WO2022250155A1 - アンチセンス核酸 - Google Patents

アンチセンス核酸 Download PDF

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WO2022250155A1
WO2022250155A1 PCT/JP2022/021818 JP2022021818W WO2022250155A1 WO 2022250155 A1 WO2022250155 A1 WO 2022250155A1 JP 2022021818 W JP2022021818 W JP 2022021818W WO 2022250155 A1 WO2022250155 A1 WO 2022250155A1
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Prior art keywords
oligonucleotide
pharmaceutically acceptable
acceptable salt
bond
seq
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PCT/JP2022/021818
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English (en)
French (fr)
Japanese (ja)
Inventor
智秋 満
成宏 浅野
恭介 日野
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Sumitomo Pharma Co Ltd
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Sumitomo Pharmaceuticals Co Ltd
Sumitomo Pharma Co Ltd
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Priority to AU2022279820A priority Critical patent/AU2022279820A1/en
Priority to US18/564,976 priority patent/US20250084411A1/en
Priority to CA3221758A priority patent/CA3221758A1/en
Priority to JP2023524257A priority patent/JPWO2022250155A1/ja
Priority to EP22811423.7A priority patent/EP4349987A1/en
Priority to KR1020237044656A priority patent/KR20240013202A/ko
Priority to CN202280038163.1A priority patent/CN117500925A/zh
Priority to MX2023014092A priority patent/MX2023014092A/es
Publication of WO2022250155A1 publication Critical patent/WO2022250155A1/ja
Anticipated expiration legal-status Critical
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    • 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
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
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    • C12N2320/00—Applications; Uses
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    • C12N2320/33—Alteration of splicing
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    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/04—Hydrolases acting on acid anhydrides (3.6) acting on acid anhydrides; involved in cellular and subcellular movement (3.6.4)
    • C12Y306/04012—DNA helicase (3.6.4.12)

Definitions

  • the present invention relates to an antisense oligonucleotide (antisense nucleic acid) that enables skipping of the 27th exon of the WRN gene, which is the causative gene of Werner's syndrome, and a pharmaceutical composition containing the oligonucleotide.
  • Werner's syndrome is a rare disease that causes premature aging, and in adolescence, common geriatric features such as graying of the hair, baldness, diabetes, heart disease, cancer, skin atrophy, and scleroderma-like changes in the skin. , juvenile cataract, premature aging, and hypogonadism. More specifically, skin atrophy/hardening, hair changes such as gray hair and baldness, and signs of aging such as cataracts appear from the twenties to thirties. Furthermore, diabetes, arteriosclerosis, malignant tumors, and the like are often complicated, and many die in their 50s. Moreover, exacerbation of intractable skin ulcers, which frequently occur on elbows, knees, heels, etc., often leads to limb amputation, impairing the quality of life (Non-Patent Document 1).
  • Werner's syndrome is an autosomal recessive rare genetic disease, and the WRN gene encoding RecQ-type DNA / RNA helicase (WRN helicase) present on the short arm of chromosome 8 (8p12) has been identified as the causative gene ( Non-Patent Document 2, Non-Patent Document 3).
  • the WRN protein consists of 1432 amino acids and has ATP-dependent DNA/RNA helicase activity and 3' ⁇ 5' exonuclease activity. It has various physiological functions such as DNA replication, base excision repair, DNA double-strand break repair, transcription, and maintenance of telomeres, and works for gene repair and maintenance of chromosomal stability. Therefore, significant genomic instability is observed in Werner's syndrome.
  • Non-Patent Document 4 Decreased fibroblast mitotic potential is considered to be one of the factors behind the delay in wound healing of skin ulcers in Werner's syndrome (Non-Patent Document 5).
  • a nuclear localization signal (NLS: Neuclear Localization Signal) is present near the C-terminus of the WRN protein, and induces nuclear localization to perform normal physiological functions (Fig. 1).
  • NLS Neuclear Localization Signal
  • Fig. 1 A nuclear localization signal
  • a premature stop codon terminal codon
  • Figs. 1 and 2 Non-Patent Document 3
  • Werner's syndrome is an intractable disease that threatens the lives and quality of life of patients, but there is currently no effective treatment for Werner's syndrome, and the creation of new therapeutic agents is eagerly awaited.
  • nucleic acid drugs have been put into practical use as new therapeutic agents.
  • Nucleic acid drugs are composed of oligonucleic acids in which ten or more to several dozen bases of nucleic acids or modified nucleic acids are linked, and act directly on messenger RNA or untranslated RNA involved in protein translation, or act on target proteins like antibody drugs. It is known to do so and is a drug produced by chemical synthesis.
  • Splicing-controlled antisense oligonucleotides which are a type of nucleic acid medicine, inhibit the binding of splicing factors to pre-mRNA (hereinafter sometimes referred to as "pre-mRNA"), thereby exons present in the vicinity can switch the splicing of (splice out) and normalize the reading frame of frameshifted aberrant RNAs.
  • pre-mRNA pre-mRNA
  • the skipping mutation c.3139-1G>C in the 26th exon of the human WRN gene is reported to be the most frequent mutation in Werner's syndrome.
  • the above skip mutation c.3139-1G>C causes a termination codon to appear in the 27th exon, creating a WRN protein in which the amino acid sequence after the termination codon containing the nuclear localization signal present at the C-terminus is deleted, Loss of natural physiological functions.
  • the present inventors focused on splicing-controlled antisense oligonucleotides and investigated the creation of antisense oligonucleotides that induce skipping of exon 27 of the human WRN gene.
  • the present invention has been made in view of the above circumstances, and the problem to be solved by the present invention is an oligonucleotide that enables skipping of the 27th exon of the human WRN gene, and a human WRN gene containing the same.
  • An object of the present invention is to provide a drug capable of skipping the 27th exon with high efficiency.
  • exon 27 skipping can be induced with high efficiency by targeting a sequence consisting of exon 27 and surrounding nucleotides in the pre-mRNA of the human WRN gene with an antisense oligonucleotide.
  • the present inventors completed the present invention based on this finding. That is, the present invention is as follows.
  • the oligonucleotide according to the present invention is an oligonucleotide that expresses a functional human WRN protein against a skip mutation in the 26th or 28th exon of the human WRN gene, or a pharmaceutically acceptable oligonucleotide thereof.
  • each nucleotide is bound by a phosphate group and/or a modified phosphate group, the oligonucleotide comprises a modified nucleic acid having at least one modified sugar;
  • the oligonucleotide has a base length of 10 to 30 mer,
  • the base sequence of the above oligonucleotide is to at least one target region composed of the same base length as the oligonucleotide in the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6
  • a base sequence having a sequence identity of 90% or more and 100% or less based on the complementary base sequence A base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted or added in the target region, or It is an oligonucleotide or a pharmaceutically acceptable salt thereof, which is a nucleotide
  • An oligonucleotide having the above structure or a pharmaceutically acceptable salt thereof enables skipping of the 27th exon of the human WRN gene.
  • the base sequence of the oligonucleotide is to at least one target region composed of the same base length as the oligonucleotide in the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6
  • a base sequence having a sequence identity of 95% or more and 100% or less based on the complementary base sequence is preferable.
  • the base sequence of the oligonucleotide is to at least one target region composed of the same base length as the oligonucleotide in the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6
  • a complementary nucleotide sequence is preferred.
  • the base length of the oligonucleotide is preferably 15-25mer.
  • the sugar constituting the oligonucleotide is D-ribofuranose
  • the sugar modification is sugar modification of the hydroxyl group at the 2'-position of D-ribofuranose.
  • the modification of the sugar constituting the oligonucleotide is a group represented by the following formula (A1).
  • Bx is a nucleobase, each independently a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine or uracil
  • X is a phosphate bond, each independently is a phosphodiester bond, a phosphorothioate bond, a phosphorodithioate bond, a phosphoramidate bond, a boranophosphate bond or an alkylphosphonate bond
  • each Y is independently a hydrogen atom, a hydroxyl group, a fluorine atom, an optionally substituted alkoxy group having 1 to 6 carbon atoms, or an optionally substituted amino group
  • Z is each independently a hydrogen atom or a carbon-oxygen double It is an alkyl group having 1 to 5 carbon atoms which may have a bond or a
  • the modification of the sugar constituting the oligonucleotide is a group represented by the following formula (A2) or (A3).
  • Bx is a nucleobase, each independently a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine or uracil;
  • X is a phosphate bond, each independently is a phosphodiester bond, a phosphorothioate bond, a phosphorodithioate bond, a phosphoramidate bond, a boranophosphate bond or an alkylphosphonate bond, and each R4 is independently a hydrogen atom, a substituted is an optionally substituted alkyl group having 1 to 6 carbon atoms, Y' is an oxygen atom or an optionally substituted nitrogen atom, R 5 and R 6 are each independently a hydrogen atom, An optionally substituted alkyl group having 1 to 6 carbon atoms, or R 5 and R 6 together
  • Modification of the sugar constituting the oligonucleotide is a group represented by the formula (A2), and each R 4 is independently a methyl group, a methoxyethyl group, or N-methylpropanamide. It is preferably a group.
  • Modification of the sugar constituting the oligonucleotide is a group represented by the formula (A3), and Y' is an oxygen atom, a hydrogen atom, a methyl group, a methoxyethyl group, or N-methylpropane. a nitrogen atom optionally substituted with an amido group, and R 5 and R 6 are each independently a hydrogen atom, an alkyl group having 1 to 2 carbon atoms, or R 5 and R 6 together Together, they form a carbonyl group or a ring having 3 to 6 carbon atoms, and n is preferably 0 or 1.
