dashed line).
  • mRNA transcript
  • Figure 3, x-marked outside the dashed line When an oligonucleotide containing DNA is introduced into a cell as ASO, a partial DNA-RNA heteroduplex is formed.
  • the antisense effect can be achieved by targeting the introns of the pre-mRNA.
  • the antisense effect may also be brought about by targeting the miRNA, in which case the function of the miRNA may be inhibited and the expression of the gene for which the miRNA normally regulates expression may be increased.
  • regulation of target transcript expression can be a reduction in the amount of target transcript.
  • the antisense oligonucleotide region in the first nucleic acid strand contains a base sequence that can hybridize to at least a part of the target transcript (for example, any target region).
  • Target regions may include 3'UTRs, 5'UTRs, exons, introns, coding regions, translation initiation regions, translation termination regions or other nucleic acid regions.
  • the "target gene” whose expression is regulated (for example, suppressed, altered, or modified) by the antisense effect is not particularly limited, and examples thereof include genes whose expression is increased in various diseases.
  • the target transcript includes mRNA transcribed from genomic DNA encoding the target gene, and further includes unmodified mRNA, unprocessed mRNA precursor, and the like.
  • the "target transcript” can include not only mRNA but also non-coding RNA (ncRNA) such as miRNA. More generally, the "transcript” can be any RNA synthesized by DNA-dependent RNA polymerase.
  • the "target transcript” is, for example, scavenger receptor B1 (scavenger receptor B1: often referred to herein as "SR-B1”), myotonic dystrophy protein kinase, Often referred to as “DMPK” herein), transthyretin (often referred to as “TTR” herein), Apolipoprotein B (often referred to herein as “ApoB”).
  • SR-B1 myotonic dystrophy protein kinase
  • TTR transthyretin
  • Apolipoprotein B often referred to herein as "ApoB”
  • Metastasis associated lung adenocarcinoma transcript 1 genes of metastasis associated lung adenocarcinoma transcript 1: often referred to herein as "Malat1", such as these non-coding RNAs or mRNAs.
  • SEQ ID NO: 1 shows the base sequence of mouse Malat1 non-coding RNA
  • SEQ ID NO: 2 shows the base sequence of human Malat1 non-coding RNA
  • SEQ ID NO: 39 shows the base sequence of mouse SR-B1 mRNA
  • SEQ ID NO: 40 shows the base sequence of human SR-B1 mRNA
  • SEQ ID NO: 41 shows the base sequence of mouse DMPK mRNA
  • SEQ ID NO: 42 shows the base sequence of human DMPK mRNA.
  • the base sequence of mRNA is replaced with the base sequence of DNA.
  • Nucleotide sequence information of these genes and transcripts can be obtained from known databases such as NCBI (National Center for Biotechnology Information) databases (eg, GenBank, Trace Archive, Sequence Read Archive, BioSample, BioProject).
  • NCBI National Center for Biotechnology Information
  • GenBank GenBank
  • Trace Archive Sequence Read Archive
  • BioSample, BioProject The target region of the target transcript may include, for example, the nucleotide sequence of positions 1317 to 1332 of SEQ ID NO: 1 in the case of mouse Malat-1 non-coding RNA.
  • nucleic acid or “nucleic acid molecule” may refer to a monomeric nucleotide or nucleoside, or may mean an oligonucleotide consisting of a plurality of monomers.
  • nucleic acid strand or “stranded” are also used herein to refer to oligonucleotides.
  • the nucleic acid strand is made into a full-length chain or a partial strand by a chemical synthesis method (for example, using an automatic synthesizer) or by an enzymatic step (for example, but not limited to, a polymerase, a ligase, or a limiting reaction). Can be made.
  • nucleobase or “base” used in the present specification is a base component (heterocyclic portion) constituting a nucleic acid, and mainly adenine, guanine, cytosine, thymine, and uracil are known.
  • the term "complementary" means that nucleobases are hydrogen-bonded to so-called Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen base pairs). It means a relationship that can form a pair (such as a pair).
  • the antisense oligonucleotide region in the first nucleic acid strand does not necessarily have to be completely complementary to at least a portion of the target transcript (eg, the transcript of the target gene), and the nucleotide sequence is It is acceptable to have at least 70%, preferably at least 80%, and even more preferably at least 90% (eg, 95%, 96%, 97%, 98%, or 99% or more) complementarity.
  • the antisense oligonucleotide region in the first nucleic acid chain is when the base sequence is complementary (typically when the base sequence is complementary to at least a portion of the target transcript). It can hybridize to the target transcript.
  • the complementary region in the second nucleic acid strand does not necessarily have to be completely complementary to at least a portion of the first nucleic acid strand, with a base sequence of at least 70%, preferably at least 80%, and more. More preferably, it is acceptable if it has at least 90% complementarity (eg, 95%, 96%, 97%, 98%, or 99% or more).
  • Complementary regions in the second nucleic acid strand can be annealed if the base sequence is complementary to at least a portion of the first nucleic acid strand.
  • Nucleotide sequence complementarity can be determined by using a BLAST program or the like. Those skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which the two strands can be annealed or hybridized in consideration of the complementarity between the strands.
  • those skilled in the art can easily design an antisense nucleic acid complementary to the target transcript, for example, based on the information on the base sequence of the target gene.
  • the hybridization conditions may be various stringent conditions such as low stringent conditions and high stringent conditions.
  • the low stringent conditions may be relatively low temperature and high salt concentration conditions, such as 30 ° C., 2 ⁇ SSC, 0.1% SDS.
  • the high stringent conditions may be relatively high temperature and low salt concentration conditions, such as 65 ° C., 0.1 ⁇ SSC, 0.1% SDS.
  • the stringency of hybridization can be adjusted by changing conditions such as temperature and salt concentration.
  • 1 ⁇ SSC comprises 150 mM sodium chloride and 15 mM sodium citrate.
  • the antisense oligonucleotide region in the first nucleic acid chain may usually be 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, or at least 13 bases long. , Not particularly limited.
  • the antisense oligonucleotide region in the first nucleic acid chain is 35 bases or less, 30 bases or less, 25 bases or less, 24 bases or less, 23 bases or less, 22 bases or less, 21 bases or less, 20 bases. It may be longer or less, 19 bases or less, 18 bases or less, 17 bases or less or 16 bases or less.
  • the antisense oligonucleotide region in the first nucleic acid chain is, for example, 8 to 35 bases long, 9 to 30 bases long, 10 to 25 bases long, 10 to 20 bases long, 11 to 18 bases long, or 12 to 16 bases long. , Or may be 13 to 22 bases long, 16 to 22 bases long, or 16 to 20 bases long.
  • the first nucleic acid chain is not particularly limited, but may be at least 9 bases long, at least 10 bases long, at least 11 bases long, at least 12 bases long, or at least 13 bases long.
  • the first nucleic acid chain has a length of 50 bases or less, 45 bases or less, 40 bases or less, 35 bases or less, 30 bases or less, 28 bases or less, 26 bases or less, 24 bases or less, 22 bases or less. , 20 bases or less, 18 bases or less, or 16 bases or less.
  • the first nucleic acid chain is, for example, 9 to 50 bases long, 10 to 40 bases long, 11 to 35 bases long, 12 to 30 bases long, or 13 to 20 bases long, or 13 to 22 bases long, 16 to 22 bases long. It may be long or 16 to 20 bases long.
  • Complementary regions in the second nucleic acid strand may usually be 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, or at least 13 bases long, but in particular. Not limited. Complementary regions in the second nucleic acid chain are 35 bases or less, 30 bases or less, 25 bases or less, 24 bases or less, 23 bases or less, 22 bases or less, 21 bases or less, 20 bases or less. , 19 bases or less, 18 bases or less, 17 bases or less, or 16 bases or less. In one embodiment, the complementary region in the second nucleic acid chain is 9 to 35 bases long, 9 to 30 bases long, 10 to 25 bases long, 10 to 20 bases long, 11 to 18 bases long or 12 to 16 bases long. It is long.
  • the complementary region in the second nucleic acid strand is 13 to 22 bases long, 16 to 22 bases long, or 16 to 20 bases long. In one embodiment, the complementary region in the second nucleic acid chain is 8 to 35 bases long, 8 to 30 bases long, 8 to 25 bases long, 8 to 20 bases long, 8 to 16 bases long, 8 to 12 bases long. Long, 8 to 10 bases long.
  • the second nucleic acid strand is not particularly limited, but is at least 5 bases long, at least 6 bases long, at least 7 bases long, 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. Alternatively, it may be at least 13 bases long.
  • the second nucleic acid chain has a length of 50 bases or less, 45 bases or less, 40 bases or less, 35 bases or less, 30 bases or less, 28 bases or less, 26 bases or less, 24 bases or less, 22 bases or less. , 20 bases or less, 18 bases or less, 16 bases or less, 14 bases or less, 12 bases or less, 10 bases or less, 9 bases or less.
  • the second nucleic acid chain is, for example, 9 to 50 bases long, 10 to 40 bases long, 11 to 35 bases long, 12 to 30 bases long, or 13 to 20 bases long, 13 to 22 bases long, 16 to 22 bases long. Or 16 to 20 bases long, or 5 to 14 bases long, 6 to 12 bases long, 6 to 10 bases long, 7 to 10 bases long, 8 to 10 bases long, 8 to 9 bases long, or 7 to 9 bases long It may be long.
  • the choice of length is generally determined by the balance between the strength of the antisense effect and the specificity of the nucleic acid strand to the target, among other factors such as cost, synthesis yield, etc.
  • the second nucleic acid strand contains or consists of a complementary region complementary to at least a part of the first nucleic acid strand.
  • the complementary region in the second nucleic acid strand can be complementary to at least a portion of the antisense oligonucleotide region in the first nucleic acid strand.
  • the complementary region in the second nucleic acid strand may be complementary to the entire antisense oligonucleotide region in the first nucleic acid strand.
  • the complementary region in the second nucleic acid strand may be complementary to other parts in addition to the antisense oligonucleotide region in the first nucleic acid strand. Examples of this embodiment are International Publication No. 2013/089283, Nishina K, et.
  • HDOs heteroduplex oligonucleotides
  • the second nucleic acid strand may further comprise at least one overhang region located on one or both of the 5'end and 3'end of the complementary region.
  • the "overhang region” is a region adjacent to a complementary region, and when the first nucleic acid strand and the second nucleic acid strand are annealed to form a double-stranded structure, the 5'end of the second nucleic acid strand is the first.
  • a second nucleic acid that extends beyond the 3'end of the nucleic acid chain and / or the 3'end of the second nucleic acid chain extends beyond the 5'end of the first nucleic acid chain, i.e., protruding from the double-stranded structure.
  • the overhang region in the second nucleic acid strand may be located on the 5'end side of the complementary region (Fig. 2A) or on the 3'end side (Fig. 2B).
  • the overhang region in the second nucleic acid strand may be located on the 5'end side and the 3'end side of the complementary region (Fig. 2C).
  • nucleoside is a combination of base and sugar.
  • the nucleobase (also known as a base) portion of a nucleoside is usually a heterocyclic base portion.
  • the "nucleotide” further comprises a phosphate group covalently attached to the sugar moiety of the nucleoside.
  • the phosphate group can be linked to the 2', 3', or 5'hydroxyl moiety of the sugar.
  • Oligonucleotides are formed by covalent bonds of adjacent nucleosides to form linear polymer oligonucleotides. Within the oligonucleotide structure, the phosphate group is generally considered to form an internucleoside bond of the oligonucleotide.
  • Nucleic acid chains can include natural and / or non-natural nucleotides.
  • Natural nucleotides include deoxyribonucleotides found in DNA and ribonucleotides found in RNA.
  • Deoxyribonucleotide and ribonucleotide may also be referred to as “DNA nucleotide” and “RNA nucleotide”, respectively.
  • Naturally nucleosides include deoxyribonucleosides found in DNA and ribonucleosides found in RNA.
  • deoxyribonucleoside and ribonucleoside may also be referred to as “DNA nucleoside” and “RNA nucleoside”, respectively.
  • Non-natural nucleotide refers to any nucleotide other than the natural nucleotide, including modified nucleotides and nucleotide mimetics.
  • non-natural nucleoside refers to any nucleoside other than the natural nucleoside, including modified nucleosides and nucleoside mimetics.
  • Modified nucleotide means a nucleotide having any one or more of a modified sugar moiety, a modified nucleoside linkage, and a modified nucleobase.
