WO2024257847A1 - Ｒｐｓ２５遺伝子の発現及び／又はその機能を調節するアンチセンスオリゴヌクレオチド - Google Patents

Ｒｐｓ２５遺伝子の発現及び／又はその機能を調節するアンチセンスオリゴヌクレオチド Download PDF

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WO2024257847A1
WO2024257847A1 PCT/JP2024/021613 JP2024021613W WO2024257847A1 WO 2024257847 A1 WO2024257847 A1 WO 2024257847A1 JP 2024021613 W JP2024021613 W JP 2024021613W WO 2024257847 A1 WO2024257847 A1 WO 2024257847A1
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antisense oligonucleotide
stranded antisense
nucleic acid
pharma
acceptable salt
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French (fr)
Japanese (ja)
Inventor
智彦 青山
成宏 浅野
友美 角谷
峻哲 川野邊
アジャヤラム セレスタ
高尾 鈴木
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Luxna Biotech Co Ltd
Sumitomo Pharma Co Ltd
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Luxna Biotech Co Ltd
Sumitomo Pharmaceuticals Co Ltd
Sumitomo Pharma Co Ltd
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Priority to JP2025528002A priority Critical patent/JPWO2024257847A1/ja
Priority to EP24823460.1A priority patent/EP4729616A1/en
Priority to CN202480039651.3A priority patent/CN121311591A/zh
Publication of WO2024257847A1 publication Critical patent/WO2024257847A1/ja
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A—HUMAN NECESSITIES
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    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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

Definitions

  • the present invention relates to antisense oligonucleotides that regulate the expression and/or function of the RPS25 gene, and to agents that regulate the expression and/or function of the RPS25 gene, including the same.
  • the Ribosomal Protein S25 (RPS25) gene is a gene that encodes one of the constituent proteins of the ribosomal 40S subunit.
  • the three-dimensional structure of the constituent protein (RPS25 protein) encoded by the RPS25 gene in the ribosomal 40S subunit has also been elucidated.
  • the RPS25 protein binds to an RNA element that enables the initiation of cap-independent translation as part of the protein synthesis process, and controls translation.
  • the RNA element to which the RPS25 protein binds is called an internal ribosome entry site (IRES). IRES is one of the cap-independent translation mechanisms that is commonly found, especially in viruses. (Non-Patent Document 2)
  • Non-Patent Document 3 Repeat associated non-ATG translation (RAN translation) was first identified in patients with spinocerebellar ataxia type 8 in 2011 (Non-Patent Document 3).
  • RAN translation refers to a mechanism in which a specific sequence is repeated to be translated into a protein (dipeptide repeat (DPR) etc.) in an ATG-independent manner. Subsequently, the involvement of RAN translation has been reported in several repeat diseases (diseases caused by the repetition of a specific gene sequence) such as amyotrophic lateral sclerosis (ALS) with a mutation in the C9orf72 gene (hereinafter sometimes referred to as "C9orf72 ALS”), Huntington's disease, and myotonic dystrophy. Research has been conducted on the relationship between DPR produced by RAN translation and pathology, and it has been reported that removal of DPR is effective in improving pathology. (Non-Patent Document 4, Non-Patent Document 5)
  • the RPS25 protein was reported as a molecule that contributes greatly to the production of dipeptide repeats by RAN translation. Knocking down the RPS25 gene suppressed the production of DPRs derived from GGGGCC repeat sequences or DPRs derived from CAG repeat sequences that are RAN translation-dependent.
  • the GGGGCC repeat sequence is known as an abnormal elongation mutation of the C9orf72 gene, which is one of the familial mutations in amyotrophic lateral sclerosis.
  • the CAG repeat sequence is known as an abnormal elongation mutation of the huntingtin gene and the ATXN2 gene.
  • Non-Patent Document 6 It was revealed that knocking down the RPS25 gene suppressed the production of DPRs derived from GGGGCC repeat sequences and also suppressed motor neuron cell death in motor neuron cells derived from induced purulipotent stem cells (iPSCs) established from patients with C9orf72 gene mutations.
  • iPSCs induced purulipotent stem cells
  • the antisense oligonucleotide against the RPS25 gene disclosed in Non-Patent Document 6 is a gapmer in which the wing portions are modified with 2'-O-methylated RNA (2'-OMe nucleic acid) and the internucleoside space is phosphorothioated.