  • At least one internucleotide bond of the oligonucleotide is preferably a phosphorothioate bond.
  • At least one internucleotide bond of the oligonucleotide is preferably a phosphodiester bond.
  • the internucleotide bond of the oligonucleotide is preferably a phosphodiester bond or a phosphorothioate bond.
  • the base sequence of the oligonucleotide is The oligonucleotide is composed of a morpholino nucleic acid represented by the following formula (A4), and in the base sequence described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 , is preferably a morpholino oligonucleic acid having a base sequence complementary to at least one target region composed of the same base length as the above oligonucleotide.
  • A4 a morpholino nucleic acid represented by the following formula (A4), and in the base sequence described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 , is preferably a morpholino oligonucleic acid having a base sequence complementary to at least one target region composed of the same base length as the above oligonucleotide.
  • Bx is a nucleobase, each independently a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine or uracil;
  • X′ is a phosphate bond; independently a phosphorodiamidate bond, a phosphorodiamidothioate bond, or a phosphorodiamidodithioate bond.
  • the base sequence of the oligonucleotide is In the nucleotide sequence of SEQ ID NO: 1, a 15 to 25 mer contiguous from the bases located at 1st to 40th, 46th, 51st, 56th, or 62nd to 63rd counted from the 5' end 108873, 108878 to 108917, 108923, 108928, 108933, or 108939 to 108940 in the target region or the nucleotide sequence set forth in SEQ ID NO: 2, counting from the 5' end
  • a base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted or added in the target region or It is preferably a nucleotide sequence that hybridizes under stringent conditions to the oligonucleotide having the target region.
  • the nucleotide sequence of the above oligonucleotide is the nucleotide sequence set forth in SEQ ID NO: 1, 1 to 32, 34 to 40, 46, 51, or 62 to 63 counted from the 5' end. 108878 to 108909, 108911 to 108917, 108923, counting from the 5′ end of the target region composed of 15 to 25 mers contiguous from the base located at the number, or the base sequence set forth in SEQ ID NO: 2, A base having a sequence identity of 90% or more and 100% or less based on the base sequence complementary to the target region composed of a 15-25mer contiguous from the bases located at 108928 or 08939-108940. An array is preferred.
  • the nucleotide sequence of the above oligonucleotide is the nucleotide sequence set forth in SEQ ID NO: 1, 1st, 3rd, 5th to 12th, 14th to 19th, 21st and 25th counted from the 5′ end.
  • the base sequences of the above oligonucleotides are SEQ ID NOs: 9-14, 16-26, 28-39, 41-42, 44-50, 52, 55-56, 69, 73, 80-105, 107-108 , 110, 112, 114, 116, 118-131, 133, and 135.
  • the above oligonucleotides have the sequence names 6-20-A, 8-20-A, 8-20-B, 8-20-C, and 9-20-A shown in Tables 2-1 to 2-7. , 10-20-A, 12-20-A, 14-20-A, 16-20-A, 17-20-A, 18-20-A, 19-20-A, 15-25-A, 10 -17-A, 10-20-B, 12-20-B, 14-20-B, 15-20-B, 16-20-B, 19-20-B, 31-20-B, 34-20 -B, 6-20-B, 7-20-B, 8-20-D, and 9-20-B.
  • the oligonucleotide is preferably a single-stranded antisense oligonucleotide.
  • the double-stranded antisense oligonucleotide according to the present invention comprises the oligonucleotide according to [19] above, A double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprising a second-stranded oligonucleotide hybridized to the single-stranded antisense oligonucleotide,
  • the base sequence of the second-strand oligonucleotide has a sequence identity of 90% or more and 100% or less based on the base sequence complementary to the base sequence of the single-stranded antisense oligonucleotide.
  • a double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof is derived from oligonucleotide according to [19] above.
  • a double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof comprising a second-stranded oligonucleotide
  • the oligonucleotide complex according to the present invention is the oligonucleotide according to any one of [1] to [19] above or a pharmaceutically acceptable salt thereof, or the two according to [20] above.
  • a strand antisense oligonucleotide or a pharmaceutically acceptable salt thereof an additional substance linked directly or via a linker bond to the oligonucleotide or the second strand oligonucleotide, or a pharmaceutically acceptable salt thereof, the additional substance is selected from the group consisting of polyethylene glycol, peptide, alkyl chain, ligand compound, antibody, protein, and sugar chain;
  • Each of the above linker bonds is independently a phosphodiester bond, a phosphorothioate bond, a phosphorodithioate bond, a phosphoramidate bond, a boranophosphate bond, an alkylphosphonate bond, a phosphorodiamidate bond, and a phosphorodiamide bond.
  • the medicament according to the present invention comprises the oligonucleotide according to any one of [1] to [19] above or a pharmaceutically acceptable salt thereof, and the double-stranded antisense oligo according to [20] above.
  • a pharmaceutical product containing, as an active ingredient, a nucleotide or a pharmaceutically acceptable salt thereof, or the oligonucleotide complex or a pharmaceutically acceptable salt thereof according to [21] above.
  • the human WRN gene exon 27 skipping agent according to the present invention includes the oligonucleotide according to any one of [1] to [19] above or a pharmaceutically acceptable salt thereof, ] or a pharmaceutically acceptable salt thereof, or the oligonucleotide complex or a pharmaceutically acceptable salt thereof according to [21] above as an active ingredient, It is a skipping agent for the 27th exon of the human WRN gene.
  • the functional WRN restorer according to the present invention comprises the oligonucleotide according to any one of [1] to [19] above or a pharmaceutically acceptable salt thereof, and the two oligonucleotides according to [20] above.
  • the 27th human WRN gene comprising, as an active ingredient, a strand antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the oligonucleotide complex according to [21] above or a pharmaceutically acceptable salt thereof. It is a functional WRN restorer that carries a nuclear localization signal by skipping the third exon.
  • the therapeutic agent for Werner's syndrome according to the present invention includes the oligonucleotide according to any one of [1] to [19] or a pharmaceutically acceptable salt thereof, and the two according to [20] above.
  • a therapeutic agent for Werner's syndrome comprising, as an active ingredient, a strand antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the oligonucleotide complex or a pharmaceutically acceptable salt thereof according to [21] above. be.
  • the prophylactic agent against Werner's syndrome according to the present invention includes the oligonucleotide according to any one of [1] to [19] or a pharmaceutically acceptable salt thereof, and the two according to [20] above.
  • a prophylactic agent against Werner's syndrome comprising, as an active ingredient, a strand antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the oligonucleotide complex or a pharmaceutically acceptable salt thereof according to [21] above. be.
  • the method for treating or preventing Werner's syndrome according to the present invention includes the oligonucleotide according to any one of [1] to [19] or a pharmaceutically acceptable salt thereof, and the oligonucleotide according to [20] above. or a pharmaceutically acceptable salt thereof, or the oligonucleotide complex or pharmaceutically acceptable salt thereof according to [21] above to an individual, It is a method for treating or preventing Werner's syndrome.
  • the present invention provides the oligonucleotide according to any one of [1] to [19] above, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of Werner's syndrome, [20] above. or a pharmaceutically acceptable salt thereof, or the oligonucleotide complex of [21] above or a pharmaceutically acceptable salt thereof.
  • the present invention provides the oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to [19] above, which is used for producing a therapeutic or preventive agent for Werner's syndrome, Providing the double-stranded antisense oligonucleotide according to [20] above or a pharmaceutically acceptable salt thereof, or the oligonucleotide complex according to [21] above or a pharmaceutically acceptable salt thereof .
  • FIG. 1 is a schematic diagram showing the structure of the human WRN gene product.
  • FIG. 2 is a schematic diagram illustrating the onset mechanism of Werner's syndrome with the 26th exon skipping mutation c.3139-1G>C.
  • FIG. 3 is a schematic diagram illustrating the exon 27 skipping mechanism using the single-stranded antisense oligonucleotide according to this embodiment.
  • FIG. 4 is a schematic diagram showing the relative positions in exon 27 of antisense oligonucleotides designed to induce WRN exon 27 skipping.
  • FIG. 5 is an electrophoresis photograph showing the skipping efficiency of exon 27 of the WRN gene in HEK293T.
  • FIG. 1 is a schematic diagram showing the structure of the human WRN gene product.
  • FIG. 2 is a schematic diagram illustrating the onset mechanism of Werner's syndrome with the 26th exon skipping mutation c.3139-1G>C.
  • FIG. 3 is a schematic diagram illustrating the ex
  • FIG. 6 is an electrophoresis photograph showing the skipping activity of exon 27 of the WRN gene in Werner's syndrome patient fibroblasts with the 26th exon skipping mutation c.3139-1G>C.
  • FIG. 7 is a fluorescence microscopy image showing the skipping activity of exon 27 of the WRN gene in Werner's syndrome patient fibroblasts having the 26th exon skipping mutation c.3139-1G>C.
  • the oligonucleotide of this embodiment is An oligonucleotide or a pharmaceutically acceptable salt thereof that expresses a functional human WRN protein for a skipping mutation in exon 26 or exon 28 of the human WRN gene,
  • each nucleotide is bound by a phosphate group and/or a modified phosphate group, the oligonucleotide comprises a modified nucleic acid having at least one modified sugar;
  • the oligonucleotide has a base length of 10 to 30 mer,
  • the base sequence of the above oligonucleotide is to at least one target region composed of the same base length as the oligonucleotide in the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6
  • an oligonucleotide having the above structure or a pharmaceutically acceptable salt thereof enables skipping of the 27th exon of the human WRN gene.