  • Modified nucleoside means a nucleoside having a modified sugar moiety and / or a modified nucleobase.
  • Nucleic acid chains containing unnatural oligonucleotides are often due to desirable properties such as enhanced cell uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity. , Preferable over natural type.
  • Modified nucleoside bond refers to a nucleoside bond that has a substitution or arbitrary variation from a naturally occurring nucleoside bond (ie, a phosphodiester bond). Modified nucleoside bonds include nucleoside bonds that contain a phosphorus atom and nucleoside bonds that do not contain a phosphorus atom. Typical phosphorus-containing nucleoside bonds include phosphodiester bond, phosphorothioate bond, phosphorodithioate bond, phosphotriester bond (methylphosphodiester bond and ethylphosphotriester bond described in US Patent Registration No. 5,955,599), and alkyl. Phosphodies (eg, methylphosphonate bonds as described in US Patent Registration Nos.
  • nucleoside bonds eg, partial structure represented by the following formula (II):
  • An internucleoside bond containing a guanidine moiety substituted with 1 to 4 C 1 to 6 alkyl groups eg, a tetramethylguanidine (TMG) moiety
  • TMG tetramethylguanidine
  • Internucleoside binding and phosphoramidate binding used in the self-neutralizing nucleic acid (ZON) described in WO 2016/081600, but are not limited thereto.
  • a phosphorothioate bond refers to an internucleoside bond in which the non-crosslinked oxygen atom of the phosphodiester bond is replaced with a sulfur atom.
  • Methods for preparing phosphorus-containing and non-phosphorus-containing bonds are well known.
  • Modified nucleoside linkages are preferably those with higher nuclease resistance than naturally occurring nucleoside linkages.
  • the nucleoside-to-nucleoside bond may be chiral-controlled.
  • chiral controlled is intended to be present in a single diastereomer with respect to a chiral center, eg, a chiral-bound phosphorus.
  • Chiral-controlled internucleoside bonds may be completely chiral pure or have high chiral purity, such as 90% de, 95% de, 98% de, 99% de, 99.5% de, 99.8. 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 the diastereomeric excess (% de) (the diastereomer of interest-the other diastereomers). Defined as (stereomeric) / (total diastereomer) x 100 (%).
  • internucleoside linkages chiral controlled phosphorothioate linkages Rp configuration or Sp configuration, substituted with 1-4 alkyl groups of C 1 ⁇ 6 guanidine moiety (e.g., tetramethylguanidine (TMG) moiety; e.g. Internucleoside linkages (eg, partial structure represented by formula (III)), including Alexander A. Lomzov et al., Biochem Biophys Res Commun., 2019, 513 (4), 807-811), and / Or it may be an internucleoside bond containing a cyclic guanidine moiety (eg, a partial structure represented by formula (II)).
  • TMG tetramethylguanidine
  • Internucleoside linkages eg, partial structure represented by formula (III)
  • it may be an internucleoside bond containing a cyclic guanidine moiety (eg, a partial structure represented by formula (II)).
  • nucleobase or “base” includes either modified or unmodified nucleobase (base) unless otherwise specified. Therefore, unless otherwise specified, the purine base may be either a modified or unmodified purine base. Unless otherwise specified, the pyrimidine base may be either a modified or unmodified pyrimidine base.
  • Modified nucleobase or “modified nucleobase” means any nucleobase other than adenine, cytosine, guanine, thymine, or uracil.
  • the "unmodified nucleobase” or “unmodified nucleobase” is the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and It means uracil (U).
  • modified nucleobases include 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine or N4-methylcytosine; N6-methyladenine or 8-bromoadenine; 2-thio-thymine; Examples include, but are not limited to, N2-methylguanine or 8-bromoguanine.
  • the modified nucleobase is preferably 5-methylcytosine.
  • Modified sugar refers to a sugar that has substitutions and / or arbitrary changes from the natural sugar moiety (ie, the sugar moiety found in DNA (2'-H) or RNA (2'-OH)).
  • Sugar modification refers to substitutions and / or arbitrary changes from the natural sugar moiety.
  • the nucleic acid chain may optionally contain one or more modified nucleosides containing modified sugars.
  • the "sugar-modified nucleoside” refers to a nucleoside having a modified sugar moiety. Such sugar-modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the nucleic acid chain.
  • the nucleoside comprises a chemically modified ribofuranose ring moiety.
  • chemically modified ribofuranose rings include, but are not limited to, bicyclic nucleic acids (branched nucleic acids, BNA) by addition of substituents (including 5'and 2'substituents) and cross-linking of non-geminal ring atoms. ), S, N (R) of ribosyl ring oxygen atom, or C (R1) (R2) (R, R1 and R2, respectively, independently H, C 1- C 12 alkyl, or protective group (Represented), and combinations thereof can be mentioned.
  • sugar-modified nucleosides are, 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'-OCH 3 (2'-OMe group or 2'-O-methyl group), and nucleosides containing 2'-O (CH 2 ) 2 OCH 3 substituents.
  • Substituents at the 2'position are also allyl, amino, azide, thio, -O-allyl, -OC 1 -C 10 alkyl, -OCF 3 , -O (CH 2 ) 2 SCH 3 , -O (CH 2 ).
  • "2'-Modified sugar” means a furanosyl sugar modified at the 2'position.
  • a nucleoside containing a 2'-modified sugar may be referred to as a "2'-sugar-modified nucleoside".
  • 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 acids (BNAs). A nucleoside containing a bicyclic sugar moiety may also be referred to as a "crosslinked nucleoside” or a "BNA nucleoside”. FIG. 4 exemplifies some of the crosslinked nucleic acids.
  • Bicyclic sugars may be sugars in which a carbon atom at the 2'position and a carbon atom at the 4'position are crosslinked 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 (BNAs) or BNA nucleosides is 4'-(CH 2 ) p -O-2', 4'-(CH 2 ) p -CH 2 -2', 4 '-(CH 2 ) p -S-2', 4'-(CH 2 ) p -OCO-2', 4'-(CH 2 ) n -N (R 3 ) -O- (CH 2 ) m- 2'[In the formula, p, m and n represent integers 1 to 4, integers 0 to 2 and integers 1 to 3, respectively; and R 3 is a hydrogen atom, an alkyl group, an alkenyl group and a cyclo.
  • a hydroxyl group a hydroxyl group protected by a protective group for nucleic acid synthesis
  • a mercapto group a mercapto group protected by a protective group for nucleic acid synthesis
  • an amino group 1 Substituted with an alkoxy group having up to 5 carbon atoms, an alkylthio group with 1 to 5 carbon atoms, a cyanoalkoxy group with 1 to 6 carbon atoms, or an alkyl group with 1 to 5 carbon atoms Represents an amino group that is Non-limiting examples of such BNAs are also known as Methyleneoxy (4'-CH 2- O-2') BNA (LNA (Locked Nucleic Acid®, 2', 4'-BNA).
  • ⁇ -L-methyleneoxy (4'-CH 2- O-2') BNA or ⁇ -D-methyleneoxy (4'-CH 2- O-2') BNA ethyleneoxy ( 4'-(CH 2 ) 2 -O-2') BNA (also known as ENA), ⁇ -D-thio (4'-CH 2 -S-2') BNA, Aminooxy (4'- CH 2 -ON (R 3 ) -2') BNA, Oxyamino (4'-CH 2 -N (R 3 ) -O-2') BNA (2', 4'-BNA
  • Me is also known as GuNA [N-Me])
  • Amin BNA also known as 2'-Amino-LNA
  • 2'-Amino-LNA eg, 3- (Bis (3-aminopropyl) amino) propanoyl substitute
  • 2'-O, 4'-C-spirocyclopropylene crosslinked nucleic acid also known as scpBNA
  • other BNAs known to those of skill in the art.
  • a "cationic nucleoside” is used at a certain pH (eg, human physiological pH (about 7.4), pH of a delivery site (eg, organella, cell, tissue, organ, organism, etc.), etc.). It is a modified nucleoside that exists as a cation form as compared to a neutral form (such as the neutral form of ribonucleoside).
  • the cationic nucleoside may contain one or more cationic modifying groups at any position of the nucleoside.
  • LNA nucleosides Bicyclic nucleosides with methyleneoxy (4'-CH 2- O-2') crosslinks are sometimes referred to as LNA nucleosides.
  • nucleobase moiety (natural, modified, or a combination thereof) may be maintained for hybridization with a suitable nucleic acid target.
  • nucleoside mimic comprises a sugar or sugar and a base at one or more positions of an oligomeric compound, as well as a structure used to replace a bond, if not necessarily.
  • oligomer compound is meant a polymer of linked monomer subunits that are hybridizable to at least a region of a nucleic acid molecule.
  • nucleoside mimetics include morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclic or tricyclic sugar mimetics, such as nucleoside mimetics having non-furanose sugar units.
  • a "nucleotide mimic” comprises a structure used to replace a nucleoside and bond at one or more positions of an oligomeric compound.
  • Peptide nucleic acid (PNA) is a nucleotide mimetic having a main chain in which N- (2-aminoethyl) glycine is bound by an amide bond instead of sugar.
  • An example of the structure of morpholinonucleic acid is shown in FIG.
  • “Imitation” refers to a group that replaces one or more of a sugar, a nucleobase, and an internucleoside bond. In general, mimetics are used in place of sugars, or combinations of sugar and nucleoside linkages, and nucleobases are maintained for hybridization to the target of choice.
  • the modification can be performed so that the nucleotides in the same strand can independently undergo different modifications.
  • the same nucleotides have modified nucleoside bonds (eg, phosphorothioate bonds) and are further modified sugars (eg, 2'-O-methyl modified sugars or bicyclics).
  • the same nucleotide can also have a modified nucleobase (eg, 5-methylcytosine) and further have a modified sugar (eg, 2'-O-methyl modified sugar or bicyclic sugar).
  • the number, type and position of unnatural nucleotides in the nucleic acid strand can affect the antisense effect provided by the nucleic acid complex and the like.
  • the choice of modification may vary depending on the sequence of the target gene, etc., but those skilled in the art will explain the literature related to the antisense method (eg, WO2007 / 143315, WO2008 / 043753, and WO2008 / 049085). Suitable embodiments can be determined by reference.
  • the measured value thus obtained is not significantly lower than the measured value of the unmodified nucleic acid complex (eg,).
  • the measured value obtained after the modification is 70% or more, 80% or more or 90% or more of the measured value of the nucleic acid complex before the modification), the related modification can be evaluated.
  • the antisense effect is measured, for example, by administering the test nucleic acid compound to a subject (eg, a mouse) and, for example, after a few days (eg, 2-7 days), 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 the amount of 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 (eg, vehicle administration).
  • a negative control eg, vehicle administration
  • the test nucleic acid compound can provide an antisense effect (eg, reduced target transcript levels).
  • the first nucleic acid chain is a gapmer. That is, the antisense oligonucleotide region in the first nucleic acid strand is a gapmer-type antisense oligonucleotide region (gapmer-type antisense oligonucleotide region).
  • the "gapmer type” is a central region (DNA gap region) containing at least 4 consecutive deoxyribonucleosides and a region containing unnatural nucleosides located on the 5'end and 3'ends thereof (gapmer type). Refers to the nucleoside composition consisting of the 5'wing region and the 3'wing region).
  • Gapmers in which unnatural nucleosides are composed of cross-linked nucleosides are particularly referred to as "BNA / DNA gapmers".
  • 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, 7 to 14 bases. It may be long or 8-12 bases long.
  • the central region in the gapmer may contain or consist of natural nucleosides, unmodified sugars (and modified or unmodified nucleoside linkages) in which the sugar moiety of the nucleic acid is unmodified, eg, 13-22 base length.
  • 16 to 22 bases, or 16 to 20 bases, or 4 to 20 bases, 5 to 18 bases, 6 to 16 bases, 7 to 14 bases or 8 to 12 bases are natural nucleosides, Or contains unmodified sugar.
  • the central region may include or consist of native nucleosides linked by modified nucleoside linkages such as, for example, phosphorothioate bonds.
  • the lengths of the 5'wing region and the 3'wing region may be independently, usually 1 to 10 bases long, 1 to 7 bases long, 2 to 5 bases long, or 2 to 3 bases long.
  • the 5'wing region and the 3'wing region may contain at least one unnatural nucleoside, and may further contain a natural nucleoside.