  • the base sequence of the antisense oligonucleotide contains a portion of the base sequence of the sense strand of the RPS25 gene.
  • Patent Document 7 a single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof that binds to the RPS25 gene and effectively regulates the expression of the RPS25 gene.
  • Antisense oligonucleotides are compounds that have a gap region made of deoxyribose and modified nucleic acids in the wing region that have been developed to improve nuclease resistance and improve binding affinity and specificity to target RNA. They act by hybridizing to the target RNA sequence and suppressing gene expression.
  • modified nucleic acids have been developed for clinical application of gapmers, and many compounds (gapmers) that use these have been developed and marketed. Due to their nature, nucleic acid drugs, including gapmers, are known to accumulate in specific organs such as the liver when exposed to the whole body.
  • toxicity of antisense oligonucleotides can be categorized into "toxicity caused by hybridization with non-target RNA (off-target toxicity in the narrow sense)” and “toxicity not caused by hybridization with RNA (off-target toxicity in the broad sense),” and various approaches have been adopted to avoid these toxicities.
  • toxicity caused by hybridization with non-target RNA off-target toxicity in the narrow sense
  • toxicity not caused by hybridization with RNA off-target toxicity in the broad sense
  • Non-Patent Document 8 a document evaluating central toxicity due to phosphorothioate modifications in antisense oligonucleotides discloses that central toxicity is reduced in phosphorothioate-modified gapmers by replacing phosphorothioate bonds in the wing portions with phosphodiester bonds.
  • delayed central toxicity In addition to acute central toxicity, it is also known that there is central toxicity that appears a certain time after administration (hereinafter sometimes referred to as “delayed central toxicity”; also referred to as “in vivo delayed neuroTox” in this specification), and from the day after exposure to the central nervous system, symptoms such as decreased spontaneous movement, abnormal gait and abnormal function of the hind limbs, tremors, weakness of the hind limbs or tail, loss of hind limb reflexes, and weight loss are observed. As far as the inventor knows, no specific solution to this delayed central toxicity has been reported.
  • Patent Document 1 reports that inhibiting the RPS25 gene suppresses the production of DPR, it is unclear whether a similar effect can be achieved by an approach using nucleic acids such as antisense.
  • the antisense oligonucleotide described in Non-Patent Document 5 suppresses the expression of the RPS25 gene, but the inhibitory effect is partial.
  • Patent Document 7 we have reported a single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof that effectively regulates the expression of the RPS25 gene, but there is a need to create an antisense oligonucleotide against the RPS25 gene that has reduced toxicity (e.g., delayed central nervous toxicity) for use as a pharmaceutical.
  • toxicity e.g., delayed central nervous toxicity
  • the present invention has been made in consideration of the above circumstances, and the problem that the present invention aims to solve is to provide a single-stranded antisense oligonucleotide that regulates the expression and/or function of the RPS25 gene with reduced delayed central nervous system toxicity, and an agent for regulating the expression and/or function of the RPS25 gene that contains the same.
  • the present invention is as follows.
  • the antisense oligonucleotide according to the first aspect of the present invention comprises: A single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof that regulates the expression and/or function of the RPS25 gene,
  • the single-stranded antisense oligonucleotide has each nucleoside linked by a phosphate group and/or a modified phosphate group;
  • the single-stranded antisense oligonucleotide comprises a gap region, a 3' wing region attached to the 3' end of the gap region, and a 5' wing region attached to the 5' end of the gap region;
  • the gap region is a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the gap region comprises at least one 5'-CP nucleic acid;
  • the 3' wing region and the 5' wing region are modified nucleic acids having a substitution at
  • the antisense oligonucleotide according to the second aspect of the present invention comprises: A single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof that regulates the expression and/or function of the RPS25 gene,
  • the single-stranded antisense oligonucleotide has each nucleoside linked by a phosphate group and/or a modified phosphate group;
  • the single-stranded antisense oligonucleotide comprises a gap region, a 3' wing region attached to the 3' end of the gap region, and a 5' wing region attached to the 5' end of the gap region;
  • the gap region is a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the gap region comprises at least one 5'-CP nucleic acid;
  • the 3' wing region and the 5' wing region are modified nucleic acids having a substitution at
  • the antisense oligonucleotide according to the third aspect of the present invention comprises: A single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof that regulates the expression and/or function of the RPS25 gene,
  • the single-stranded antisense oligonucleotide has each nucleoside linked by a phosphate group and/or a modified phosphate group;
  • the single-stranded antisense oligonucleotide comprises a gap region, a 3' wing region attached to the 3' end of the gap region, and a 5' wing region attached to the 5' end of the gap region;
  • the gap region is a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the gap region comprises at least one 5'-CP nucleic acid;
  • the 3' wing region and the 5' wing region are modified nucleic acids having a substitution at
  • modified nucleic acids are located in the gap region, the 5' wing region and the 3' wing region.