  • the oligonucleotide or a pharmaceutically acceptable salt thereof is understood as "an oligonucleotide that skips exon 27 of the human WRN gene or a pharmaceutically acceptable salt thereof.”
  • You can also The present invention relates to exon 27 skipping of pre-mRNA with shifted amino acid reading frame caused by splicing abnormality caused by skipping mutation c.3139-1G>C of exon 26 of human WRN gene. Werner's syndrome symptoms, including intractable skin ulcers, can be improved by inducing and eliminating misalignment of the reading frame.
  • the oligonucleotides according to the present embodiment may be referred to as "antisense oligonucleotides", “single-stranded antisense oligonucleotides” and the like, but are not intended to be limited to these.
  • single-stranded antisense oligonucleotide or “antisense oligonucleotide” (hereinafter sometimes referred to as "ASO") means target gene mRNA, pre-mRNA, or ncRNA (non-co- It means an oligonucleotide or a pharmacologically acceptable salt thereof that is complementary to the target RNA) (hereinafter, these three may be collectively referred to as "target RNA”).
  • ASO target gene mRNA, pre-mRNA, or ncRNA (non-co- It means an oligonucleotide or a pharmacologically acceptable salt thereof that is complementary to the target RNA) (hereinafter, these three may be collectively referred to as "target RNA”).
  • Antisense oligonucleotides are composed of DNA, RNA and/or analogues thereof.
  • Antisense oligonucleotides suppress the action of target mRNA, pre-mRNA or ncRNA by forming a double strand with target mRNA, pre-mRNA or ncRNA.
  • the antisense oligonucleotide has a base sequence that is completely complementary to the target mRNA, pre-mRNA, or ncRNA base sequence, and has one or several bases in the complementary base sequence. are deleted, substituted, inserted or added, and those containing bases forming fluctuating base pairs in the base sequence.
  • the antisense oligonucleotides of the present invention may further contain modified nucleotides known in the art, other than the "modified nucleic acids in which the sugar moiety is a modified sugar" (modified nucleotides with sugar modification) described later.
  • Modified nucleotides known in the art include, in addition to sugar-modified modified nucleotides, for example, phosphate group-modified modified nucleotides and nucleobase-modified modified nucleotides described later.
  • both ends of the antisense oligonucleotide in this embodiment is not particularly limited, and for example, it may be -OH or -OR (where R is an alkyl chain, a phosphate, or It may be an additional substance described later.).
  • the single-stranded antisense oligonucleotide in this embodiment may be in a single-stranded form, or may be hybridized with a second-stranded oligonucleotide described later to take a double-stranded form.
  • a double-stranded oligonucleotide consisting of the single-stranded antisense oligonucleotide and a second-stranded oligonucleotide hybridized to the single-stranded antisense oligonucleotide is referred to as a "double-stranded antisense oligonucleotide.” It is sometimes called
  • oligonucleotide means a nucleotide polymer in which 2 to 30 identical or different nucleotides are linked by phosphodiester bonds or other bonds.
  • the above oligonucleotide can also be understood to be composed of a nucleobase portion, a phosphate portion, and a sugar portion or a morpholino ring portion as shown in the structural formula below.
  • oligonucleotides are roughly classified into natural oligonucleotides and non-natural oligonucleotides.
  • naturally occurring oligonucleotide is meant an oligonucleotide composed of naturally occurring nucleotides.
  • non-naturally occurring oligonucleotide means an oligonucleotide containing at least one modified nucleotide described below as a structural unit.
  • the "unnatural oligonucleotide” preferably includes a modified sugar derivative in which the sugar moiety is modified; a phosphorothioate derivative in which one non-bridging oxygen atom of the phosphodiester bond is replaced with a sulfur atom; phosphorodithioate derivatives in which two non-bridging oxygen atoms are replaced by sulfur atoms; ester derivatives in which the phosphodiester bond is triesterified; phosphoamide derivatives in which the phosphodiester bond is amidated; Acid-esterified boranophosphate derivatives; Alkyl phosphonate (e.g., methyl phosphonate, methoxypropyl phosphonate, etc.) derivatives in which the non-bridging oxygen atom of the phosphodiester bond is substituted with an alkyl group; Phosphodiester bond is substituted with an amide bond modified amide derivatives: Modified base derivatives in which the nucleobase is modified.
  • a phosphorothioate derivative in which
  • the non-natural oligonucleotide is a crosslinked modified sugar derivative in which the sugar moiety is modified; esterified ester derivative; Alkylphosphonate derivatives in which non-bridging oxygen atoms of acid diester bonds are substituted with alkyl groups are included.
  • nucleoside means a compound in which a purine base or a pyrimidine base and a sugar are linked. Nucleosides occurring in nature are sometimes referred to as “natural nucleosides”. Non-naturally occurring modified nucleosides are sometimes referred to as “modified nucleosides.” In particular, modified nucleosides in which sugar moieties have been modified are sometimes referred to as “modified sugar nucleosides”.
  • nucleotide means a compound in which a phosphate group is bound to the sugar of the above nucleoside.
  • Naturally occurring nucleotides are sometimes referred to as “natural nucleotides”.
  • Non-naturally occurring modified nucleotides are sometimes referred to as “modified nucleotides” or “modified nucleic acids.”
  • modified nucleotides or “modified nucleic acids” include compounds in which a phosphate group is bound to the sugar moiety of the above-mentioned modified nucleosides, and compounds in which a modified phosphate group described later is bound to the sugar moiety of natural nucleosides.
  • sugar modification means modification of the sugar portion of the above nucleotide.
  • a modified sugar moiety is sometimes specifically referred to as a "modified sugar”.
  • Modified nucleotides with sugar modification can be used as modified nucleic acids, for example, 2'-O-alkylated nucleic acids, 2'-F nucleic acids, 5'-methylated nucleic acids, 2',4'-BNA (Bridged Nucleic Acid, hereinafter sometimes referred to as "LNA"), AmNA (Amido-bridged nucleic acid), GuNA (Guanidino-bridged nucleic acid), scpBNA (2 '-O, 4'-C-Spirocyclopropylene bridged nucleic acid), ENA (2'-O, 4'-C-Ethylene-Bridged Nucleic Acid), S-cEt (2', 4'-constrained Ethyl Nucleic Acid), etc.
  • Examples of 2'-O-alkylated nucleic acids include the symbols “A(M)”, “A(m)”, “C(M)”, “5(m)”, and “G(M)” described later. , “G(m)”, “U(M)”, and “T(m)”.
  • LNAs include, for example, structures represented by symbols “A(L)”, “5(L)”, “G(L)”, and “T(L)”, which will be described later.
  • AmNA includes, for example, structures represented by symbols “A(Y)”, “5(Y)", “G(Y)", and “T(Y)” described later.
  • GuNA includes, for example, structures represented by symbols "A(Gx)”, “5(Gx)”, “G(Gx)”, and “T(Gx)” described later.
  • Examples of scpBNA include those containing structures represented by symbols “A(S)”, “5(S)”, “G(S)”, and “T(S)” described later.
  • the modification of the sugar constituting the oligonucleotide is preferably a group represented by the following formula (A1). In another aspect of this embodiment, it can also be understood that the modified sugar constituting the oligonucleotide is represented by the following formula (A1).
  • Bx is a nucleobase, each independently a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine or uracil
  • X is a phosphate bond
  • each independently is a phosphodiester bond, a phosphorothioate bond, a phosphorodithioate bond, a phosphoramidate bond, a boranophosphate bond or an alkylphosphonate bond
  • each Y is independently a hydrogen atom, a hydroxyl group, a fluorine atom, an optionally substituted alkoxy group having 1 to 6 carbon atoms, or an optionally substituted amino group
  • Z is each independently a hydrogen atom or a carbon-oxygen double It is an alkyl group having 1 to 5 carbon atoms which may have a bond or a cyclic structure, or some carbon atoms of the above alkyl group and Y together form a ring.
  • the "optionally substituted alkoxy group having 1 to 6 carbon atoms” includes, for example, an unsubstituted alkoxy group having 1 to 6 carbon atoms, a hydroxyl group, a fluorine atom, and a straight chain having 1 to 6 carbon atoms. or from the group consisting of a branched alkoxy group, a cyclic alkoxy group having 3 to 8 carbon atoms, a carbamoyl group, and an alkylamide group having 1 to 8 carbon atoms (a group represented by —(CO)—NR 7 R 8 )
  • R 7 and R 8 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 8 carbon atoms.
  • the "optionally substituted amino group” includes, for example, an unsubstituted amino group, and a linear or branched alkyl group having 1 to 6 carbon atoms (the alkyl group has 1 to 6 carbon atoms or a group represented by —(CO)—NR 9 R 10 ), a cyclic alkyl group having 3 to 8 carbon atoms, a cyclic alkyl group having 1 to 6 carbon atoms, Examples include an acyl group and an amino group substituted with a substituent selected from the group consisting of an amidine group substituted with a linear or branched alkyl group having 1 to 6 carbon atoms at 1 to 3 positions.
  • R 9 and R 10 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms.
  • alkyl group having 1 to 5 carbon atoms which may have a carbon-oxygen double bond or a cyclic structure include, for example, an unsubstituted alkyl group having 1 to 5 carbon atoms, a carbon-oxygen double bond, and an alkyl group having 1 to 5 carbon atoms and a cycloalkyl group having 3 to 5 carbon atoms (such as a cyclopropyl group).
  • the number of carbon atoms in the "alkyl group having 1 to 5 carbon atoms and having a carbon-oxygen double bond” may include the carbon atoms forming the carbon-oxygen double bond.