  • the Gapmer-type antisense oligonucleotide region contains a 5'wing region containing 2 or 3 bridged nucleosides, a 3'wing region containing 2 or 3 bridged nucleosides, and a BNA / containing a DNA gap region between them. It may have a DNA gapmer type nucleoside composition.
  • the crosslinked nucleoside may contain a modified nucleobase (eg, 5-methylcytosine).
  • the gapmer may be an "LNA / DNA gapmer" in which the crosslinked nucleoside is composed of an LNA nucleoside.
  • the 5'wing region and the 3'wing region may be, for example, an unnatural nucleoside linked by a modified nucleoside bond such as a phosphorothioate bond, for example a 2'-O-methyl modified nucleoside.
  • the nucleoside of the first nucleic acid chain comprises or consists of a deoxyribonucleoside, eg, 70% or more, 80% or more, 90% or more, or 95% or more of the nucleoside of the first nucleic acid chain is a deoxyribonucleoside. is there.
  • the nucleoside-linked bond in the first nucleic acid chain may be a naturally occurring nucleoside-linked bond and / or a modified nucleoside-linked bond.
  • At least one, at least two, or at least three nucleoside linkages from the 5'end of the first nucleic acid strand may be modified nucleoside linkages.
  • the at least one, at least two, or at least three nucleoside linkages from the 3'end of the first nucleic acid strand may be modified nucleoside linkages.
  • the binding between two nucleosides from the end of a nucleic acid chain is the binding between nucleosides closest to the end of the nucleic acid chain and the binding between nucleosides adjacent to this and located in the direction opposite to the end of the nucleic acid chain. Point. Modification between modified nucleosides in the terminal region of the nucleic acid chain is preferable because it can suppress or inhibit undesired degradation of the nucleic acid chain.
  • Modified nucleoside linkages are at least 70%, at least 80%, at least 85%, at least 90%, at least 93%, at least 95%, at least 98% of the nucleoside linkages of the antisense oligonucleotide region in the first nucleic acid strand. Or it may be 100%. Moreover, the bond between modified nucleosides may be a phosphorothioate bond.
  • the nucleoside in the first nucleic acid chain may be a natural nucleoside (including deoxyribonucleoside, ribonucleoside, or both) and / or an unnatural nucleoside.
  • the second nucleic acid strand has at least one sugar-unmodified central region (first exposed region) complementary to a part of the first nucleic acid strand (for example, a part of the central region of the first nucleic acid strand), for example 1. Includes ⁇ 5, 1-4, 1-3, 1-2, or 1, or at least 2.
  • first exposed region a sugar-unmodified central region
  • the first exposed region may be completely complementary to a part of the first nucleic acid strand, or may have one or more mismatched bases.
  • the first exposed region may have 1 to 3, 1 to 2, or 1 mismatched base (non-complementary base) with respect to a part of the first nucleic acid chain.
  • the first exposed region consists of one or two to three consecutive unmodified ribonucleosides linked by internucleoside bonds.
  • the term "sugar-unmodified ribonucleoside” means a ribonucleoside in which the sugar moiety of a natural ribonucleoside does not contain a sugar modification, for example, a 2'-position substitution and / or any change.
  • the first exposed region may contain at least one modified base and / or modified nucleoside bond.
  • the sugar-unmodified ribonucleoside in the first exposed region comprises a natural ribonucleoside, eg, all of the sugar-unmodified ribonucleosides are natural ribonucleosides, or a portion thereof, such as one or two.
  • the sugar-unmodified ribonucleoside is a natural ribonucleoside.
  • the second nucleic acid strand comprises at least one first exposed region, eg, 1-10, 1-5, 1-4, 1-3, 1-2, or 1. At least 2 pieces, for example 2-10 pieces, 2-5 pieces, 3-4 pieces, 2-3 pieces or 2 pieces, or at least 3 pieces, for example 3-10 pieces, 3-5 pieces, 3-4 pieces or 3 Including.
  • the first exposed region consists of two or three contiguous sugar-unmodified ribonucleosides linked by internucleoside bonds. In one embodiment, the first exposed region consists of three contiguous sugar-unmodified ribonucleosides linked by internucleoside linkages.
  • the present embodiment may have the effect that the cleavage activity by RNase H is maintained higher than that of the first exposed region consisting of one or two consecutive sugar-unmodified ribonucleosides. This embodiment may have the effect of maintaining and improving activity in the RNase H-dependent and / or independent pathway as compared to the central region consisting of sugar-unmodified ribonucleosides.
  • the first exposed region comprises a nucleoside containing a modified or unmodified purine base.
  • the cleavage activity by RNase A is suppressed to be low while maintaining the cleavage activity by RNase H as compared with the first exposed region which does not contain a nucleoside containing a purine base (for example, consisting of a nucleoside containing a pyrimidine base).
  • the activity in the RNase H-dependent and / or independent pathway is maintained and improved as compared with the first exposed region containing no purine base-containing nucleoside (for example, consisting of a pyrimidine base-containing nucleoside). Can have the effect of being done.
  • the second nucleic acid strand has at least one defense region, eg, 1-10, 1-5, 1-4, 1-3, 1-2, or 1, at least 2. Includes, eg 2-10, 2-5, 3-4, 2-3 or 2, or at least 3, eg 3-10, 3-5, 3-4 or 3 ..
  • the term "defensive region” refers to a region that is resistant to cleavage by a nucleolytic enzyme.
  • the nucleolytic enzyme is not limited. Examples of nucleolytic enzymes include RNase A and RNase H.
  • the defensive region may be, for example, a region resistant to RNase A, a region resistant to RNase H, or a region resistant to any of RNase A and RNase H.
  • the resistance to cleavage by a nucleolytic enzyme does not have to be complete resistance, as long as it has the same base sequence and the resistance is increased as compared with the region containing unmodified ribonucleoside.
  • the defensive region is from one or two or more (a) deoxyribonucleosides linked by internucleoside bonds, (b) sugar-modified nucleosides and / or (c) nucleosides with modified nucleoside bonds on the 3'side. Become.
  • the defense region may contain at least one modified base and / or modified nucleoside bond.
  • Deoxyribonucleosides and / or sugar-modified nucleosides in the defense region are preferably deoxyribonucleosides, 2'-sugar-modified nucleosides (eg, nucleosides containing 2'-O-methyl groups), crosslinked nucleosides (eg, LNA nucleosides) and /.
  • a cationic nucleoside more preferably a deoxyribonucleoside, a 2'-sugar modified nucleoside (eg, a nucleoside containing a 2'-O-methyl group) and / or a crosslinked nucleoside (eg, an LNA nucleoside), even more preferred.
  • a deoxyribonucleoside and / or a 2'-sugar modified nucleoside eg, a nucleoside containing a 2'-O-methyl group
  • particularly preferably a deoxyribonucleoside and / or a nucleoside containing a 2'-O-methyl group. is there.
  • Nucleosides of nucleosides having an intermodified nucleoside bond on the 3'side of the defense region are preferably deoxyribonucleosides, ribonucleosides, 2'-sugar modified nucleosides (eg, nucleosides containing 2'-O-methyl groups), crosslinked nucleosides. (Eg, LNA nucleosides) and / or cationic nucleosides, more preferably deoxyribonucleosides, ribonucleosides, 2'-sugar-modified nucleosides (eg, nucleosides containing 2'-O-methyl groups) and / or crosslinked nucleosides.
  • LNA nucleosides even more preferably deoxyribonucleosides, ribonucleosides and / or 2'-sugar-modified nucleosides (eg, nucleosides containing 2'-O-methyl groups), even more preferably deoxyribos.
  • a nucleoside having a modified nucleoside interbond on the 3'side of the defense region preferably has a phosphorothioate bond, a phosphorothioate bond on the 3'side, and a guanidine moiety substituted with 1 to 4 alkyl groups of C 1 to 6 (preferably.
  • An internucleoside bond containing a TMG moiety preferably a partial structure represented by formula (III)
  • / or a nucleoside interbond containing a cyclic guanidine moiety preferably a partial structure represented by formula (II)
  • It is a nucleoside having a nucleoside, and more preferably a nucleoside having a phosphorothioate bond on the 3'side.
  • the 3'side modified nucleoside bond may be chirally controlled to an Rp or Sp configuration.
  • a nucleoside having a modified nucleoside bond on the 3'side means a nucleoside having a modified nucleoside bond on at least the 3'side, and the nucleoside having a modified nucleoside bond on the 5'side also has a bond. May be good.
  • the defense region may be completely complementary to a portion of the first nucleic acid strand (eg, a portion of the central region of the first nucleic acid strand) or has one or more mismatched bases. You may.
  • the defense region may have 1 to 3, 1 to 2, or 1 mismatched base (non-complementary base) with respect to a part of the first nucleic acid chain.
  • the defensive region comprises (a) a deoxyribonucleoside containing a pyrimidine base, (b) a sugar-modified nucleoside and / or (c) a nucleoside having a modified nucleoside interlinkage on the 3'side.
  • the pyrimidine base may be either modified or unmodified.
  • at least one of the defense regions, or all of the defense regions contains modified or unmodified pyrimidine bases. It may contain a nucleoside having a bond.
  • the cleavage activity of RNase A on the second nucleic acid chain is suppressed to be lower than that in the case where the nucleoside containing a pyrimidine base is contained in the sugar-unmodified region (for example, the first exposed region or the second exposed region). Can have an effect.
  • the activity in the RNase H-dependent and / or independent pathway is higher than that in the case where the nucleoside containing a pyrimidine base is contained in a sugar-unmodified region (for example, a first exposed region or a second exposed region). It can have the effect of being maintained and improved.
  • the sugar-modified nucleoside in the defense region is a sugar-modified ribonucleoside.
  • the second nucleic acid strand consists of a first exposed region and a defensive region. In a further embodiment, the second nucleic acid strand consists of alternating first exposed and defensive regions. In a further embodiment, at least one of the defense regions, or all of the defense regions, contains (a) deoxyribonucleosides, (b) sugar-modified nucleosides and / or (c) 3'sides containing modified or unmodified pyrimidine bases. Includes nucleosides with modified nucleoside interlinks.
  • the second nucleic acid strand has at least one second exposed region, eg, 1-5, 1-4, 1-3, 1-2, which is complementary to a portion of the first nucleic acid strand. Includes, one, or at least two.
  • the second exposed region consists of four or more consecutive sugar-unmodified ribonucleosides linked by nucleoside linkages, and the base of the sugar-unmodified ribonucleoside in the second exposed region contains a purine base.
  • the purine base may be either modified or unmodified.
  • the number of sugar-unmodified ribonucleosides constituting the second exposed region is, for example, 4 to 10, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 4 to 5, or 4 It may be an individual.
  • the second exposed region may contain at least one modified base and / or modified nucleoside bond.
  • the second exposed region may be completely complementary to a portion of the first nucleic acid strand (eg, a portion of the central region of the first nucleic acid strand) or has one or more mismatched bases. You may be doing it.
  • the second exposed region may have 1 to 3, 1 to 2, or 1 mismatched base (non-complementary base) with respect to a part of the first nucleic acid chain.
  • the sugar-unmodified ribonucleoside in the second exposed region comprises a natural ribonucleoside, eg, all of the sugar-unmodified ribonucleosides are natural ribonucleosides, or a portion thereof, such as one or two.
  • the sugar-unmodified ribonucleoside is a natural ribonucleoside.
  • the second nucleic acid strand has at least one second exposed region, eg, 1-10, 1-5, 1-4, 1-3, 1-2, or 1. At least 2 pieces, for example 2-10 pieces, 2-5 pieces, 3-4 pieces, 2-3 pieces or 2 pieces, or at least 3 pieces, for example 3-10 pieces, 3-5 pieces, 3-4 pieces or 3 Including.
  • the second nucleic acid strand is at least one, for example, 1 to 10, 1 to 6, 1 to 5, 1 to 4, in total of the first exposed region and / or the second exposed region. 1, 1-3, 1-2, or 1, at least 2, eg 2-10, 2-5, 3-4, 2-3 or 2, or at least 3, eg Includes 3-10 pieces, 3-5 pieces, 3-4 pieces or 3 pieces.
  • the second nucleic acid strand consists of a first exposed region, a second exposed region, and a protective region. In a further embodiment, the second nucleic acid strand consists of alternating first or second exposed regions and defense regions. In a further embodiment, at least one of the defense regions, or all of the defense regions, contains (a) deoxyribonucleosides, (b) sugar-modified nucleosides and / or (c) 3'sides containing modified or unmodified pyrimidine bases. Includes nucleosides with modified nucleoside interlinks.