  • the gap region contains at least one 5'-CP nucleic acid (5'-cyclopropyl nucleic acid).
  • Modified nucleic acids having a substituent at the 2'-position located in the 5'-wing region and the 3'-wing region include, as non-bridged modified nucleic acids, 2'-MOE nucleic acid (2'-O-methoxyethyl nucleic acid), 2'-OMe nucleic acid (2'-O-methyl nucleic acid), and MCE (2'-O-(2-N-methylcarbamoyl)ethyl nucleic acid), and as bridged modified nucleic acids, 2',4'-BNA/LNA (2',4'-Bridged Nucleic Acid/Locked Nucleic Acid, hereinafter sometimes referred to as "LNA"), AmNA (amide-bridged modified nucleic acid, Amido-bridged
  • the antisense oligonucleotide of the present invention is preferably selected from the group consisting of 2'-O,4'-C-spirocyclopropylene bridged nucleic acid (scpBNA
  • the antisense oligonucleotide of the present invention is expected to have high binding affinity to RPS25 mRNA or pre-mRNA while reducing delayed central nervous system toxicity.
  • the antisense oligonucleotide of the present invention is a so-called gapmer type single-stranded antisense oligonucleotide, it functions as a catalyst in the decomposition reaction of the RPS25 gene by RNase, which will be described later, and is therefore considered to have a sustained desired effect even when administered in a small amount.
  • the base sequence of the single-stranded antisense oligonucleotide has a sequence identity of 95% to 100% based on a base sequence complementary to at least one target region of the base sequence set forth in SEQ ID NO: 1, the base sequence having the same base length as the single-stranded antisense oligonucleotide.
  • the base sequence of the single-stranded antisense oligonucleotide is a base sequence complementary to at least one target region in the base sequence set forth in SEQ ID NO: 1, the target region having the same base length as the single-stranded antisense oligonucleotide.
  • the number of bases in the gap region is 5 to 20 mer; the 3' wing region is a 3-5 mer modified nucleic acid having a substituent at the 2' position, The 5' wing region is a 3-5 mer modified nucleic acid having a substituent at the 2' position.
  • the 5'-CP nucleic acid is positioned at least at the second position counting from the 5' side of the gap region.
  • the 5'-CP nucleic acid is arranged in a ninth or more position relative to the gap region.
  • the 5'-CP nucleic acid is arranged in a position that is one-fifth or more of the gap region.
  • the 5'-CP nucleic acid is arranged in a continuous 2-4 mer sequence at least at one location in the gap region.
  • the 5'-CP nucleic acid is located on the 5' end side of the gap region.
  • the 5'-CP nucleic acid is arranged on the 5'-end side and the 3'-end side of the gap region.
  • the base length of the single-stranded antisense oligonucleotide is 15 to 20 mer.
  • the single-stranded antisense oligonucleotide has a base length of 18 to 20 mer.
  • the modified nucleic acid having a substituent at the 2'-position in the 3'-wing region comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA;
  • the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.
  • the modified nucleic acid having a substituent at the 2'-position in the 3'-wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA;
  • the modified nucleic acid having a substituent at the 2' position in the 5' wing region is comprised of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.
  • the modified nucleic acid having a substituent at the 2'-position in the 3'-wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA;
  • the modified nucleic acid having a substituent at the 2' position in the 5' wing region is comprised of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA.
  • At least one internucleoside bond in the single-stranded antisense oligonucleotide is a phosphorothioate bond.
  • At least one internucleoside bond in the single-stranded antisense oligonucleotide is a phosphodiester bond.