  • the "alkyl group having 1 to 5 carbon atoms and having a carbon-oxygen double bond" may include an aldehyde group.
  • Z is an aldehyde group and Y is an optionally substituted amino group
  • the ring formed together by Y and Z may have a structure containing an amide bond.
  • the modification of the sugar constituting the oligonucleotide is preferably a group represented by the following formula (A2) or (A3).
  • the modified sugar constituting the oligonucleotide is represented by the following formula (A2) or formula (A3).
  • Bx is a nucleobase, each independently a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine or uracil
  • X is a phosphate bond
  • each independently is a phosphodiester bond, a phosphorothioate bond, a phosphorodithioate bond, a phosphoramidate bond, a boranophosphate bond or an alkylphosphonate bond
  • each R4 is independently a hydrogen atom, substituted an alkyl group having 1 to 6 carbon atoms which may be substituted
  • Y′ is an oxygen atom or an optionally substituted nitrogen atom
  • R 5 and R 6 are each independently a hydrogen atom, a an alkyl group having 1 to 6 carbon atoms which may be substituted, or R 5 and R 6 together form a carbonyl group or a ring
  • n is 0 or 1.
  • the modification of the sugar constituting the oligonucleotide is a group represented by the formula (A2), and the R 4 is each independently a methyl group, a methoxyethyl group, or N-methylpropanamide group (--CH 2 CH 2 --CONH--CH 3 , methyliminocarbonylethyl group).
  • the modification of the sugar constituting the oligonucleotide is a group represented by the formula (A3), and Y' is an oxygen atom, a hydrogen atom, a methyl group, or a methoxyethyl group. , or a nitrogen atom optionally substituted with an N-methylpropanamide group, and R 5 and R 6 are each independently a hydrogen atom, an alkyl group having 1 to 2 carbon atoms, or R 5 and R 6 together form a carbonyl group or a ring having 3 to 6 carbon atoms, and n is preferably 0 or 1.
  • nucleotide modifications known in the art other than sugar modifications can be used as modified nucleic acids for producing the single-stranded antisense oligonucleotides of the present invention.
  • Phosphate group modification and nucleobase modification which will be described later, are known as modifications of nucleotides.
  • Such nucleotide modifications are described, for example, in J. Am. Med. Chem. (2016) 59:9645-9667. (Non-Patent Document 5) and the like. Modification of these nucleotides can be performed based on methods known in the art described in the documents cited above.
  • the “phosphate group” means a naturally occurring phosphodiester bond (a bond indicated by the symbol “ ⁇ ” described below) in which the phosphate moiety of the nucleotide is bonded.
  • phosphate group modification means that the phosphate moiety of the above nucleotide is modified.
  • a modified phosphate moiety may be particularly referred to as a "modified phosphate group".
  • binding modes containing the modified phosphate group include phosphorothioate bonds (bonds represented by the symbol “ ⁇ ” described later), phosphorodithioate bonds, phosphoramidate bonds, or boranophosphate bonds, alkyl A phosphonate bond, a phosphorodiamidate bond (a bond indicated by the symbol "*” described later), a phosphorodiamide thioate bond, a phosphorodiamide dithioate bond, and the like can be mentioned.
  • nucleobase modification means that the nucleobase portion of the above nucleotide is modified.
  • a modified nucleobase portion may be specifically referred to as a "modified nucleobase”.
  • Modified nucleobases include, for example, 5-methylcytosine, 5-hydroxymethylcytosine, 5-propynylcytosine and the like.
  • the analogue of DNA or RNA mentioned above means a molecule having a structure similar to that of DNA or RNA. Examples thereof include peptide nucleic acid (pNA), morpholino nucleic acid and the like.
  • the nucleic acid used in the present invention is not limited to modification of the sugar portion of the nucleic acid, and morpholino nucleic acid and peptide nucleic acid may be used. That is, as another aspect of the single-stranded antisense oligonucleotide of the present invention, in the sequence described in the following column ⁇ Base sequence of single-stranded antisense oligonucleotide>, the oligonucleotide is DNA or RNA.
  • the DNA or RNA analogs include at least peptide nucleic acids and morpholino nucleic acids. Synthesis of the antisense oligonucleotides of the present invention, including peptide nucleic acids and morpholino nucleic acids, is carried out according to standard methods. Morpholino oligonucleic acids are described, for example, in International Publication No. 1991/009033 (Patent Document 4), International Publication No. 2009/064471 (Patent Document 5), J. Am. Am. Chem. Soc (2016) 138:15663-15672 (Non-Patent Document 8). Morpholino nucleic acids include, for example, those containing structures represented by the symbols "A(N)", “C(N)", “G(N)”, and "T(N)” described below.
  • the oligonucleotide is preferably a morpholino oligonucleic acid composed of a morpholino nucleic acid represented by the following formula (A4).
  • Bx is a nucleobase, each independently a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine or uracil
  • X' is a phosphate bond, each independently phosphorodiamidate linkage, phosphorodiamidothioate linkage, or phosphorodiamidodithioate linkage.
  • ncRNA is a generic term for RNAs that are not involved in protein translation.
  • examples of the ncRNA include ribosomal RNA, transfer RNA, miRNA, Natural Antisense Transcript (NAT) and the like.
  • the nucleobase portion of the above oligonucleotide includes thyminyl group, cytosinyl group, adenynyl group, guanynyl group, 5-methylcytosinyl group, uracilyl group, 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidine-1.
  • the nucleobase moiety includes thyminyl group, cytosinyl group, adenynyl group, guanynyl group, 5-methylcytosinyl group, and uracilyl group.
  • uracil (U) and thymine (T) are interchangeable. Both uracil (U) and thymine (T) can base pair with adenine (A) on the complementary strand. The same applies to the nucleobase portion of antisense oligonucleotides.
  • target RNA refers to RNA to which the above single-stranded antisense oligonucleotide binds.
  • the target RNA in this embodiment means the pre-mRNA of the WRN gene.
  • the target RNA include the pre-mRNA of the human WRN gene having the nucleotide sequence set forth in SEQ ID NO: 2, and the exon 26 skipping mutation of the human WRN gene having the nucleotide sequence set forth in any one of SEQ ID NOS: 3-5. and an mRNA precursor having exon 28 skipping mutation of the human WRN gene having the nucleotide sequence set forth in SEQ ID NO:6.
  • binding to target RNA means that the nucleobase of the single-stranded antisense oligonucleotide forms a double-stranded nucleic acid with the nucleobase of the target RNA due to complementarity with the target RNA.
  • the double-stranded nucleic acid may be formed in at least part of the target RNA.
  • the strength of binding to the target RNA can be measured, for example, by a thermostability index.
  • the thermostability index include the melting temperature (Tm value) of the double-stranded nucleic acid.
  • the Tm value is preferably 40 to 90°C, more preferably 50 to 70°C.
  • the target region means a region in the pre-mRNA of the WRN gene that binds to the single-stranded antisense oligonucleotide.
  • the target region includes a target region consisting of the indicated base sequence and a region on the WRN pre-mRNA.
  • the pre-mRNA means a primary transcript of RNA transcribed from DNA. That is, the pre-mRNA is RNA containing an exon region, an intron region and an untranslated region (UTR).
  • the pre-mRNA can also be understood as RNA before post-transcriptional splicing. When the above-mentioned pre-mRNA is spliced, it becomes mRNA.
  • Binding to the target region means that the single-stranded antisense oligonucleotide of the present invention forms a double strand with the target region.
  • the single-stranded antisense oligonucleotide of the present invention does not necessarily need to form a double strand with the entire target region, and may form a double strand with a partial region of the target region. That is, the single-stranded antisense oligonucleotides of the present invention preferably have complete complementarity with the target region, but as long as they bind to the target region of WRN, they have at least a portion of the target region. It is sufficient if it is complementary to the region of
  • the part of the target region means a 10 to 15 nucleotide base length region of the target region.
  • Complementary to at least part of the target region means to be complementary to the bases of at least part of the target region on the target RNA.
  • it also includes being complementary to the bases of the region on the pre-mRNA corresponding to at least a part of the region.
  • the present inventors focused on splicing-regulated antisense oligonucleotides and investigated the creation of antisense oligonucleotides that induce skipping of exon 27 of the human WRN gene.
  • a single-stranded antisense oligonucleotide is a compound that enables the abnormal reading frame to be restored to an abnormal RNA due to frameshift or the like, and allows the target region, the exon, to be skipped. Therefore, the structure of the antisense oligonucleotide is designed so that it can bind to the target region of the target RNA, and in addition, a sugar moiety-modified nucleic acid is arranged so as not to be recognized by nucleases such as RNaseH.
  • the base sequence of the single-stranded antisense oligonucleotide according to this embodiment is (A) In the nucleotide sequence of SEQ ID NO: 1, a 10 to 30 mer contiguous from the bases located at 1st to 40th, 46th, 51st, 56th, or 62nd to 63rd counted from the 5' end (preferably 15 to 25 mer), or in the base sequence described in SEQ ID NO: 2, 108873, 108878 to 108917, 108923, 108928, 108933, or A base sequence having a sequence identity of 90% or more and 100% or less based on a base sequence complementary to a target region composed of 15 to 25 mers contiguous from bases located at positions 108939 to 108940, (B) a nucleotide sequence complementary to a nucleotide sequence in which one or several nucleotides have been deleted, substituted,
  • each base sequence shown in the sequence listing is used to show only the sequence information of the nucleobase portion.
  • Structural information of oligonucleotides including sugar moieties and phosphate moieties in addition to the above nucleobase moieties shall be shown in the format shown in Tables 2-1 to 2-7 below.