  • the second nucleic acid strand consists of a second exposed region and a defensive region. In a further embodiment, the second nucleic acid strand consists of alternating second exposed and defensive regions. In a further embodiment, at least one of the defense regions, or all of the defense regions, contains (a) deoxyribonucleosides, (b) sugar-modified nucleosides and / or (c) 3'sides containing modified or unmodified pyrimidine bases. Includes nucleosides with modified nucleoside interlinks.
  • At least one defense region of the second nucleic acid strand is linked to the first exposed region and / or the second exposed region by nucleoside binding at the 5'end and 3'end.
  • the first exposed region and / or the second exposed region is chirally controlled to at least one, eg, one or two modified nucleoside linkages, eg, a phosphorothioate bond, eg, an Rp or Sp configuration. bond, substituted with 1-4 alkyl groups of C 1 ⁇ 6 guanidine moiety (e.g., TMG partial) internucleoside linkages including (eg, the partial structure represented by formula (III)) and / or cyclic guanidine Includes a moiety-containing internucleoside bond (eg, a partial structure represented by formula (II)).
  • the second nucleic acid strands are linked by modified or unmodified nucleoside linkages, (1) said first exposed region and / or (2) said said second exposed region, and (3) (3) ( a) Deoxyribonucleosides and / or (b) sugar-modified nucleosides, including or consisting of, for example, sugar-modified ribonucleosides.
  • the second nucleic acid strand further comprises the following sugar-unmodified terminal regions at the 5'end and / or the 3'end.
  • the length of the sugar-unmodified terminal region at the 5'and / or 3'end is independent, usually 1-10 bases long, 1-7 bases long, 2-5 bases long or 2 It is ⁇ 3 bases long.
  • the second nucleic acid chain is linked by a modified or unmodified nucleoside interlinkage, said first exposed region and / or second exposed region, and a sugar-modified ribonucleoside (and optionally a sugar-unmodified end). Area) includes or consists of.
  • the second nucleic acid strand is at least one, eg, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 or at least 2. It contains a sugar-unmodified terminal region containing a sugar-unmodified ribonucleoside at the 5'end and / or the 3'end. Since the toxicity of the nucleic acid complex tends to increase as the amount of sugar modification increases, the number of sugar-modified nucleic acids in the second nucleic acid chain is reduced by making the terminal region sugar-unmodified, and as a result, the toxicity is reduced. obtain.
  • the sugar-unmodified terminal region may contain a modified base and / or a modified nucleoside bond.
  • the sugar-unmodified terminal region complements a portion of the first nucleic acid strand (eg, the 5'wing region, the central region, the 3'wing region, or any part of the first nucleic acid strand). It is a target.
  • the sugar-unmodified terminal region may be completely complementary to a part of the first nucleic acid chain, or may have one or more mismatched bases.
  • the second exposed region may have 1 to 3, 1 to 2, or 1 mismatched base (non-complementary base) with respect to a part of the first nucleic acid chain.
  • the sugar-unmodified ribonucleoside in the sugar-unmodified terminal region comprises a natural ribonucleoside, eg, all of the sugar-unmodified ribonucleosides are natural ribonucleosides, or a portion thereof, such as 1-5. Natural ribonucleosides are 1 to 4, 1 to 3, or 1 or 2 sugar-unmodified ribonucleosides.
  • the sugar-unmodified terminal region comprises a nucleoside containing a modified or unmodified purine base and / or a modified or unmodified pyrimidine base.
  • the sugar-unmodified terminal region comprises a nucleoside containing a modified or unmodified purine base.
  • the second nucleic acid strands are linked by modified or unmodified nucleoside linkages, (1) at least one first exposed region and / or at least one second exposed region, (2) at least one. It consists of two defense regions and (3) 5'and 3'terminal sugar-unmodified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 1-10 first exposed regions and / or 1-10 second exposed regions, (1) 2) It consists of 1 to 10 defense regions, and (3) 5'end and 3'end sugar-unmodified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 1-5 first exposed regions and / or 1-5 second exposed regions, (1).
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 1-3 first exposed regions and / or 1-3 second exposed regions, (1) It consists of 2) 1 to 3 defense regions and (3) 5'and 3'terminal sugar-unmodified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 2-10 first exposed regions and / or second exposed regions, (2) 2-10. It consists of individual defense regions and (3) 5'and 3'terminal sugar-unmodified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 2-6 first exposed regions and / or second exposed regions, (2) 2-6. It consists of individual defense regions and (3) 5'and 3'terminal sugar-unmodified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 2-5 first exposed regions and / or second exposed regions, (2) 2-5. It consists of individual defense regions and (3) 5'and 3'terminal sugar-unmodified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 2-4 first exposed regions and / or second exposed regions, (2) 2-4.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 2-3 first exposed regions and / or 2nd exposed regions, (2) 2-3. It consists of individual defense regions and (3) 5'and 3'terminal sugar-unmodified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 3-10 first exposed regions and / or second exposed regions, (2) 3-10. It consists of individual defense regions and (3) 5'and 3'terminal sugar-unmodified terminal regions.
  • the second nucleic acid strands are linked by modified or unmodified nucleoside linkages, (1) 3-6 first exposed regions and / or 2nd exposed regions, (2) 3-6. It consists of the defense region of (3) and the sugar-unmodified terminal region at the 5'end and the 3'end.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 3-5 first exposed regions and / or second exposed regions, (2) 3-5. It consists of individual defense regions and (3) 5'and 3'terminal sugar-unmodified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 3-4 first exposed regions and / or second exposed regions, (2) 3-4.
  • the second nucleic acid strand has (a) a first exposed region or a second exposed region, and (b) a defense region alternately arranged.
  • the second nucleic acid strand has a sugar-modified terminal region containing at least one deoxyribonucleoside, a sugar-modified nucleoside and / or a nucleoside having a modified nucleoside-linked bond on the 3'side, 5'end and / or 3'. Included at the end.
  • the second nucleic acid strand comprises a sugar-modified terminal region containing at least one deoxyribonucleoside and / or sugar-modified nucleoside at the 5'end and / or 3'end.
  • the sugar-modified terminal region is complementary to a part of the first nucleic acid chain (for example, a 5'wing region, a central region, a 3'wing region, or a part thereof of the first nucleic acid chain).
  • the sugar-modified terminal region may be completely complementary to a part of the first nucleic acid chain, or may have one or more mismatched bases.
  • the second exposed region may have 1 to 3, 1 to 2, or 1 mismatched base (non-complementary base) with respect to a part of the first nucleic acid chain.
  • the sugar-modified terminal region comprises a deoxyribonucleoside containing a pyrimidine base, a sugar-modified nucleoside and / or a nucleoside having a modified nucleoside interlink on the 3'side.
  • the pyrimidine base may be either modified or unmodified.
  • the sugar-modified terminal region comprises a deoxyribonucleoside containing a purine base, a sugar-modified nucleoside and / or a nucleoside having a modified nucleoside interlinkage on the 3'side.
  • the sugar-modified terminal region comprises a deoxyribonucleoside containing a purine base and / or a sugar-modified nucleoside.
  • the purine base may be either modified or unmodified.
  • the second nucleic acid strand comprises or will contain said first exposed region, second exposed region, defense region, and / or sugar modified terminal region linked by modified or unmodified nucleoside interlinkage.
  • the lengths of the sugar-modified terminal regions at the 5'and / or 3'ends are independent, usually 1-10 bases long, 1-7 bases long, 2-5 bases long or 2-5 bases long. It is 3 bases long.
  • the sugar-modified terminal region is a nucleoside in which all of the nucleosides have a deoxyribonucleoside, a sugar-modified nucleoside and / or a modified nucleoside-linked bond on the 3'side (preferably all of the nucleosides are deoxyribonucleosides and / or sugar-modified.
  • Nucleosides or some of them, such as 1, 1-2, 1-3, 1-4, 1-5, 1-6 or 1-7 are deoxyribonucleosides, sugar-modified nucleosides.
  • nucleoside having a modified nucleoside interlinkage on the 3'side preferably all of the nucleosides are deoxyribonucleosides and / or sugar-modified nucleosides.
  • nucleosides in the sugar-modified terminal region any proportion of the nucleosides, sugar-modified nucleosides and / or nucleosides having a modified nucleoside-linked bond on the 3'side (preferably all of the nucleosides are deoxyribonucleosides and /).
  • sugar-modified nucleoside preferably all of the nucleosides are deoxyribonucleosides and /.
  • 10% or more, 30% or more, 50% or more, 70% or more, 80% or more, 90% or more are deoxyribonucleosides, sugar-modified nucleosides and / or nucleosides having a modified nucleoside-linked bond on the 3'side (preferably, All of the nucleosides are deoxyribonucleosides and / or sugar-modified nucleosides).
  • Deoxyribonucleosides and / or sugar-modified nucleosides are preferably deoxyribonucleosides, 2'-sugar-modified nucleosides (eg, nucleosides containing 2'-O-methyl groups), crosslinked nucleosides (eg, LNA nucleosides) and / or cations. It is a sex nucleoside, more preferably a deoxyribonucleoside, a 2'-sugar modified nucleoside (eg, a nucleoside containing a 2'-O-methyl group) and / or a crosslinked nucleoside (eg, LNA nucleoside''), even more preferred.
  • 2'-sugar-modified nucleosides eg, nucleosides containing 2'-O-methyl groups
  • crosslinked nucleosides eg, LNA nucleoside''
  • Is a deoxyribonucleoside a nucleoside containing a 2'-O-methyl group and / or an LNA nucleoside, more preferably a deoxyribonucleoside and / or an LNA nucleoside, and particularly preferably an LNA nucleoside.
  • a nucleoside having a modified nucleoside bond on the 3'side is preferably a nucleoside having a phosphorothioate bond on the 3'side.
  • the second nucleic acid strands are linked by modified or unmodified nucleoside linkages, (1) at least one first exposed region and / or at least one second exposed region, (2) at least one. It consists of two defense regions and (3) 5'and 3'terminal sugar-modified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 1-10 first exposed regions and / or 1-10 second exposed regions, (1) It consists of 2) 1 to 10 defense regions and (3) 5'and 3'terminal sugar-modified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 1-5 first exposed regions and / or 1-5 second exposed regions, (1).
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 1 to 4 first exposed regions and / or 1 to 4 second exposed regions, (1). It consists of 2) 1 to 4 defense regions and (3) 5'and 3'terminal sugar-modified terminal regions. In one embodiment, the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 1-3 first exposed regions and / or 1-3 second exposed regions, (1) It consists of 2) 1 to 3 defense regions and (3) 5'and 3'terminal sugar-modified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 2-5 first exposed regions and / or 1-5 second exposed regions, (1). It consists of 2) 2 to 5 defense regions and (3) 5'and 3'terminal sugar-modified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 2-4 first exposed regions and / or 1-4 second exposed regions, (1). It consists of 2) 2-4 defense regions and (3) 5'and 3'terminal sugar-modified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 2-3 first exposed regions and / or 1-3 second exposed regions, (1) It consists of 2) 2-3 defense regions and (3) 5'and 3'terminal sugar-modified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 3-5 first exposed regions and / or 1-5 second exposed regions, (1). It consists of 2) 3-5 defense regions and (3) 5'and 3'terminal sugar-modified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 3-4 first exposed regions and / or 1-4 second exposed regions, (1).
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 2-10 first exposed regions and / or second exposed regions, (2) 2-10. It consists of individual defense regions and (3) 5'and 3'terminal sugar-unmodified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 2-6 first exposed regions and / or second exposed regions, (2) 2-6. It consists of individual defense regions and (3) 5'and 3'terminal sugar-unmodified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 2-5 first exposed regions and / or second exposed regions, (2) 2-5. It consists of individual defense regions and (3) 5'and 3'terminal sugar-unmodified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 2-4 first exposed regions and / or second exposed regions, (2) 2-4. It consists of individual defense regions and (3) 5'and 3'terminal sugar-unmodified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 2-3 first exposed regions and / or 2nd exposed regions, (2) 2-3.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 3-10 first exposed regions and / or second exposed regions, (2) 3-10. It consists of individual defense regions and (3) 5'and 3'terminal sugar-unmodified terminal regions.