  • the ratio of phosphorothioate bonds in the internucleoside bonds constituting the single-stranded antisense oligonucleotide is 50% to 80%.
  • the proportion of phosphorothioate bonds in the internucleoside bonds constituting the single-stranded antisense oligonucleotide is 50% to 70%.
  • the bond between the 5'-CP nucleic acid and the nucleoside adjacent to the 3' side of the 5'-CP nucleic acid is a phosphorothioate bond (excluding the case where the 5'-CP nucleic acid is located at the 3' end of the gap region).
  • the bond between the 5'-CP nucleic acid and the nucleoside adjacent to the 3' side of the 5'-CP nucleic acid is a phosphorothioate bond (excluding the case where the 5'-CP nucleic acid is located at the 3' end of the gap region and the case where the nucleoside adjacent to the 3' side of the 5'-CP nucleic acid is a 5'-CP nucleic acid).
  • the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 5' side of the 5'-CP nucleic acid is a phosphodiester bond.
  • the base sequence of the single-stranded antisense oligonucleotide is A base sequence having a sequence identity of 90% to 100% based on a base sequence complementary to a target region consisting of a continuous 14-22 mer from bases located at positions 123, 124, 185, 186, 263, 264, 324, 325, 442, 443, or 447 to 453 from the 5' end in the base sequence set forth in SEQ ID NO: 1; A base sequence complementary to a base sequence in which one or several bases are deleted, substituted, inserted or added in the target region; or It is a base sequence that hybridizes under stringent conditions to an oligonucleotide having the above-mentioned target region.
  • the base sequence of the single-stranded antisense oligonucleotide is A base sequence having a sequence identity of 90% to 100% based on a base sequence complementary to a target region consisting of a continuous 14-22mer from the bases located at positions 324, 325, or 448 to 453 from the 5' end in the base sequence set forth in SEQ ID NO: 1; A base sequence complementary to a base sequence in which one or several bases are deleted, substituted, inserted or added in the target region; or It is a base sequence that hybridizes under stringent conditions to an oligonucleotide having the above-mentioned target region.
  • the base sequence of the single-stranded antisense oligonucleotide has a sequence identity of 90% to 100% based on a base sequence complementary to a target region consisting of a continuous 18-22mer from the bases located at positions 448 to 453 from the 5' end in the base sequence set forth in SEQ ID NO: 1, the 3' wing region is a 3-5 mer, The 5' wing region is a 3-5 mer.
  • the base sequence of the single-stranded antisense oligonucleotide is a base sequence that has 90% to 100% sequence identity with respect to a base sequence complementary to a target region consisting of 18 to 20 consecutive bases from the 450th to 451st or 453rd bases counting from the 5' end in the base sequence set forth in SEQ ID NO: 1.
  • the base sequence of the single-stranded antisense oligonucleotide is a base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 5 to 47 and 49 to 56.
  • the base sequence of the single-stranded antisense oligonucleotide is a base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 6, 9, 12, 15 to 18, 22, 24, 27 to 29, 31 to 36, 38, 40 to 42, 47, and 49 to 54.
  • the base sequence of the single-stranded antisense oligonucleotide is a base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 41, 47, 50, and 53 to 55.
  • the antisense oligonucleotide complex according to the present invention comprises a single-stranded antisense oligonucleotide according to any one of [1] to [33] above or a pharma- ceutical acceptable salt thereof, and an additional substance bound to the single-stranded antisense oligonucleotide, or a pharma- ceutically acceptable salt thereof,
  • the additional substance is selected from the group consisting of polyethylene glycol, peptides, alkyl chains, nucleic acids, ligand compounds, antibodies, proteins, and sugar chains.
  • the pharmaceutical product according to the present invention contains, as an active ingredient, any one of the single-stranded antisense oligonucleotides [1] to [33] above or a pharma- ceutical acceptable salt thereof, or the antisense oligonucleotide complex [34] above or a pharma-ceutical acceptable salt thereof.
  • the single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof, or the antisense oligonucleotide complex or a pharma- ceutically acceptable salt thereof, is administered so as to be exposed to the central nervous system.
  • the single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof, or the antisense oligonucleotide complex or a pharma- ceutically acceptable salt thereof is administered to a subject susceptible to delayed central nervous system toxicity.