  • sequence identity refers to the optimal alignment when two base sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm is introduction of gaps in one or both of the sequences)) means the ratio (%) of identical bases to all overlapping base sequences.
  • sequence identity of base sequences can be easily confirmed by those skilled in the art. For example, NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) can be used.
  • the base sequence of the single-stranded antisense oligonucleotide according to this embodiment has a sequence identity of 95% or more and 100% or less with the base sequence complementary to the above-mentioned predetermined target region in the base sequence shown in SEQ ID NO: 1. more preferably 98% or more and 100% or less sequence identity, and even more preferably 100% sequence identity.
  • nucleotide sequence in which one or several bases are deleted, substituted, inserted or added includes, for example, deleted, substituted, inserted or added by deletion, substitution, insertion or addition
  • a base sequence having a sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more with respect to the base sequence of the previous sequence can be mentioned.
  • the specific number of "one or several bases” includes the above-mentioned deletion, substitution, insertion or addition independently at 1, 2, 3, 4 or 5 positions. You can do it, or you can have a combination of multiple things.
  • stringent conditions are 6 ⁇ SSC (composition of 1 ⁇ SSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 0.5% SDS and 5 ⁇ Incubate at room temperature for 12 hours in a solution containing Denhardt, 100 ⁇ g/mL denatured salmon sperm DNA and 50% (v/v) formamide, and then wash with 0.5 ⁇ SSC at a temperature of 50° C. or higher.
  • more stringent conditions for example, incubation at 45° C. or 60° C. for 12 hours, washing with 0.2 ⁇ SSC or 0.1 ⁇ SSC, temperature conditions of 60° C. or 65° C. or higher during washing It also includes more stringent conditions such as washing with
  • the base sequence of the single-stranded antisense oligonucleotide is 1 to 40, 46, 51, and 56 counted from the 5' end in the base sequence set forth in SEQ ID NO: 1. 108873, 108878 to 108917 counting from the 5′ end in the target region composed of 15 to 25 mers contiguous from the bases located at positions 62 to 63, or the nucleotide sequence set forth in SEQ ID NO: 2 , 108923, 108928, 108933, or 108939-108940.
  • the base sequence of the single-stranded antisense oligonucleotide is 1 to 32, 34 to 40, and 46 counted from the 5' end in the base sequence set forth in SEQ ID NO: 1.
  • 108878 to 108909 counting from the 5′ end in the target region composed of 15 to 25 mers contiguous from the bases located at positions 51, 62 to 63, or the nucleotide sequence set forth in SEQ ID NO: 2 , 108911 to 108917, 108923, 108928, or 108939 to 108940, and is more preferably complementary to the target region.
  • the base sequence of the single-stranded antisense oligonucleotide is 1, 3, 5 to 12, and 14 counted from the 5′ end in the base sequence set forth in SEQ ID NO: 1. No. 19, 21, 25, 29, 30, 31, 34, 46, or a target region composed of a 15-25 mer contiguous from the bases located at 51, or SEQ ID NO: 2 108878, 108880, 108882 to 108889, 108891 to 108896, 108898, 108902, 108906 to 108908, 108911, 108923, or More preferably, it is a nucleotide sequence complementary to the target region, which consists of 15 to 25 mers contiguous from the base located at position 108928.
  • binding of the single-stranded antisense oligonucleotide of the present invention to the target region of the WRN gene means direct binding of the single-stranded antisense oligonucleotide of the present invention to the WRN pre-mRNA. contain.
  • One aspect of the single-stranded antisense oligonucleotide of the present invention is a single-stranded antisense oligonucleotide capable of skipping exon 27 of the WRN gene, which is any of those listed in Tables 1-1 to 1-4.
  • the single-stranded oligonucleotide is complementary to the target region in the pre-mRNA of the human WRN gene. If the single-stranded antisense oligonucleotide contains the nucleotide sequences listed in Tables 1-1 to 1-4, it extends 1 to 5 bases on the 3' side and/or 5' side.
  • the above-mentioned target region can be said to be a region (for example, a region having a secondary structure of mRNA to which antisense nucleotides are likely to bind) particularly related to expression regulation of the human WRN gene in the human WRN pre-mRNA.
  • the symbols “A′”, “C′”, “G′” and “T′” are natural nucleosides (a, c, g, and t) or modified nucleosides (including modified sugar nucleosides).
  • the symbol “A′” is selected from A(M) or A(m) described below, and the symbol “C′” is selected from C (M) or 5 (m), the symbol “G'” is selected from G (M) or G (m) described later, and the symbol “T'” is U (M) described later , or T(m).
  • the symbol “A'” is selected from A(L), A(Y), A(Gx), or A(S) described below, and the symbol “C'” is 5 (x), 5(L), 5(Y), 5(Gx), or 5(S), and the symbol “G'” is G(L), G(Y), G(Gx ), or G(S), and the symbol “T′” is selected from T(L), T(Y), T(Gx), or T(S), which will be described later.
  • the symbol “A′” is A (N) described later
  • the symbol “C′” is C (N) described later
  • the symbol “G′” is G (N) described later.
  • the symbol “T'” is T(N), which will be described later.
  • a single-stranded antisense oligonucleotide according to this embodiment may be in the form of a pharmacologically acceptable salt.
  • pharmaceutically acceptable salt means a salt of the single-stranded antisense oligonucleotide of the present invention, which is a physiologically acceptable salt of the single-stranded antisense oligonucleotide of the present invention, That is, salts that retain the desired biological activity of the single-stranded antisense oligonucleotide and do not retain undesired toxicological effects.
  • double-stranded antisense oligonucleotides and antisense oligonucleotide complexes which will be described later.
  • the single-stranded antisense oligonucleotides may be in the form of pharmaceutically acceptable salts.
  • “Pharmaceutically acceptable salt” as used herein means the pharmacologically acceptable salt described above and which is either an acid addition salt or a base addition salt.
  • Acid addition salts include, for example, inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate and phosphate, as well as citrate, oxalate and phthalate.
  • base addition salts include inorganic base salts such as sodium salt, potassium salt, calcium salt, magnesium salt, barium salt and aluminum salt, trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine and ethanol.
  • organic base salts such as amine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, dicyclohexylamine and N,N-dibenzylethylamine; Furthermore, salts (amino acid salts) with basic amino acids or acidic amino acids such as arginine, lysine, ornithine, aspartic acid or glutamic acid are included. The same applies to double-stranded antisense oligonucleotides and antisense oligonucleotide complexes, which will be described later.
  • the single-stranded antisense oligonucleotides according to this embodiment are composed of natural oligonucleotides and/or non-natural oligonucleotides.
  • the single-stranded antisense oligonucleotides are preferably in single-stranded form.
  • the single-stranded antisense oligonucleotide may hybridize with a second-stranded oligonucleotide described below to form a double-stranded form (double-stranded antisense oligonucleotide).
  • the base sequence of the second-strand oligonucleotide has a sequence identity of 90% or more and 100% or less based on the base sequence complementary to the base sequence of the single-stranded antisense oligonucleotide. is preferred.
  • Exon skipping can be induced by binding to either or both the 5' and 3' splice sites of an exon, or a single-stranded antisense oligonucleotide targeting the interior of an exon. . That is, the single-stranded antisense oligonucleotide binds to the target region of the target RNA and inhibits splicing of the exon, making it possible to skip the target exon and functioning based on the expressed mRNA. induces protein translation.
  • the single-stranded antisense oligonucleotide skips the 27th exon of the human WRN gene with high efficiency by the mechanism described above (regulating the maturation of the pre-mRNA of WRN). It can be suitably used for this purpose, including the case where it acts via More specifically, the single-stranded antisense oligonucleotide binds to exon 27, which is the target region of the target RNA (mRNA precursor of WRN gene) (upper part of FIG. 3), and inhibits exon 27 splicing.
  • the termination codon in exon 27 that appeared due to the exon 26 skipping mutation seen in Werner's syndrome patients was skipped, and the WRN mRNA in which exons 25 and 28 were linked (corrected to the reading frame of the WRN gene in healthy subjects: in-frame ) is generated (FIG. 3 middle), and a functional WRN protein having a nuclear localization signal contained in exon 34 is translated based on the mRNA.
  • the single-stranded antisense oligonucleotide regulates functional WRN gene expression even in transdermal, intravenous, and intradermal administration, which are administration routes commonly used in clinical applications. effect can be exhibited.
  • highly efficient skipping of the 27th exon of the human WRN gene means at least skipping of the 27th exon of the human WRN gene with high efficiency, and as a result, a functional WRN It means at least to restore expression of the protein.
  • natural nucleotides whose sugar moiety is deoxyribose include deoxyadenosine monophosphate, deoxyguanosine monophosphate, thymidine monophosphate, deoxycytidine monophosphate, deoxy-5-methylcytidine monophosphate, and the like.
  • natural nucleotides constituting the single-stranded antisense oligonucleotides include those having structural formulas corresponding to the symbols a, g, t, c and 5(x) described later.
  • non-natural nucleotides whose sugar moiety is deoxyribose include 2-thio-thymidine-phosphate, 2-aminoadenosine-phosphate, 7-deazaguanosine-phosphate and the like.
  • the base length of the single-stranded antisense oligonucleotide of the present invention is 10 to 30 mer, preferably 15 to 25 mer, more preferably 18 to 22 mer, still more preferably 16 to 20 mer, particularly preferably 18-20 mers.
  • the base length of the single-stranded antisense oligonucleotide of the present invention is 15 to 25 mer, 18 to 22 mer, 16 to 20 mer, or 18 to 20 mer
  • the binding to the pre-mRNA of WRN is particularly strong, and the human WRN gene can more effectively modulate highly efficient skipping in the 27th exon of .