  • the second nucleic acid strands are linked by modified or unmodified nucleoside linkages, (1) 3-6 first exposed regions and / or 2nd exposed regions, (2) 3-6. It consists of the defense region of (3) and the sugar-unmodified terminal region of (3) 5'end and 3'end.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 3-5 first exposed regions and / or second exposed regions, (2) 3-5. It consists of individual defense regions and (3) 5'and 3'terminal sugar-unmodified terminal regions.
  • the second nucleic acid strand is linked by modified or unmodified nucleoside linkages, (1) 3-4 first exposed regions and / or second exposed regions, (2) 3-4. It consists of individual defense regions and (3) 5'and 3'terminal sugar-unmodified terminal regions.
  • (a) the first exposed region or the second exposed region, and (b) the defense region are alternately arranged in the second nucleic acid chain.
  • the modified nucleoside linkages of the first and / or second nucleic acid strands have a pH (eg, human physiological pH (about 7.4)), delivery site (eg, organella, cell, tissue, organ). , The pH of the organism, etc.), the modified nucleoside bond is in the anion form (eg, -OP (O) (O - )-O- (anion form of the natural phosphate bond), -OP (O). Includes unloaded (neutral or cationic) nucleoside interbonds that are present in neutral or cationic form as compared to (S -)-O-((anionic form of phosphorothioate bond), etc.).
  • a pH eg, human physiological pH (about 7.4)
  • delivery site eg, organella, cell, tissue, organ.
  • the modified nucleoside bond is in the anion form (eg, -OP (O) (O - )-O- (anion form of the natural phosphate bond), -
  • the modified nucleoside linkage of the first and / or second nucleic acid strand comprises a neutral nucleoside linkage.
  • Interbonding comprises a cationic internucleoside bond.
  • an unloaded electro-nucleoside interlink eg, a neutral internucleoside bond
  • the unloaded electronucleoside bond is, for example, the methylphosphonates described in US Patent Registration Nos. 5,264,423 and 5,286,717.
  • the unloaded electronucleoside bond comprises a triazole moiety or an alkin moiety.
  • the unloaded electronucleoside bond comprises a cyclic guanidine moiety and /. or guanidine moieties (preferably, TMG partial) substituted with 1-4 C 1 ⁇ 6 alkyl group in.
  • one embodiment comprising, modified internucleoside including cyclic guanidine moiety bonds in formula (II) It has a partial structure represented.
  • the guanidine moiety substituted with 1 to 4 alkyl groups of C 1 to 6 has a partial structure represented by the formula (III).
  • internucleoside linkage neutral containing guanidine moiety substituted with cyclic guanidine moiety and / or one to four alkyl groups of C 1 ⁇ 6 are chiral controlled.
  • the present The disclosure relates to a composition comprising an oligonucleotide containing at least one neutral nucleoside bond and at least one phosphorothioate nucleoside bond.
  • neutral internucleotide binding is characteristic compared to equivalent nucleic acids that do not contain neutral internucleotide binding.
  • / or activity can be improved, for example, improved delivery, improved resistance to exonucleases and endonucleases, improved cell uptake, improved endosome prolapse, and / or improved nuclear uptake, etc. To do.
  • the first and / or second nucleic acid strands have one, two, three, four, five, six, seven, eight, nine modified nucleoside linkages, respectively. , 10, 11, 12, 13, 14, 14, 15, 16, 17, 18, 18, 19, 20, 21, 22, 23, 24, 25, It may contain 26, 27, 28, 29, 30, 35, 40, 45, 50, or more.
  • the first and / or second nucleic acid strands have at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 50% of the modified nucleoside bonds, respectively. It may contain 60%, at least 70%, at least 80%, at least 90%, or more.
  • the first exposed region, the second exposed region, the defense region, the sugar-unmodified terminal region and / or the sugar-modified terminal region of the second nucleic acid chain each have one modified nucleoside bond, 1-2. 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12 1 to 13 pieces, 1 to 14 pieces, 1 to 15 pieces, 1 to 16 pieces, 1 to 17 pieces, 1 to 18 pieces, 1 to 19 pieces, 1 to 20 pieces, 1 to 21 pieces, 1 to 22 pieces It may contain individual pieces.
  • the first exposed region, the second exposed region, the defense region, the sugar-unmodified terminal region and / or the sugar-modified terminal region of the second nucleic acid chain have at least 5% and at least 10% of modified nucleoside binding, respectively. , At least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more.
  • the first and / or second nucleic acid strands have one, two, three, four, five, six, seven, eight chiral-controlled internucleoside linkages, respectively.
  • the first and / or second nucleic acid strands have at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50 of chiral-controlled internucleoside linkages, respectively. %, At least 60%, at least 70%, at least 80%, at least 90%, or more.
  • the first exposed region, the second exposed region, the defense region, the sugar-unmodified terminal region and / or the sugar-modified terminal region of the second nucleic acid chain each have one chiral-controlled internucleoside bond.
  • the first exposed region, the second exposed region, the defense region, the sugar-unmodified terminal region and / or the sugar-modified terminal region of the second nucleic acid chain each have at least 5% of chiral-controlled internucleoside binding. , At least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more.
  • the first and / or second nucleic acid strands have one, two, three, or four unloaded electronucleoside linkages (preferably neutral nucleoside linkages), respectively. 5, 6, 7, 8, 9, 10, 10, 11, 12, 13, 14, 14, 15, 16, 17, 18, 18, 19, 20, 21, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more.
  • the first and / or second nucleic acid strands have at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, and at least 50% unloaded electronucleoside binding, respectively. , At least 60%, at least 70%, at least 80%, at least 90%, or more.
  • the first exposed region, the second exposed region, the defense region, the sugar-unmodified terminal region and / or the sugar-modified terminal region of the second nucleic acid chain are each unloaded electronucleoside-linked (preferably medium).
  • Sexual nucleoside bond 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10 pieces, 1-11 pieces, 1-12 pieces, 1-13 pieces, 1-14 pieces, 1-15 pieces, 1-16 pieces, 1-17 pieces, 1-18 pieces, 1-19 pieces, It may contain 1 to 20, 1 to 21, and 1 to 22 pieces.
  • the first exposed region, the second exposed region, the defensive region, the sugar-unmodified terminal region and / or the sugar-modified terminal region of the second nucleic acid chain each have at least 5% of unloaded electronucleoside binding. It may contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more.
  • any of the above regions (first exposed region, protective region, second exposed region, sugar unmodified terminal region, and / or sugar modified terminal region) in the second nucleic acid chain is inside or adjacent to that region.
  • guanidine moieties eg, TMG moieties
  • C 1 ⁇ 6 guanidine moiety substituted with an alkyl group e.g., TMG partial
  • internucleoside linkages including e.g., the partial structure represented by formula (III)
  • internucleoside linkages containing a cyclic guanidine moiety e.g. , A partial structure represented by the formula (II)
  • an internucleoside bond containing a chiral-controlled cyclic guanidine moiety in an Rp or Sp configuration eg, a partial structure represented by the formula (II)).
  • the at least one, at least two, or at least three nucleoside linkages from the 5'end of the second nucleic acid strand may be modified nucleoside linkages. At least one of the 3 'end of the second nucleic acid strand, at least two, or at least three internucleoside linkages, modified linkages, for example, substituted with phosphorothioate linkages ,, 1-4 alkyl group C 1 ⁇ 6 Nucleoside-linked bond (eg, partial structure represented by formula (III)) containing the guanidine moiety (eg, TMG moiety) and / or nucleoside-linked bond containing the cyclic guanidine moiety (eg, represented by formula (II)). Partial structure).
  • the binding between modified nucleosides may be chirally controlled to the Rp or Sp configuration.
  • the second nucleic acid chain may not contain a sugar-unmodified region other than the first exposed region and / or the second exposed region (and the sugar-unmodified terminal region if present). That is, the sugar of the nucleic acid in the region other than the first exposed region and / or the second exposed region (and the sugar-unmodified terminal region if present) is a modified sugar, for example, a 2'-modified sugar.
  • bases in regions other than the first exposed region and / or the second exposed region (and the sugar-unmodified terminal region, if present) include modified and / or unmodified pyrimidine bases.
  • all of the bases are modified and / or unmodified pyrimidine bases, or some of them, such as 1-20, 1-15, 1-10, 1-5, 1-2, or One is a modified and / or unmodified pyrimidine base.
  • Modified and / or unmodified pyrimidine bases are preferably cytosine, uracil, thymine, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, N4-methylcytosine, and / or 2 -Thio-thymine, more preferably cytosine, uracil, thymine, and / or 5-methylcytosine, particularly preferably cytosine.
  • Modified and / or unmodified pyrimidine bases include cytosine, uracil, thymine, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, N4-methylcytosine, and / Or 2-thio-thymine, more preferably cytosine, uracil, thymine, and / or 5-methylcytosine, particularly preferably cytosine.
  • the base in the first exposed region comprises a purine base, eg, all of the bases are modified and / or unmodified purine bases, or some, eg, one or two bases are modified. And / or unmodified purine bases.
  • RNase A cleaves the phosphodiester bond on the 3'side of the modified and / or unmodified pyrimidine base, so that the base in the first exposed region is a modified and / or unmodified pyrimidine base. This is because this part can be cut by RNase A. Therefore, the base in the first exposed region may contain modified and / or unmodified purine bases to reduce unwanted degradation by RNase A.
  • the base in the first exposed region does not include, for example, any of the modified and / or unmodified pyrimidine bases C, UC and UU, eg, none of the three sequences.
  • the base in the first exposed region comprises a modified and / or unmodified purine base.
  • all of the bases are modified and / or unmodified purine bases, or some, such as 1-3, 1-2 or 1 are modified and / or unmodified purine bases.
  • the base in the second exposed region comprises a modified and / or unmodified purine base.
  • all of the bases are modified and / or unmodified purine bases, or some of them, such as 1-7, 1-6, 1-5, 1-4, 1-3, 1 ⁇ 2 or 1 are modified and / or unmodified purine bases.
  • the bases in the first exposed region and / or the second exposed region contain an arbitrary proportion of modified and / or unmodified purine bases.
  • modified and / or unmodified purine bases are preferably adenine, guanine, N6-methyladenine, 8-bromoadenine, N2-methylguanine and / or 8-bromoguanine, more preferably adenine and / or guanine. is there.
  • the second nucleic acid strand comprises a sugar-modified nucleoside containing a modified and / or unmodified pyrimidine base and / or a deoxyribonucleoside.
  • all of the bases are modified and / or unmodified pyrimidine bases, or some of them, such as 1-20, 1-15, 1-10, 1-5, 1-2, or One is a modified and / or unmodified pyrimidine base.
  • the second nucleic acid strand comprises a sugar-modified nucleoside containing a modified and / or unmodified pyrimidine base and / or a deoxyribonucleoside in any proportion.
  • the sugar-modified pyrimidine bases are preferably cytosine, uracil, thymine, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, N4-methylcytosine, and / or 2-thio-.
  • Thymine more preferably cytosine, uracil, thymine, and / or 5-methylcytosine, particularly preferably cytosine.
  • the defense region of the second nucleic acid strand comprises a modified and / or unmodified pyrimidine base.
  • all of the bases are modified and / or unmodified pyrimidine bases, or some of them, such as 1-20, 1-15, 1-10, 1-5, 1-2, or One is a modified and / or unmodified pyrimidine base.
  • the defense region of the second nucleic acid strand comprises a modified and / or unmodified pyrimidine base. For example, 10% or more, 30% or more, 50% or more, 70% or more, 80% or more, and 90% or more of the second nucleic acid chain are pyrimidine bases.
  • the sugar-modified pyrimidine bases are preferably cytosine, uracil, thymine, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, N4-methylcytosine, and / or 2-thio-.
  • Thymine more preferably cytosine, uracil, thymine, and / or 5-methylcytosine, particularly preferably cytosine.
  • the position of the first exposed region and / or the second exposed region in the second nucleic acid strand is not limited. In one embodiment, at least one, eg, all first and / or second exposed regions, is located 3'from the center of the second nucleic acid strand.
  • “the first exposed region and / or the second exposed region is located 3'side from the center of the second nucleic acid strand” means that the center of the first exposed region and / or the second exposed region, 5 Includes that the'end, or 3'end is located 3'side from the center of the second nucleic acid strand.