  • the expression and/or function regulator of the RPS25 gene according to the present invention contains, as an active ingredient, any one of the single-stranded antisense oligonucleotides [1] to [33] above or a pharma- ceutical acceptable salt thereof, or the antisense oligonucleotide complex [34] above or a pharma-ceutical acceptable salt thereof.
  • the inhibitor of dipeptide repeat production by RNA translation of the present invention contains, as an active ingredient, any one of the single-stranded antisense oligonucleotides [1] to [33] above or a pharma- ceutical acceptable salt thereof, or the antisense oligonucleotide complex [34] above or a pharma-ceutical acceptable salt thereof.
  • the therapeutic agent for repeat disease according to the present invention contains, as an active ingredient, any one of the single-stranded antisense oligonucleotides [1] to [33] above or a pharma- ceutical acceptable salt thereof, or the antisense oligonucleotide complex [34] above or a pharma-ceutical acceptable salt thereof.
  • the preventive agent for repeat disease according to the present invention contains, as an active ingredient, any one of the single-stranded antisense oligonucleotides [1] to [33] above or a pharma- ceutical acceptable salt thereof, or the antisense oligonucleotide complex [34] above or a pharma-ceutical acceptable salt thereof.
  • the repeat disease is at least one selected from the group consisting of C9orf72 ALS, C9orf72 FTLD, Huntington's disease, spinocerebellar ataxia, dentatorubral-pallidoluysian atrophy, spinal-bulbar muscular atrophy, Friedreich ataxia, fragile X-associated tremor ataxia syndrome, and myotonic dystrophy.
  • the method for treating or preventing a repeat disease comprises administering to an individual the single-stranded antisense oligonucleotide of any one of [1] to [33] above or a pharma- ceutical acceptable salt thereof, or the antisense oligonucleotide complex of [34] above or a pharma-ceutical acceptable salt thereof.
  • the repeat disease is at least one selected from the group consisting of C9orf72 ALS, C9orf72 FTLD, Huntington's disease, spinocerebellar ataxia, dentatorubral-pallidoluysian atrophy, spinal-bulbar muscular atrophy, Friedreich ataxia, fragile X-associated tremor ataxia syndrome, and myotonic dystrophy.
  • the present invention provides a single-stranded antisense oligonucleotide according to any one of [1] to [33] above or a pharma- ceutical acceptable salt thereof, or an antisense oligonucleotide conjugate according to [34] above or a pharma-ceutical acceptable salt thereof, for use in the treatment or prevention of C9orf72 ALS.
  • the present invention provides a single-stranded antisense oligonucleotide according to any one of [1] to [33] above or a pharma- ceutical acceptable salt thereof, or an antisense oligonucleotide complex according to [34] above or a pharma-ceutical acceptable salt thereof, for use in producing a therapeutic or prophylactic agent for C9orf72 ALS.
  • the present invention makes it possible to provide a single-stranded antisense oligonucleotide that regulates the expression and/or function of the RPS25 gene and has reduced delayed central nervous system toxicity, as well as an agent for regulating the expression and/or function of the RPS25 gene that contains the same.
  • FIG. 1 is a schematic diagram showing an example of the configuration of a single-stranded antisense oligonucleotide according to this embodiment.
  • FIG. 2 is a schematic diagram illustrating the mechanism by which the expression of the RPS25 gene is suppressed when the single-stranded antisense oligonucleotide according to this embodiment is used.
  • the single-stranded antisense oligonucleotide of this embodiment is a single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof that regulates the expression and/or function of the RPS25 gene,
  • the single-stranded antisense oligonucleotide has each nucleoside linked by a phosphate group and/or a modified phosphate group;
  • the single-stranded antisense oligonucleotide comprises a gap region, a 3' wing region attached to the 3' end of the gap region, and a 5' wing region attached to the 5' end of the gap region;
  • the gap region is a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the gap region comprises at least one 5'-CP nucleic acid;
  • the "RPS25 gene” can be defined by Mol. Gen. Genet. (1979) 169: 1-6 (Non-Patent Document 9) and Curr. Opin. Struct. Biol. (2014) 24: 165-169 (Non-Patent Document 10). Synonyms of "RPS25” include 40S ribosomal protein S25, ribosomal protein S25, small ribosomal subunit protein eS25, Rps25, 2810009D21Rik, ribosomal protein s25, ribosomal protein S25, S25, eS25, and ribosomal protein.