  • each nucleotide is bound by a phosphate group and/or a modified phosphate group, and may be bound by a phosphodiester bond or a phosphorothioate bond. preferable.
  • the double-stranded antisense oligonucleotide according to this embodiment includes the above single-stranded antisense oligonucleotide, and a second-stranded oligonucleotide that hybridizes to the single-stranded antisense oligonucleotide, or a pharmaceutically acceptable salt thereof.
  • the base sequence of the second-strand oligonucleotide has a sequence identity of 90% or more and 100% or less based on the base sequence complementary to the base sequence of the single-stranded antisense oligonucleotide. is preferred.
  • the double-stranded antisense oligonucleotide can be dissociated in solution and separated into the single-stranded antisense oligonucleotide and the second-stranded oligonucleotide.
  • the isolated single-stranded antisense oligonucleotides are capable of binding to the target RNA described above.
  • the single-stranded antisense oligonucleotide can also be understood as a "first-strand oligonucleotide" in relation to the second-strand oligonucleotide.
  • the first-strand oligonucleotide has an antisense strand against the target RNA described above, but the first-strand oligonucleotide and the second-strand A double-stranded oligonucleotide consisting of an oligonucleotide will be referred to as a "double-stranded antisense oligonucleotide" for convenience.
  • the single-stranded antisense oligonucleotides of the present invention can be produced by solid-phase synthesis by the phosphoramidite method. For example, a commercially available automatic nucleic acid synthesizer is first used to synthesize a single-stranded oligonucleotide having a predetermined base sequence on a solid-phase carrier. Next, the single-stranded oligonucleotide synthesized from the solid support is cleaved using a basic substance or the like and deprotected to obtain a crude single-stranded oligonucleotide.
  • the single-stranded antisense oligonucleotide of the present invention can be produced not only by the production method described above, but also by appropriately changing the base sequence of the nucleic acid, modification site, etc. according to methods known to those skilled in the art.
  • AmNA, GuNA, and scpBNA are described in International Publication No. 2011/052436 (Patent Document 1), International Publication No. 2014/046212 (Patent Document 2), and International Publication No. 2015/125783 (Patent Document 3), respectively. ) can be produced by the method described in .
  • PMO morpholino oligonucleic acid
  • the double-stranded antisense oligonucleotide of the present invention is first prepared using the same manufacturing method as for the above-mentioned single-stranded antisense oligonucleotide, based on the base sequence complementary to the single-stranded antisense oligonucleotide. Oligonucleotides with sequence identity (second strand oligonucleotides) are produced. It can then be produced by hybridizing the single-stranded antisense oligonucleotide and the second-stranded oligonucleotide.
  • the antisense oligonucleotide complex is the oligonucleotide (preferably, single-stranded antisense oligonucleotide) or a pharmaceutically acceptable salt thereof, or the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof; an adduct attached to the oligonucleotide or the second strand oligonucleotide; have Said adducts are selected from the group consisting of polyethylene glycol, peptides, alkyl chains (eg saturated aliphatic hydrocarbons etc.), ligand compounds, antibodies, proteins and sugar chains (eg carbohydrates, polysaccharides etc.).
  • the oligonucleotide complex comprises the oligonucleotide or a pharmaceutically acceptable salt thereof, or the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof; an additional substance linked directly or via a linker bond to the oligonucleotide or the second strand oligonucleotide, or a pharmaceutically acceptable salt thereof, the additional substance is selected from the group consisting of polyethylene glycol, peptide, alkyl chain, ligand compound, antibody, protein, and sugar chain;
  • Each of the above linker bonds is independently a phosphodiester bond, a phosphorothioate bond, a phosphorodithioate bond, a phosphoramidate bond, a boranophosphate bond, an alkylphosphonate bond, a phosphorodiamidate bond, and a phosphorodiamide bond.
  • a thioate bond or a phosphorodiamide dithioate bond is independently a
  • the antisense oligonucleotide conjugate is the oligonucleotide (preferably single-stranded antisense oligonucleotide) or a pharmaceutically acceptable salt thereof; an adduct attached to the oligonucleotide, wherein the adduct is selected from the group consisting of polyethylene glycol, peptides, alkyl chains, ligand compounds, antibodies, proteins, and sugar chains.
  • the antisense oligonucleotide conjugate comprises the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof; and an adduct attached to the single-stranded antisense oligonucleotide or the second-strand oligonucleotide, wherein the adduct is polyethylene glycol, peptide, alkyl chain (e.g., saturated aliphatic hydrocarbon, etc.). , ligand compounds, antibodies, proteins, and sugar chains (eg, carbohydrates, polysaccharides, etc.).
  • the "addition substance” is a substance that binds to the single-stranded antisense oligonucleotide or the second-strand oligonucleotide, and means a substance that is used to impart a predetermined action.
  • the additional substance may be bound to the 5' end of the single-stranded antisense oligonucleotide, may be bound to the 3' end, or may be bound to both the 5' end and the 3' end. may be In addition, the additional substance may be bound to the 5' end of the second strand oligonucleotide, may be bound to the 3' end, or may be bound to both the 5' end and the 3' end.
  • the additional substance is preferably attached to either the 5' end or the 3' end of the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide.
  • the additional substance may be directly covalently bound to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide.
  • the additional substance may be bound to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide via a linker substance.
  • the linker substance include linkers composed of alkyls, polyethylene glycols, peptides, disulfides, phosphate bonds, etc. and/or combinations thereof.
  • the linker substance can also be understood as a linker bond.
  • peptides used as the above addition substances include, but are not limited to, the following. CPPs (cell penetrating peptides), nuclear translocating peptides, TAT (trans-activator of transcription protein), polyarginine, glucagon-like peptide-1 analogous peptides, synthetic cyclic RGD peptides, skin-permeable peptides.
  • ligand compound used as the above addition substance examples include, but are not limited to, the following. N-acetylgalactosamine (GalNAc), sugars (glucose, mannose, etc.), lipids (cholesterol, etc.), vitamins (folic acid, vitamin A, vitamin E, etc.), amino acids.
  • GaNAc N-acetylgalactosamine
  • sugars glucose, mannose, etc.
  • lipids cholesterol, etc.
  • vitamins folic acid, vitamin A, vitamin E, etc.
  • amino acids amino acids
  • antibodies used as the above-mentioned additional substances include, but are not limited to, the following. anti-insulin receptor antibody, anti-transferrin receptor antibody, anti-LDL receptor-related protein antibody, anti-CD22 antibody, anti-CD30 antibody, anti-HER2 antibody.
  • proteins used as the above addition substances include, but are not limited to, the following. albumin.
  • examples of the combination of the addition substance and the linker substance include, but are not limited to, those represented by the following structural formulas.
  • the 27th exon skipping agent of the human WRN gene comprises the single-stranded antisense oligonucleotide, the double-stranded antisense oligonucleotide, or the antisense-oligonucleotide complex of the present invention as an active ingredient.
  • the single-stranded antisense oligonucleotides of the present invention can skip the 27th exon of the human WRN gene by binding to the pre-mRNA of WRN. Any administration method and formulation known in the art can be used for the administration method and formulation of the skipping agent.
  • the functional WRN restorer is the oligonucleotide or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the oligonucleotide complex It is a functional WRN restorer having a nuclear localization signal obtained by skipping the 27th exon of the human WRN gene, which contains a protein or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the single-stranded antisense oligonucleotides of the present invention can skip the 27th exon of the human WRN gene by binding to the pre-mRNA of WRN.
  • the termination codon in exon 27 that appears in the human WRN gene is skipped, and exon 25 and exon 28 are joined to form a WRN mRNA.
  • a functional WRN protein having a nuclear localization signal contained in exon 34 is translated based on the mRNA.
  • the pharmaceutical composition according to this embodiment comprises the single-stranded antisense oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, Alternatively, it contains the above antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof as an active ingredient.
  • Any administration method and formulation known in the art can be used for the administration method and formulation of the pharmaceutical composition of this embodiment.
  • the pharmaceutical composition may be referred to as "pharmaceutical composition such as antisense oligonucleotide".
  • the pharmaceutical composition is used for treating or preventing Werner's syndrome.
  • the pharmaceutical composition can be used for the treatment or prevention of Werner's syndrome, which is expected to improve the symptoms of Werner's syndrome by skipping the 27th exon of the human WRN gene with high efficiency.
  • the therapeutic agent for Werner's syndrome includes the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, Alternatively, it contains the above antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the preventive agent against Werner's syndrome includes the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, Alternatively, it contains the above antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof as an active ingredient.
  • An individual in the present invention means a mammal. Preferred are humans, monkeys, marmosets, dogs, pigs, rabbits, guinea pigs, rats and mice. Humans are more preferred.
  • the administration method and dosage form are not particularly limited. That is, any administration method and formulation known in the art can be used as the administration method and formulation for the antisense oligonucleotides and the like of the present invention.
  • administration methods include oral administration and parenteral administration.
  • Parenteral administration includes eye drops, vaginal administration, intrarectal administration, intranasal administration, transdermal administration, intravenous injection, infusion, subcutaneous administration, intraperitoneal or intramuscular injection, pulmonary administration by inhalation or inhalation, marrow administration. Examples include intracavitary administration and intracerebroventricular administration.
  • Formulations of the antisense oligonucleotides of the present invention contain excipients, binders, wetting agents, disintegrants, lubricants, diluents, flavoring agents, fragrances, solubilizers, suspending agents, and emulsifying agents. , stabilizers, preservatives, thickeners, and other pharmaceutical additives can be mixed as necessary.