  • the first exposed region and / or the second exposed region (and the sugar-unmodified terminal region, if present) can be cleaved by RNase H. Whether or not it is cleaved by RNase H can be determined, for example, by reacting a nucleic acid chain with RNase H, performing electrophoresis, and measuring the concentration of undegraded bands, the details of which are described in Examples. That's right.
  • the fact that the first exposed region consisting of one or two or three consecutive sugar-unmodified ribonucleosides linked by an internucleoside bond can be cleaved by RNase H means that the cleavage activity by RNase H is at least four.
  • the first exposed region and / or the second exposed region may have activity in RNase H-dependent and / or independent pathways.
  • the first nucleic acid chain and / or the second nucleic acid chain may contain a cationic nucleoside in whole or in part.
  • the cationic nucleoside is a 2'-Amino-LNA nucleoside (eg, 3- (Bis (3-aminopropyl) amino) propanoyl substituted nucleoside), an aminoalkyl modified nucleoside (eg, 2'-O-methyl).
  • the second nucleic acid chain may have a functional portion bound to it.
  • the bond between the second nucleic acid chain and the functional moiety may be a direct bond or an indirect bond via another substance, but in certain embodiments, it is a covalent bond or an ionic bond.
  • the second nucleic acid chain is preferably directly bonded to the functional portion by hydrogen bonding or the like, and a covalent bond is more preferable from the viewpoint of obtaining a more stable bond.
  • Desired functions include labeling function, purification function and delivery function to the target.
  • the portion that imparts the labeling function include compounds such as fluorescent protein and luciferase.
  • the portion that imparts the purification function include compounds such as biotin, avidin, His tag peptide, GST tag peptide, and FLAG tag peptide.
  • the second nucleic acid strand functions from the viewpoint of delivering the first nucleic acid strand to the target site with high specificity and efficiency and suppressing the expression of the target gene by the nucleic acid very effectively.
  • the sex moiety it is preferable that a molecule having an activity of delivering the double-stranded nucleic acid complex in a certain embodiment to the target site is bound. Examples of parts that provide delivery function to the target include lipids, antibodies, aptamers, ligands for specific receptors, and the like.
  • the first and / or second nucleic acid strand (preferably the second nucleic acid strand) is bound to a functional moiety.
  • the first and / or second nucleic acid chains are bound to lipids.
  • Lipids include tocopherols, cholesterol, fatty acids, phospholipids and their relatives; folic acid, vitamin C, vitamin B1, vitamin B2; estradiol, androstan and their relatives; steroids and their relatives; LDLR, SRBI or LRP1 / 2 ligands; FK-506 and cyclosporin; lipids described in PCT / JP2019 / 12077, PCT / JP2019 / 10392 and PCT / JP2020 / 035117, but are not limited thereto.
  • analog refers to a compound 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. Whether or not one compound is an analog of another compound can be determined by those skilled in the art.
  • Tocopherol can be selected from the group consisting of ⁇ -tocopherol, ⁇ -tocopherol, ⁇ -tocopherol, and ⁇ -tocopherol.
  • Examples of tocopherol analogs include various unsaturated analogs of tocopherols, such as ⁇ -tocotrienols, ⁇ -tocotrienols, ⁇ -tocotrienols, and ⁇ -tocotrienols.
  • the tocopherol is ⁇ -tocopherol.
  • Cholesterol analogs refer to, but are not limited to, various cholesterol metabolites and analogs that are alcohols with a sterol skeleton, including, but not limited to, cholestanol, lanosterol, celebrosterol, dehydrocholesterol, and coprostanol. Including.
  • the lipid may be linked to the 5'end, 3'end, or both ends of the first nucleic acid chain and / or the second nucleic acid chain (preferably the second nucleic acid chain).
  • the lipid may be linked to a nucleotide inside the first nucleic acid chain and / or the second nucleic acid chain (preferably the second nucleic acid chain).
  • the first nucleic acid chain and / or the second nucleic acid chain (preferably the second nucleic acid chain) contains two or more lipids, which may be linked to multiple positions of the second nucleic acid chain, and / or the first.
  • the lipid may be linked to the 5'end and the 3'end of the first nucleic acid chain and / or the second nucleic acid chain (preferably the second nucleic acid chain) one by one (preferably at the 5'end).
  • the bond between the first nucleic acid chain and / or the second nucleic acid chain (preferably the second nucleic acid chain) and the lipid may be a direct bond or an indirect bond mediated by another substance. Good. However, in certain embodiments, the lipid is directly attached to the first and / or second nucleic acid chain (preferably the second nucleic acid chain) via covalent bonds, ionic bonds, hydrogen bonds, and the like. A covalent bond is more preferable, and a covalent bond is more preferable from the viewpoint that a more stable bond can be obtained.
  • the lipid may also be bound to the first nucleic acid chain and / or the second nucleic acid chain (preferably the second nucleic acid chain) via a cleavable linking group (linker).
  • “Cleaveable linking group (linker)” means a linking group that is cleaved under physiological conditions, for example, intracellularly or in an animal body (eg, in a human body).
  • the cleavable linker is selectively cleaved by an endogenous enzyme such as a nuclease.
  • Cleavable linkers include one or both esters of amides, esters, phosphodiesters, phosphate esters, disulfide bonds, and natural DNA linkers.
  • Non-cleavable linker means a linking group that is not cleaved under physiological conditions, eg, in cells or in animals (eg, in humans). Examples of the non-cleavable linker include a phosphorothioate bond and a linker consisting of a modified or unmodified deoxyribonucleoside linked by a phosphorothioate bond or a modified or unmodified ribonucleoside.
  • the linker is a nucleic acid such as DNA or an oligonucleotide
  • the chain length is not limited, but may be 2 to 20 bases, 3 to 10 bases, or 4 to 6 bases.
  • linker As a specific example of the linker, there is a linker represented by the following formula (I).
  • L 2 is a substituted or unsubstituted C 1 to C 12 alkylene group (eg, propylene, hexylene, dodecylene), a substituted or unsubstituted C 3 to C 8 cycloalkylene group (eg, cyclohexyl).
  • C 1 to C 12 alkylene group eg, propylene, hexylene, dodecylene
  • C 3 to C 8 cycloalkylene group eg, cyclohexyl
  • the linker represented by formula (I) is an alkylene group of C 3 to C 6 in which L 2 is not substituted (eg, propylene, hexylene),-(CH 2 ) 2- O-(. CH 2) 2 -O- (CH 2 ) 2 -O- (CH 2) 3 -, or - (CH 2) 2 -O- ( CH 2) 2 -O- (CH 2) 2 -O- (CH 2 ) 2 -O- (CH 2 ) 3- , L 3 is -NH-, and L 4 and L 5 are bonds.
  • L 2 is not substituted (eg, propylene, hexylene),-(CH 2 ) 2- O-(. CH 2) 2 -O- (CH 2 ) 2 -O- (CH 2) 3 -, or - (CH 2) 2 -O- ( CH 2) 2 -O- (CH 2) 2 -O- (CH 2 ) 2 -O- (CH 2 ) 3-
  • L 3 is -NH-
  • the binding position and type of binding in the first nucleic acid chain and / or the second nucleic acid chain (preferably the second nucleic acid chain) of the functional portion are lipid and the first nucleic acid chain and / or the second nucleic acid chain (preferably the second nucleic acid chain).
  • the binding to the nucleic acid chain) is as described above.
  • a person skilled in the art can produce a first nucleic acid chain and a second nucleic acid chain constituting a nucleic acid complex by appropriately selecting a known method.
  • each base sequence of the nucleic acid is designed based on the information of the base sequence of the target transcript (or, in some cases, the base sequence of the target gene), and a commercially available automatic nucleic acid synthesizer (Applied) is used.
  • Nucleic acids were synthesized by using products from Applied Biosystems, Inc., products from Beckman Coulter, Inc., etc.), and then the resulting oligonucleotides were reversed phase. It can be produced by purifying using a column or the like.
  • the nucleic acids produced by this method are mixed in a suitable buffer solution, denatured at about 90 ° C. to 98 ° C. for several minutes (eg 5 minutes), and then the nucleic acids are annealed at about 30 ° C. to 70 ° C. for about 1-8 hours.
  • the nucleic acid complex can be produced in this way. Preparation of annealed nucleic acid complexes is not limited to such time and temperature protocols. Suitable conditions for facilitating chain annealing are well known in the art.
  • the nucleic acid complex to which the functional moiety is bound may be produced by carrying out the above synthesis, purification and annealing using a nucleic acid species to which the functional moiety is bound in advance, or the functional moiety may be prepared. It may be bound to the nucleic acid later. Numerous methods for linking functional moieties to nucleic acids are well known in the art. Alternatively, the nucleic acid chain can be obtained by ordering from a manufacturer (for example, Gene Design Co., Ltd.) by specifying the base sequence and the modification site or type.
  • the present invention relates to a composition comprising the above nucleic acid complex for suppressing the expression of a target gene or a target transcript by an antisense effect.
  • the present composition may be a pharmaceutical composition.
  • the present composition may be for treating or preventing a disease associated with upregulation of a target gene, such as a neurological disease, a central nervous system disease, a metabolic disease, a tumor, or an infectious disease, in a subject.
  • the subject can be an animal, including a human. However, animals other than humans are not particularly limited, and various livestock, poultry, pets, laboratory animals, etc. can be subjects.
  • the present composition can be formulated by a known pharmaceutical method.
  • the composition comprises capsules, tablets, pills, liquids, powders, granules, fine granules, film coatings, pellets, lozenges, sublinguals, peptizers, buccal agents, pastes.
  • Syrups suspensions, elixirs, emulsions, coatings, ointments, plasters, cataplasms, transdermal agents, lotions, inhalants, aerosols, eye drops, injections and suppositories It can be used orally or parenterally in the form of a drug.
  • a pharmaceutically acceptable carrier or solvent or a carrier or solvent acceptable for foods and beverages can be appropriately incorporated.
  • Such carriers or solvents include, specifically, sterile water, physiological saline, vegetable oils, bases, emulsifiers, suspending agents, surfactants, pH regulators, stabilizers, flavors, fragrances, etc.
  • Excipients, vehicles, preservatives, binders, diluents, tonics, sedatives, bulking agents, disintegrants, buffers, coatings, lubricants, colorants, sweeteners, thickeners, flavors Agents, solubilizers, and other additives can be mentioned.
  • the dose of this composition can be appropriately selected according to the subject's age, body weight, symptoms and health condition, dosage form, and the like.
  • the dose of the composition is, for example, the nucleic acid complex 0.0000001 mg / kg / day to 1000000 mg / kg / day, 0.00001 mg / kg / day to 10000 mg / kg / day or 0.001 mg / kg / day to 500 mg / kg. / Day may be.
  • the composition may be a single dose or multiple doses. In the case of multiple doses, it can be administered daily or at appropriate time intervals (for example, at intervals of 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month), for example, 2 to 20 times.
  • the single dose of the above nucleic acid complex is, for example, 0.001 mg / kg or more, 0.005 mg / kg or more, 0.01 mg / kg or more, 0.025 mg / kg or more, 0.1 mg / kg or more, 0.5 mg / kg or more.
  • 1mg / kg or more 1mg / kg or more, 2.5mg / kg or more, 0.5mg / kg or more, 1.0mg / kg or more, 2.0mg / kg or more, 3.0mg / kg or more, 4.0mg / kg or more, 5mg / kg or more, 10mg / kg Above, 20mg / kg or above, 30mg / kg or above, 40mg / kg or above, 50mg / kg or above, 75mg / kg or above, 100mg / kg or above, 150mg / kg or above, 200mg / kg or above, 300mg / kg or above, 400mg / kg Or more, or 500 mg / kg or more, for example, any amount contained in the range of 0.001 mg / kg to 500 mg / kg (for example, 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, 1 mg). / kg, 5 mg / kg, 10
  • the nucleic acid complex of the present invention may be administered at a dose of 0.01 to 10 mg / kg (for example, about 6.25 mg / kg) four times at a frequency of twice a week.
  • the nucleic acid complex may be administered at a dose of 0.05 to 30 mg / kg (for example, about 25 mg / kg) 2 to 4 times at a frequency of 1 to 2 times a week, for example, twice a week.
  • toxicity can be reduced and the load on the subject can be reduced as compared with a single administration of a higher dose.
  • the nucleic acid complex of the present invention can be administered subcutaneously to reduce toxicity and load on a subject as compared to intravenous administration.
  • the nucleic acid complex of the present invention has excellent solubility in water, the Japanese Pharmacopoeia Dissolution Test Second Solution, or the Japanese Pharmacopoeia Disintegration Test Second Solution, and has pharmacokinetics (eg, blood drug half-life).
  • pharmacokinetics eg, blood drug half-life.
  • Intracerebral transferability, metabolic stability, CYP inhibition) and low toxicity eg, acute toxicity, chronic toxicity, genetic toxicity, reproductive toxicity, cardiotoxicity, drug interaction, carcinogenicity, phototoxicity, etc. Therefore, it is more excellent as a drug) and has few side effects (for example, suppression of sedation, avoidance of stratified necrosis) and other excellent properties as a drug.
  • parenteral administration there is no specific limitation on the preferable administration form of the composition.
  • oral administration or parenteral administration may be used.
  • parenteral administration include intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, tracheal / bronchial administration, rectal administration, intrathecal administration, intraventricular administration, nasal administration, and muscle.
  • parenteral administration include intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, tracheal / bronchial administration, rectal administration, intrathecal administration, intraventricular administration, nasal administration, and muscle.
  • Examples include internal administration and administration by blood transfusion. It can also be administered by intramuscular injection, intravenous drip, or implantable continuous subcutaneous administration.
  • Subcutaneous administration is suitable because it can be self-injected by the patient himself.
  • the amount of the nucleic acid complex contained in one dose of the composition is, for example, 0.001 mg / kg or more, 0.005 mg / kg or more, 0.01mg / kg or more, 0.025mg / kg or more, 0.1mg / kg or more, 0.5mg / kg or more, 1mg / kg or more, 2.5mg / kg or more, 5mg / kg or more, 10mg / kg or more, 20mg / kg or more, 30 mg / kg or more, 40 mg / kg or more, 50 mg / kg or more, 75 mg / kg or more, 100 mg / kg or more, 150 mg / kg or more, 200 mg / kg or more, 300 mg / kg or more, 400 mg / kg or more, or 500 mg / kg or more Can be.
  • any amount contained in the range of 0.001 mg / kg to 500 mg / kg (for example, 0.001 mg / kg, 0.01 mg / kg, 0.1 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 50 mg / kg). kg, 100 mg / kg, or 200 mg / kg) can be appropriately selected.
  • a method for treating and / or preventing a disease comprises administering the above nucleic acid complex or composition to a subject.
  • Example 1 Cleavage activity of a double-stranded nucleic acid complex containing a second nucleic acid chain containing a continuous trisaccharide unmodified ribonucleoside by RNase H / A
  • a double-stranded nucleic acid complex containing a first nucleic acid strand containing an antisense oligonucleotide and a complementary strand (second nucleic acid strand) containing a continuous trisaccharide unmodified ribonucleoside in which cholesterol is bound is treated with RNase A or RNase H. The cutting efficiency was examined.
  • the second nucleic acid chain and cholesterol are bound by the same 5'terminal structure as chol # 1-cRNA (DNA) (mMalat1) shown in International Publication No. 2018/056442.
  • LNA / DNA gapmer (ASO (mMalat1) 1) (first nucleic acid strand) targeting malat1 non-coding RNA was prepared.
  • This LNA / DNA gapmer is a 16-base long oligonucleotide containing 3 LNA nucleosides at the 5'end and 3 LNA nucleosides at the 3'end, and 10 DNA nucleosides in between.
  • This LNA / DNA gapmer has a base sequence complementary to positions 1317 to 1332 of mouse malat1 non-coding RNA (SEQ ID NO: 1).
  • a complementary RNA strand (Chol-cRNA (mMalat1)) (second nucleic acid strand) having a base sequence complementary to this ASO and covalently bound to cholesterol at the 5'end was prepared.
  • the second chain consists of three 2'-O-methyl-modified ribonucleosides at both ends, seven 2'-O-methyl-modified ribonucleosides between them, and three consecutive sugar-unmodified repositions.
  • a double-stranded nucleic acid agent was prepared by annealing the above ASO with the second nucleic acid strand. Specifically, these are dissolved in PBS, mixed in equal molar amounts, the solution is heated at 98 ° C. for 5 minutes, then cooled to 37 ° C. and held for 1 hour, whereby the nucleic acid chain is annealed and described above. Double-stranded nucleic acid preparations were prepared.
  • oligonucleotides used in Example 1 are shown in Table 1 and FIG. All oligonucleotides were manufactured by Gene Design Co., Ltd. (Osaka, Japan).
  • RNase A Dulbeccoline phosphate buffered saline (Ca, Mg-free) (Nacalai Tesque) was used. In the Buffer, RNase A was reacted with 0.035 U against 10 ⁇ M 5 ⁇ L of nucleic acid.
  • the enzyme was reacted by PCR at 37 ° C. for 20 minutes using a PCR device (LifeECO: thermal cycler). Then, the enzymatic reaction was stopped in liquid nitrogen.
  • FIG. 8 shows a graph showing the concentration of the undecomposed band based on the electrophoresis result in terms of the relative intensity level (%) with respect to the untreated.
  • FIG. 9 shows a graph showing the concentration of the undecomposed band in the relative intensity level (%) with respect to the untreated band after the test.
  • 3windows-1 to 8 show the result of each of Chol-cRNA 3windows-1 to 8 shown in Table 1 and the complex of ASO.
  • RNase A resistance correlates with whether or not the triple unmodified ribonucleoside contains C / U, and this result shows that RNase A is a PO bond on the 3'side of C / U, which is a pyrimidine base. Also agrees with the fact that it disconnects.
  • RNase H when there was a 3-continuous sugar-unmodified ribonucleoside on the 3'side of the second nucleic acid chain (3windows 4-8), it was easily cleaved.
  • Example 2 Gene-suppressing effect on cells
  • a double-stranded nucleic acid complex containing a first nucleic acid strand containing an antisense oligonucleotide and a cholesterol-bound complementary strand (second nucleic acid strand) was introduced into cells, and its gene-suppressing effect was examined.
  • RNAimax Thermo Fisher Scientific
  • RNAimax Thermo Fisher Scientific
  • CDNA was synthesized using RNA using PrimeScript RT Master Mix (TAKARA-Bio) according to the manufacturer's protocol.
  • Quantitative RT-PCR was performed according to a conventional method using LightCycler 480 Probes Master (Roche Life Sciences). The primers and probes used in quantitative RT-PCR are as follows.
  • Probes Master, cDNA, primer and probe were mixed and PCR was performed at 95 ° C for 10 minutes for 1 cycle, 95 ° C for 10 seconds / 60 ° C for 30 seconds / 72 ° C for 1 second for 45 cycles, and 40 ° C for 30 seconds for 1 cycle.
  • FIG. 10 shows the relative RNA expression level when the expression level of Malat1 / Actb ( ⁇ -actin) in PBS treatment is 1 when the cells are treated with various double-stranded nucleic acid complexes.
  • Chol-HDO is a double-stranded complex of ASO and Chol-cRNA (mMalat1)
  • Chol-HDO with full OMe cRNA is a double-stranded complex of ASO and Chol-cRNA (mMalat1) full OMe
  • 3 windows 1 -8 shows the results of each of Chol-cRNA 3windows-1 to 8 shown in Table 1 and the double-stranded complex of ASO.
  • the antisense effect was higher when all 10 nucleic acids in the second nucleic acid strand complementary to the gap region of the first nucleic acid strand, which is the control, were sugar-unmodified ribonucleosides (Chol-HDO).
  • the antisense effect was low when all 10 nucleic acids in the second nucleic acid strand complementary to the gap region of the nucleic acid strand were 2'-O-methyl-modified ribonucleoside (Chol-HDO with full OMe cRNA).
  • the antisense effect observed in Chol-HDO with full OMe cRNA is a cleavage-independent effect, and the difference in antisense effect between 3windows 1-2 and 3windows 3-8 is an activity that depends on RNase H cleavage. It is thought to be derived from.
  • RNase A or RNase H is a double-stranded nucleic acid complex containing a first nucleic acid strand containing an antisense oligonucleotide and a complementary strand (second nucleic acid strand) containing a 1- or continuous disaccharide unmodified ribonucleoside in which cholesterol is bound. The cutting efficiency was examined.
  • nucleic acid agent Preparation of nucleic acid agent
  • ASO and 1 or continuous disaccharide unmodified ribonucleosides were prepared based on Example 1. After dissolving these in PBS, they are mixed in equal molar amounts, the solution is heated at 98 ° C. for 5 minutes, then cooled to 37 ° C. and held for 1 hour, thereby annealing the nucleic acid strand and the above double-stranded nucleic acid.
  • the agent was prepared.
  • Fig. 13 shows the results of electrophoresis after cleavage by RNase A
  • Fig. 14 shows the concentration of undegraded bands based on the results of electrophoresis in terms of relative intensity level (%) with respect to Chol-HDO with full OMe cRNA. Shown.
  • all 10 nucleic acids in the second nucleic acid strand complementary to the gap region of the first nucleic acid strand are sugar-unmodified ribonucleosides (Chol-HDO), and the complementary strand is complemented by RNase A. It was disconnected.
  • all 10 nucleic acids in the second nucleic acid strand complementary to the gap region of the first nucleic acid strand are 2'-O-methyl-modified ribonucleosides (Chol-HDO with full OMe cRNA) by RNase A. The complementary strand was not cleaved.
  • RNase A when one sugar-unmodified ribonucleoside is contained, RNase A is cleaved when the sugar-unmodified ribonucleoside is present at the C position in the second nucleic acid chain (1). Only window-3), and when the sugar-unmodified ribonucleoside was present at the U position (1window 1, 2, 6), it was not cleaved.
  • RNase A when two consecutive sugar-unmodified ribonucleosides were contained, RNase A cleaved the sugar-unmodified ribonucleosides of CA (2window-3), UC (2window-2), or UU (2window-1). It was the case when it was in position.
  • Example 4 In vivo inhibitory effect of Dmpk mRNA expression by a double-stranded nucleic acid complex containing a second nucleic acid strand having a sugar-modified ribonucleoside at the C / U position
  • DMPK Antisense oligonucleotide ASO
  • CU OMe cholesterol-bound complementary strand Chol-cRNA
  • Default complementary strand as a control Chol-cRNA
  • mDMPK targets the mouse DMPK gene and targets its transcript DMPK mRNA (GenBank accession number NM_032418, SEQ ID NO: 46).
  • Chol-cRNA (Default) has a sequence complementary to ASO (mDMPK), and has 3 2'-O-methyl-modified ribonucleosides at both ends and 10 sugar-unmodified ribonucleosides between them. It contains a nucleoside and has cholesterol bound to its 5'end (Fig. 15A).
  • the Chol-cRNA (CUOMe) is all C and U of the 10 sugar-unmodified ribonucleosides located in the center of the Chol-cRNA (Default) except the U position located on the 3'side. Has a 2'-O-methyl modification at position (Fig. 15B).
  • ASO (mDMPK) and Chol-cRNA (Default) or ASO (mDMPK) and Chol-cRNA (CUOMe) are dissolved in PBS, mixed in equal molar amounts, and the solution is mixed at 98 ° C. The mixture was heated for 5 minutes and then cooled to 37 ° C. and held for 1 hour, whereby the nucleic acid strand was annealed to prepare a double-stranded nucleic acid complex.
  • the prepared double-stranded nucleic acid complex is referred to as "Chol-HDO (Default)” or "Chol-HDO (CUOMe)".
  • mice injected with a single dose of PBS alone were also prepared as a negative control group.
  • mice were perfused with PBS, and then the mice were dissected to dissect the myocardium (Heart), quadriceps femoris (Quadriceps), triceps femoris (Back), tibialis anterior (TA), The gastrocnemius (GC), triceps brachii (TB), kidney, and liver were removed. Subsequently, mRNA was extracted from each tissue according to the protocol using a high-throughput fully automatic nucleic acid extractor MagNA Pure 96 (Roche Life Sciences). The cDNA was synthesized according to the protocol of Transcriptor Universal cDNA Master (Roche Life Sciences).
  • Quantitative RT-PCR was performed by TaqMan (Roche Life Sciences).
  • the primers used in the quantitative RT-PCR used were products designed and manufactured by Thermo Fisher Scientific based on various gene numbers.
  • the PCR conditions (temperature and time) were 40 cycles repeated, with 1 cycle at 95 ° C. for 15 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 1 second.