  • compositions such as antisense oligonucleotides of the present invention
  • formulations such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders may be used. can be done.
  • compositions such as an antisense oligonucleotide of the present invention
  • formulations such as powders, granules, suspensions or solutions dissolved in water or non-aqueous media, capsules, powders, tablets, etc. can be used.
  • compositions such as an antisense oligonucleotide of the present invention are administered parenterally, intravenously, subcutaneously, or intradermally, preparations such as sterile aqueous solutions can be used.
  • the effective dose of the single-stranded antisense oligonucleotide of the present invention can be arbitrarily determined according to the sex, age, weight, symptoms, etc. of the individual to be administered. Furthermore, it can be arbitrarily determined depending on the administration method, route, frequency and the like. For example, dosage may be 0.01 to 100 mg/kg. It is preferably 0.1 to 50 mg/kg, more preferably 0.1 to 10 mg/kg.
  • the method for skipping the 27th exon of the human WRN gene in this embodiment includes the above single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the above double-stranded antisense oligonucleotide or a pharmaceutical agent thereof. administering a pharmaceutically acceptable salt, the antisense oligonucleotide complex, or a pharmaceutically acceptable salt thereof as an active ingredient to cells, tissues, or individuals expressing the WRN gene.
  • the method of administering the single-stranded antisense oligonucleotide or the like to a cell, tissue, or individual may be performed in vitro or in vivo.
  • the administration route described above is used for in vivo administration.
  • cells expressing the WRN gene include fibroblasts, adipocytes, mesenchymal stem cells, keratinocytes, muscle cells, vascular endothelial cells, smooth muscle cells, and the like.
  • the single-stranded antisense oligonucleotide or its pharmaceutically acceptable salt, the double-stranded antisense oligonucleotide or its pharmaceutically acceptable A step of administering a salt, or the antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof as an active ingredient to an individual suffering from Werner's syndrome.
  • the antisense oligonucleotides according to this embodiment have been described above.
  • the single-stranded antisense oligonucleotide having the structure described above enables highly efficient skipping of the 27th exon of the human WRN gene.
  • the treatment time is preferably 48 hours to 7 days.
  • Cells to be used may be cells expressing the WRN gene, and examples thereof include HEK293T cells and fibroblasts derived from Werner's syndrome patients (hereinafter referred to as "Werner's syndrome patient fibroblasts").
  • Cells treated with antisense oligonucleotides may be harvested immediately after treatment, or may be cultured continuously after removing the antisense oligonucleotides.
  • the present invention is not limited to the above-described embodiments.
  • the above single-stranded antisense oligonucleotides include the following embodiments.
  • each nucleotide is bound by a phosphate group and/or a modified phosphate group, the oligonucleotide comprises a modified nucleic acid having at least one modified sugar;
  • the oligonucleotide has a base length of 10 to 30 mer,
  • the base sequence of the above oligonucleotide is Having a sequence identity of 90% or more and 100% or less based on the base sequence complementary to at least one target region composed of the same base length as the oligonucleotide in the base sequence set forth in SEQ ID NO: 1 base sequence, A base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted
  • the base sequence of the single-stranded antisense oligonucleotide is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, at least one composed of the same base length as the single-stranded antisense oligonucleotide
  • SEQ ID NO: 1 SEQ ID NO: 2
  • SEQ ID NO: 3 SEQ ID NO: 4
  • SEQ ID NO: 5 SEQ ID NO: 6
  • SEQ ID NO: 6 at least one composed of the same base length as the single-stranded antisense oligonucleotide
  • the base sequences of the single-stranded antisense oligonucleotide are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5. , or a base sequence complementary to at least one target region composed of the same base length as the single-stranded antisense oligonucleotide in the base sequence set forth in SEQ ID NO:6.
  • Another aspect of the single-stranded antisense oligonucleotide of the present invention is
  • the sugar constituting the oligonucleotide is D-ribofuranose
  • the modification of the sugar is the sugar modification of the hydroxyl group at the 2'-position of D-ribofuranose.
  • the base length of the above oligonucleotide is 15-25mer.
  • Another aspect of the single-stranded antisense oligonucleotide of the present invention is Modification of the sugar constituting the oligonucleotide is a group represented by the following formula (A1).
  • Bx is a nucleobase, each independently a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine or uracil
  • X is a phosphate bond, each independently is a phosphodiester bond, a phosphorothioate bond, a phosphorodithioate bond, a phosphoramidate bond, a boranophosphate bond or an alkylphosphonate bond
  • each Y is independently a hydrogen atom, a hydroxyl group, a fluorine atom, an optionally substituted alkoxy group having 1 to 6 carbon atoms, or an optionally substituted amino group
  • Z is each independently a hydrogen atom or a carbon-oxygen double It is an alkyl group having
  • Another aspect of the single-stranded antisense oligonucleotide of the present invention is At least one internucleotide linkage of the oligonucleotide is a phosphorothioate linkage.
  • Another aspect of the single-stranded antisense oligonucleotide of the present invention is At least one internucleotide bond of the oligonucleotide is a phosphodiester bond.
  • the base sequence of the above oligonucleotide is 1st to 12th, 14th to 21st, 24th, 25th, 29th to 31st, 34th, 36th, 41st, 46th counting from the 5′ end of the base sequence shown in SEQ ID NO: 1
  • the base sequence of the above oligonucleotide is In the nucleotide sequence of SEQ ID NO: 1, a 15 to 25 mer contiguous from the bases located at 1st to 40th, 46th, 51st, 56th, or 62nd to 63rd counted from the 5' end 108873, 108878 to 108917, 108923, 108928, 108933, or 108939 to 108940 in the target region or the nucleotide sequence set forth in SEQ ID NO: 2, counting from the 5' end A nucleotide sequence having a sequence identity of 90% or more and 100% or less based on a nucleotide sequence complementary to a target region composed of consecutive 15-25mers, A base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted or added in the target region, or It is a nucleotide sequence that hybridizes under string
  • the base sequence of the oligonucleotide is the base sequence shown in SEQ ID NO: 1, 1st, 3rd to 12th, 14th to 19th, 21st, 25th, 29th, 30th counted from the 5' end , 31st, 34th, 46th, or 31st, 31st, 34th, 46th, or 90% or more and 100% or less sequence identity based on the base sequence complementary to the target region composed of 15-25mers contiguous from the base located at 51st It is a base sequence having a unique property.
  • nucleotide sequence of the oligonucleotide is located at 1st to 32nd, 34th to 40th, 46th, 51st, or 62nd to 63rd counted from the 5' end in the nucleotide sequence shown in SEQ ID NO: 1.
  • the base sequence of the oligonucleotide is the base sequence shown in SEQ ID NO: 1, 1st, 3rd, 6th to 12th, 14th to 19th, 21st, 25th, 29th counting from the 5' end , 30th, 31st, 34th, 46th, or 90% or more and 100% or less based on the base sequence complementary to the target region composed of a 15-25mer contiguous from the base located at 30th, 31st, 34th, 46th, or 51st is a base sequence having the sequence identity of
  • the base sequence of the oligonucleotide is the base sequence shown in SEQ ID NO: 1, 1st, 3rd, 5th to 12th, 14th to 19th, 21st, 25th, 29th counted from the 5' end , 30th, 31st, 34th, 46th, or a target region composed of a 15-25mer contiguous from the base located at 51th, or in the base sequence shown in SEQ ID NO: 2, counting from the 5' end 108878, 108880, 108882 to 108889, 108891 to 108896, 108898, 108902, 108906 to 108908, 108911, 108923, or a continuous 15 to 25 mer from the base located at 108928
  • Another aspect of the single-stranded antisense oligonucleotide of the present invention is Modification of the sugar constituting the oligonucleotide is a group represented by the following formula (A2) or formula (A3).
  • Bx is a nucleobase, each independently a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine or uracil;
  • X is a phosphate bond, each independently is a phosphodiester bond, a phosphorothioate bond, a phosphorodithioate bond, a phosphoramidate bond, a boranophosphate bond or an alkylphosphonate bond, and each R4 is independently a hydrogen atom, a substituted is an optionally substituted alkyl group having 1 to 6 carbon atoms, Y' is an oxygen atom or an optionally substituted nitrogen atom, R 5 and R 6 are each independently a hydrogen atom, An optionally substituted al
  • the base sequence of the oligonucleotide is one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 9, 11, 14-22, 24-29, 31, 35, 39-41, 44, 52, and 53. be.
  • the base sequences of the above oligonucleotides are SEQ ID NOs: 9-14, 16-26, 28-39, 41-42, 44-50, 52, 55-56, 69, 73, 80-105, 107-108, 110, It is one base sequence selected from the group consisting of 112, 114, 116, 118-131, 133, and 135 base sequences.
  • Another aspect of the single-stranded antisense oligonucleotide of the present invention is The nucleotide sequence of the above oligonucleotide is a single-stranded antisense oligonucleotide.
  • Single-stranded antisense oligonucleotides containing 2'-OMe (O-methyl), 2'-MOE (O-methoxyethyl), AmNA, scpBNA, and GuNA were prepared using an automated nucleic acid synthesizer (type nS-8, Inc.). (manufactured by Genedesign), and synthesized on a 0.2 ⁇ mol scale. Chain length extension was performed by standard phosphoramidite protocols. At this time, CPG resin was used as the solid phase carrier.
  • DDTT ((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazaoline-3-thione) and the like were used for sulfurization for phosphorothioated (PS) skeleton formation.