  • the obtained amplification product is quantified by quantitative RT-PCR, and based on the result, the expression level of mRNA (DMPK) / mRNA (ACTB; internal standard gene) is calculated to obtain the relative expression level. It was. The mean value and standard error of the relative expression level were calculated.
  • FIGS. 16 and 17 show the gastrocnemius (GC), triceps brachii (TB), and tibialis anterior (tibialis anterior) muscles of the double-stranded nucleic acid complex Chol-HDO (Default) and Chol-HDO (CU OMe). It shows the effect of suppressing the expression of the target mDMPK gene in TA), triceps anterior (Back), quadriceps femoris (Quadriceps), and myocardium (Heart).
  • FIG. 17 shows the effect of double-stranded nucleic acid complexes Chol-HDO (Default) and Chol-HDO (CU OMe) on the expression of the target mDMPK gene in the kidney and liver. Error bars indicate each standard error.
  • Chol-HDO (CUOMe) showed a stronger suppression effect than Chol-HDO (Default) in all of the gastrocnemius muscle, triceps brachii muscle, tibialis anterior muscle, latissimus dorsi muscle, quadriceps femoris muscle, and myocardium. .. From this result, by adding sugar modification to the ribonucleoside at the C / U position of the second nucleic acid chain, resistance to cleavage by RNase A is acquired, and as a result, more efficient expression suppression of the target gene becomes possible. It has been suggested.
  • Chol-HDO (CUOMe) showed the same expression-suppressing effect as Chol-HDO (Default) in the kidney and liver.
  • Example 5 In vivo inhibitory effect of human SOD1 mRNA expression by a double-stranded nucleic acid complex containing a second nucleic acid strand having a sugar-modified ribonucleoside at the C / U position]
  • the in vivo inhibitory effect of the nucleic acid complex on the expression of mRNA in tissues will be examined.
  • Antisense oligonucleotide ASO (SOD1) targeting the SOD1 gene, cholesterol-bound complementary strand Chol-cRNA (CU OMe) with 2'-O-methyl modification at the C / U position, and complementary strand as a control Chol-cRNA (Default) was prepared.
  • SOD1 Antisense oligonucleotide ASO
  • CU OMe cholesterol-bound complementary strand Chol-cRNA
  • Default complementary strand as a control Chol-cRNA
  • the sequences of ASO (mDMPK), Chol-cRNA (Default), and Chol-cRNA (CU OMe) are shown below.
  • hSOD1 is one of 17mers having a base sequence complementary to positions 679 to 695, which targets the human SOD1 gene and targets its transcript, hSOD1 mRNA (GenBank accession number NM_000454.4). It is composed of a double-stranded LNA / DNA gapmer (SEQ ID NO: 67). More specifically, this LNA / DNA gapmer is composed of 3 LNA nucleosides from the 5'end and 3'ends, respectively, and 10 of them are composed of DNA nucleosides (Fig. 18).
  • Chol-cRNA (Default) has a sequence complementary to ASO (hSOD1), and has 3 2'-O-methyl-modified ribonucleosides at both ends and 10 sugar-unmodified ribonucleosides between them. It contains a nucleoside and has cholesterol bound to its 5'end (Fig. 18A).
  • the Chol-cRNA (CUOMe) has 2'-O-methyl modification at all C and U positions among the 10 sugar-unmodified ribonucleosides located in the center of the Chol-cRNA (Default). (Fig. 18B).
  • ASO (hSOD1) and Chol-cRNA (Default) or ASO (hSOD1) and Chol-cRNA (CUOMe) are dissolved in PBS, mixed in equal molar amounts, and the solution is mixed at 98 ° C. The mixture was heated for 5 minutes and then cooled to 37 ° C. and held for 1 hour, whereby the nucleic acid strand was annealed to prepare a double-stranded nucleic acid complex.
  • the prepared double-stranded nucleic acid complex is referred to as "Chol-HDO (Default)” or "Chol-HDO (CUOMe)".
  • mice to which the double-stranded nucleic acid complex was administered male SOD1G93A transgenic mice (G93A Tg mice) weighing 20 g and aged 4 to 5 weeks were used. 2-4 mice were used for each condition.
  • the double-stranded nucleic acid complex was subcutaneously injected into mice at a dose of 50 mg / kg in a single dose.
  • mice injected with a single dose of PBS alone were also prepared as a negative control group.
  • mice were perfused with PBS, and then the mice were dissected to remove the myocardium (Heart), quadriceps femoris (Quadriceps), diaphragm (Diaphragm), and intrinsic back muscle (Back). Subsequently, mRNA was extracted from each tissue according to the protocol using a high-throughput fully automatic nucleic acid extractor MagNA Pure 96 (Roche Life Sciences). The cDNA was synthesized according to the protocol of Transcriptor Universal cDNA Master (Roche Life Sciences). Quantitative RT-PCR was performed by TaqMan (Roche Life Sciences).
  • the primers used in the quantitative RT-PCR used were products designed and manufactured by Thermo Fisher Scientific based on various gene numbers.
  • the PCR conditions (temperature and time) were 40 cycles repeated, with 1 cycle at 95 ° C. for 15 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 1 second.
  • the obtained amplification product was quantified by quantitative RT-PCR, and based on the result, the expression level of mRNA (SOD1) / mRNA (ACTB; internal standard gene) was calculated to obtain the relative expression level. It was. The mean value and standard error of the relative expression level were calculated.
  • FIG. 19 shows the results.
  • FIG. 19 shows targets in the double-stranded nucleic acid complex Chol-HDO (Default), Chol-HDO (CU OMe) in the intrinsic back muscle (Back), quadriceps femoris (Quadriceps), diaphragm (Diaphragm) and myocardium (Heart). It shows the effect of suppressing the expression of the hSOD1 gene. Error bars indicate each standard error.
  • Chol-HDO (CUOMe) showed a stronger expression-suppressing effect than Chol-HDO (Default) in all of the intrinsic back muscle, quadriceps femoris, diaphragm, and myocardium. From this result, by adding sugar modification to the ribonucleoside at the C / U position of the second nucleic acid chain, resistance to cleavage by RNase A is acquired, and as a result, more efficient expression suppression of the target gene becomes possible. It has been suggested.
  • Example 6 In vivo inhibitory effect of mouse Malat1 RNA expression by a double-stranded nucleic acid complex containing a second nucleic acid strand having a deoxyribonucleoside at the C / U position]
  • Antisense oligonucleotide ASO (mMalat1) that targets the mouse Malat1 gene, complementary strand Chol-cRNA (CT DNA) with DNA at the C / U position to which cholesterol is bound, and complementary strand Chol-cRNA (Default) as a control. ) was prepared.
  • the sequences of ASO (mMalat1), Chol-cRNA (Default), and Chol-cRNA (CT DNA) are shown below.
  • the above ASO is one of 16mer having a base sequence complementary to positions 5032 to 5047, which targets the mouse Malat1 gene and targets its transcript, Malat1 ncRNA (GenBank accession number NR_002847.3). It is composed of a double-stranded LNA / DNA gapmer (SEQ ID NO: 49). More specifically, this LNA / DNA gapmer is composed of 3 LNA nucleosides from the 5'end and 3'ends, respectively, and 10 of them are composed of DNA nucleosides (Fig. 20).
  • Chol-cRNA (Default) has a sequence complementary to ASO (mMalat1), and has 3 2'-O-methyl-modified ribonucleosides at both ends and 10 sugar-unmodified ribonucleosides between them. It contains a nucleoside and has cholesterol bound to its 5'end (Fig. 20A).
  • the Chol-cRNA (CT DNA) has deoxyribonucleosides at all C and U positions among the 10 sugar-unmodified ribonucleosides located in the center of the Chol-cRNA (Default) (Fig. 20B).
  • ASO (mMalat1) and Chol-cRNA (Default) or ASO (mMalat1) and Chol-cRNA (CT DNA) are dissolved in PBS, mixed in equal molar amounts, and the solution is mixed at 98 ° C. The mixture was heated for 5 minutes and then cooled to 37 ° C. and held for 1 hour, whereby the nucleic acid strand was annealed to prepare a double-stranded nucleic acid complex.
  • the prepared double-stranded nucleic acid complex is referred to as "Chol-HDO (Default)” or "Chol-HDO (CT DNA)".
  • mice injected with a single dose of PBS alone were also prepared as a negative control group.
  • mice were perfused with PBS, after which the mice were dissected and the cervical spinal cord was removed. Subsequently, mRNA was extracted from each tissue according to the protocol using a high-throughput fully automatic nucleic acid extractor MagNA Pure 96 (Roche Life Sciences). The cDNA was synthesized according to the protocol of Transcriptor Universal cDNA Master (Roche Life Sciences). Quantitative RT-PCR was performed by TaqMan (Roche Life Sciences). The primers used in the quantitative RT-PCR used were products designed and manufactured by Thermo Fisher Scientific based on various gene numbers. The PCR conditions (temperature and time) were 40 cycles repeated, with 1 cycle at 95 ° C. for 15 seconds, 60 ° C.
  • RNA mMalat1 / mRNA (ACTB; internal standard gene) was calculated to obtain the relative expression level. It was. The mean value and standard error of the relative expression level were calculated.
  • FIG. 21 shows the results.
  • FIG. 21 shows the effect of double-stranded nucleic acid complexes Chol-HDO (Default) and Chol-HDO (CT DNA) on the expression of the target mMalat1 gene in the cervical spinal cord. Error bars indicate each standard error.
  • Chol-HDO (CT DNA) also showed a stronger expression-suppressing effect than Chol-HDO (Default) in the cervical spinal cord. From this result, by substituting ribonucleoside at the C / U position of the second nucleic acid chain with deoxyribonucleoside, resistance to cleavage by RNase A is acquired, and as a result, more efficient expression suppression of the target gene is possible. It was suggested that
  • Example 7 Stability of a double-stranded nucleic acid complex containing a second nucleic acid chain containing a modified ribonucleoside in mouse and human sera
  • a double-stranded nucleic acid complex containing a first nucleic acid strand containing an antisense oligonucleotide and a complementary strand (second nucleic acid strand) containing a modified ribonucleoside is mixed with mouse or human serum and double-stranded in the serum. Stability was evaluated.
  • nucleic acid agent (Preparation of nucleic acid agent) Nucleic acids similar to those in Example 1 were prepared and prepared. The sequences, chemical modifications and structures of the oligonucleotides used in Example 1 are shown in Table 7 and FIGS. 22A and 22B. All oligonucleotides were manufactured by Gene Design Co., Ltd. (Osaka, Japan).
  • Double-stranded nucleic acid 10 ⁇ M 10 ⁇ L annealed according to the above was prepared.
  • Serum was prepared from blood collected from Crl: CD1 (ICR) mice and human blood.
  • 30 ⁇ L of mouse serum or human serum was mixed with 10 ⁇ L of nucleic acid 10 ⁇ M, and incubated with a 37 ° C. incubator. After 0hr, 2hr, or 24hr incubation, Add Reaction Stop Buffer (50 mM Tris-HCl, pH7.5, 48.5 mM Borate, 20 mM EDTA, 10% SDS, final volume 60 ⁇ L) and add to liquid nitrogen for reaction. Was stopped.
  • Reaction Stop Buffer 50 mM Tris-HCl, pH7.5, 48.5 mM Borate, 20 mM EDTA, 10% SDS, final volume 60 ⁇ L
  • FIG. 23 shows the electrophoresis results after incubation with mouse serum of a double-stranded complex of ASO (mDmpk) alone, ASO (mDmpk) and cRNA (default, CU OMe, AG OMe, CU PS).
  • FIG. 25 shows the electrophoresis results after incubation of ASO (mDmpk) alone and a double-stranded complex of ASO (mDmpk) and cRNA (default, CU OMe, CT DNA) with human serum.
  • FIGS. 24 and 26 show the graphs showing the concentration of the double-stranded band after 24 hr incubation in terms of the relative intensity level (%) with respect to the double-stranded band after 0 hr incubation.
  • FIGS. 23 and 24 those in which C and U in the second nucleic acid chain were 2'-O-methyl modified ribonucleosides had improved stability in mouse serum.
  • changing the phosphodiester bond (PO) on the 3'side of ribonucleosides C and U in the second nucleic acid chain to a phosphorothioate bond (PS) also improved the stability in mouse serum.
  • FIGS. 25 and 26 those in which C and U or T in the second nucleic acid chain were 2'-O-methyl modified ribonucleoside or DNA had improved stability in human serum. ..

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