  • Antisense oligonucleotides containing AmNA and scpBNA are assumed that the hydroxyl group at the terminal 5'-position is not protected with a DMTr (4,4'-dimethoxytrityl) group and the 3'-position is supported on a solid phase. Obtained.
  • Tables 2-1 to 2-7 below list the single-stranded antisense oligonucleotides produced by the above method.
  • R 1 , R 2 and R 3 each independently represent a hydrogen atom, a linear or branched C 1-6 alkyl group, or a C 3-7 cyclo Indicates an alkyl group.
  • R 1 and R 3 in GuNA represented by “Gx” above are hydrogen atoms, and R 2 is a methyl group, it is represented as “Gm”
  • R 1 is a hydrogen atom
  • R 2 and R 3 are methyl groups
  • R 1 and R 3 are hydrogen atoms and R 2 is a tert-butyl group
  • GtB a tert-butyl group
  • ⁇ Exon skipping evaluation of the 27th human WRN gene>> The 27th exon skipping evaluation of the human WRN gene was performed by confirming the induction of exon 27 skipping in HEK293T cells and Werner's syndrome patient fibroblasts lacking exon 26. Gene expression evaluation in this example was carried out by exon 26 (HEK293T with a normal WRN gene) or 25 (Werner's syndrome lacking exon 26) of the WRN gene for complementary DNA (cDNA) obtained by reverse transcription reaction. This means evaluating the sequence structure of the 27th exon of the WRN gene by measuring the amplified amount using the PCR method in 29 regions from patient fibroblasts). Specific procedures for each expression evaluation are described below.
  • Cells expressing the human WRN gene were treated with antisense oligonucleotides for 2 days using a method such as lipofection or direct addition.
  • Cells used were cells expressing the WRN gene (for example, HEK293T cells or Werner's syndrome fibroblasts lacking exon 26).
  • Cells treated with antisense oligonucleotides were either harvested immediately after treatment, or harvested after removal of the antisense oligonucleotides and continued culture.
  • RNA extracted from the collected cells Reverse transcription reaction is performed on the total RNA extracted from the collected cells, and the obtained cDNA covers the region from exon 26 to exon 29 of the WRN gene (when using HEK293T cells having a normal WRN gene), or A region from exon 25 to exon 29 of the WRN gene (when exon 26-deleted Werner's syndrome patient fibroblasts were used) was amplified using the PCR method. These amplifications were set with an annealing temperature of 60°C.
  • the cDNA of HEK293T cells transfected with antisense oligonucleotides not only yielded amplification products of the same size as those obtained from cells not transfected with antisense oligonucleotides, but also showed smaller size after 48 hours of culture. of amplification product was obtained. Determination of the base sequence of this amplified product by a conventional method showed that it consisted of exons 26, 28 and 29. Only small size transcripts, ie those with exon 27 skipping, were obtained under these conditions.
  • the present inventors confirmed that the designed antisense oligonucleotide effectively induces exon 27 skipping in the splicing reaction of the pre-mRNA transcribed from the gene with the normal human WRN gene structure.
  • the present inventors also designed antisense oligo Nucleotides were confirmed to effectively induce exon 27 skipping.
  • the present invention will now be described in more detail based on various tests.
  • Human embryonic kidney HEK293T cells (ATCC® CRL-3216TM) were cultured in culture medium at 37° C., 5% CO 2 .
  • As a culture medium for HEK293T cells one having the following composition was used.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • HEK293T cells (12000 cells/well) were seeded in a 96-well plate and cultured overnight at 37° C. and 5% CO 2 .
  • each single-stranded antisense oligonucleotide (50 nM) diluted with phosphate-buffered saline (PBS) was transfected into the above cells by lipofection.
  • PBS phosphate-buffered saline
  • RNA was extracted total RNA using Taqman Fast Cells-to-CT Kit (manufactured by Thermo Fisher Scientific, Cat#4399003).
  • cDNA Complementary DNA obtained from this reverse transcription reaction was used to perform PCR spanning exon 25 to exon 29 of the WRN gene using specific pre-designed primers (see below).
  • the PCR program used is as follows. 94°C, 1 minute: heat denaturation [98°C, 10 seconds; 60°C, 5 seconds; 68°C, 40 seconds] x 35 cycles: PCR amplification
  • the reaction product of the above PCR reaction was separated by 3% agarose gel electrophoresis, and a gel photograph was taken with a photography system combining an LED transilluminator gel (Wako) and iPhone 8 (Apple).
  • Fig. 5 shows the experimental results of measuring the skipping activity of exon 27 of the human WRN gene using each single-stranded antisense oligonucleotide (Fig. 4) against the human WRN pre-mRNA obtained by the method described above.
  • Werner's syndrome patient fibroblasts (Coriell Cell Repositories, Cat#AG12795) were cultured in culture medium at 37° C., 5% CO 2 .
  • a culture medium for Werner's syndrome patient fibroblasts one having the following composition was used.
  • the PCR program used is as follows. 94°C, 1 minute: heat denaturation [98°C, 10 seconds; 60°C, 5 seconds; 68°C, 40 seconds] x 35 cycles: PCR amplification
  • the reaction product of the above PCR reaction was separated by 3% agarose gel electrophoresis, the amplified PCR fragment was detected by LED Transilluminator Gel View (Wako), and the gel containing the PCR fragment was excised.
  • the amplified PCR fragment was purified and analyzed by sequencing, it was confirmed that the Werner's syndrome patient fibroblasts were homozygous for c.3139-1G>C of the WRN gene.
  • Each single-stranded antisense oligonucleotide (final concentration 1 to 50 nM) diluted with PBS was transfected into the above-mentioned cells by the lipofection method.
  • As a negative control group cells transfected with PBS in which the single-stranded antisense oligonucleotides were not dissolved were used.
  • Cells treated with antisense oligonucleotides were cultured in culture medium at 37° C., 5% CO 2 for 48 hours or 240 hours. Thereafter, the culture medium was removed, and reverse transcription reaction was performed on the extracted total RNA using Taqman Fast Cells-to-CT Kit (manufactured by Thermo Fisher Scientific, Cat#4399003).
  • Complementary DNA (cDNA) obtained from this reverse transcription reaction was used to perform PCR spanning exon 25 to exon 29 of the WRN gene using specific pre-designed primers (see below).
  • the PCR program used is as follows.
  • the reaction product of the above PCR reaction was separated by 3% agarose gel electrophoresis, and a gel photograph was taken with a photography system combining an LED transilluminator gel (Wako) and iPhone 8 (Apple).
  • Fig. 6 shows the experimental results of measuring the skipping activity of exon 27 of the human WRN gene using each single-stranded antisense oligonucleotide against the human WRN pre-mRNA obtained by the method described above.
  • Werner's syndrome patient fibroblasts the region from exon 25 to exon 29 of WRN cDNA was amplified. band was obtained. Determination of the nucleotide sequence of this amplified product by sequencing revealed that this band consisted of exon 25, exon 27, exon 28 and exon 29. This result was in good agreement with the patient's genetic analysis results.
  • a single-stranded antisense oligonucleotide (final concentration: 50 nM) consisting of the base sequence of SEQ ID NO: 28 diluted with PBS was transfected into the above cells by lipofection.
  • a negative control group cells transfected with PBS in which the single-stranded antisense oligonucleotides were not dissolved were used.
  • the transfected cells were cultured in culture medium at 37° C., 5% CO 2 for 48 hours.
  • the cells were fixed with 4% paraformaldehyde (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Cat # 163-20145) for 15 minutes, and 0.5% Triton-X (manufactured by Nacalai Tesque, Cat # 12967-45). Incubated for 10 minutes. Subsequently, the incubated cells were blocked with PBS containing 5% goat serum for 1 hour and incubated overnight in anti-WRN antibody (Cell Signaling, Cat#4666) solution. Furthermore, the cells were incubated for 1 hour in an anti-mouse IgG antibody (A32766, manufactured by Thermo Fisher Scientific) solution.
  • the cells were incubated in a solution of Hoechst (registered trademark) 33342 nucleic acid stain (manufactured by Thermo Fisher Scientific, Cat#H3570) for 15 minutes and observed under a fluorescence microscope.
  • Hoechst registered trademark
  • 33342 nucleic acid stain manufactured by Thermo Fisher Scientific, Cat#H3570
  • FIG. 7 shows the induction of functional WRN protein expression by skipping activity of exon 27 of the WRN gene by single-stranded antisense oligonucleotides against human WRN pre-mRNA, obtained by the method described above. 7, the functional WRN protein translocates into the nucleus in cells transfected with a single-stranded antisense oligonucleotide consisting of the base sequence of SEQ ID NO: 28 (“ASO: No. 28” in FIG. 7). I found out.
  • HepG2 cells a human liver cancer-derived cell line, were cultured in growth medium at 37°C, 5% CO2 conditions.
  • the growth medium used had the following composition.
  • the cells (1.5 ⁇ 10 4 cells/well) were seeded in a 96-well plate on the day before the experiment. After culturing the seeded cells overnight under conditions of 37° C. and 5% CO 2 , each single-stranded antisense oligonucleotide (final concentration 3-30 nM) diluted with PBS was lipofected into the above cells. was transfected into After addition, the cells were cultured for 24 hours at 37°C and 5% CO2 . As a negative control group, cells transfected with PBS in which the single-stranded antisense oligonucleotides were not dissolved were used.
  • Caspase activity was then assessed by adding the Caspase-Glo 3/7 Assay System (Promega, Cat#G8093) to the growth medium.
  • Table 6 shows the caspase activity in each single-stranded antisense oligonucleotide determined by the method described above.

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