  • single-stranded antisense oligonucleotide or “antisense oligonucleotide” (hereinafter, sometimes referred to as "ASO") means an oligonucleotide complementary to the mRNA, pre-mRNA, or ncRNA (non-coding RNA) of a target gene (hereinafter, these three may be collectively referred to as "target RNA”), or a pharmacologically acceptable salt thereof.
  • ASO antisense oligonucleotides
  • Antisense oligonucleotides form a double strand with the target mRNA, pre-mRNA, or ncRNA, thereby suppressing the action of the target mRNA, pre-mRNA, or ncRNA.
  • Antisense oligonucleotides include those having a completely complementary base sequence to the base sequence of the target mRNA, pre-mRNA, or ncRNA, those having a base sequence in which one or several bases are deleted, substituted, inserted, or added in the complementary base sequence, and those containing a base that forms a wobble base pair in the base sequence.
  • the antisense oligonucleotide of the present invention may further contain modified nucleotides known in the art other than the "modified nucleic acid whose sugar moiety is a modified sugar" (sugar-modified modified nucleotides) described below.
  • modified nucleotides known in the art include phosphate-modified modified nucleotides and nucleic acid base-modified modified nucleotides described below.
  • both ends of the antisense oligonucleotide in this embodiment is not particularly limited, and may be, for example, -OH or -OR (wherein R represents an alkyl chain, a phosphate ester, or an additional substance described later).
  • the single-stranded antisense oligonucleotide in this embodiment may be in the form of a single strand, or may hybridize with a second strand oligonucleotide described later to form a double strand.
  • the double-stranded oligonucleotide consisting of the single-stranded antisense oligonucleotide and the second strand oligonucleotide hybridized to the single-stranded antisense oligonucleotide may be referred to as a "double-stranded antisense oligonucleotide".
  • oligonucleotide refers to a polymer of nucleosides in which 2 to 30 identical or different nucleosides are linked together by phosphodiester bonds or other bonds.
  • the oligonucleotide can also be understood to be composed of a nucleic acid base portion, a phosphate portion, and a sugar portion, as shown in the following structural formula:
  • oligonucleotides are broadly classified into natural oligonucleotides and non-natural oligonucleotides.
  • Natural oligonucleotides refers to oligonucleotides made of naturally occurring nucleotides.
  • Non-natural oligonucleotides refers to oligonucleotides containing at least one modified nucleotide as a constituent unit, as described below.
  • Non-natural oligonucleotides preferably include modified sugar derivatives in which the sugar portion is modified; phosphorothioate derivatives 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 of the phosphodiester bond are replaced with sulfur atoms; ester derivatives in which the phosphodiester bond is tri-esterified; phosphoamide derivatives in which the phosphodiester bond is amidated; boranophosphate derivatives in which the phosphodiester bond is boronated; alkylphosphonate (e.g., methylphosphonate, methoxypropylphosphonate, etc.) derivatives in which the non-bridging oxygen atom of the phosphodiester bond is replaced with an alkyl group; amide derivatives in which the phosphodiester bond is replaced with an amide bond; and modified base derivatives in which the nucleic acid base is modified.
  • the above-mentioned non-natural oligonucleotide includes a cross-linked modified sugar derivative in which the sugar portion is modified; a phosphorothioate derivative in which one non-bridging oxygen atom of the phosphodiester bond is replaced with a sulfur atom; an ester derivative in which the phosphodiester bond is esterified; and an alkylphosphonate derivative in which the sugar portion is modified with a modified sugar (e.g., a cross-linked sugar) described below and one non-bridging oxygen atom of the phosphodiester bond is replaced with a sulfur atom or the non-bridging oxygen atom of the phosphodiester bond is replaced with an alkyl group.
  • a modified sugar e.g., a cross-linked sugar
  • nucleoside refers to a compound in which a purine base or a pyrimidine base is bound to a sugar.
  • a naturally occurring nucleoside may be referred to as a "natural nucleoside”.
  • a modified nucleoside that does not exist in nature may be referred to as a "modified nucleoside”.
  • a modified nucleoside in which the sugar moiety is particularly modified may be referred to as a "modified sugar nucleoside”.
  • nucleotide refers to a compound in which a phosphate group is bound to the sugar of the nucleoside.
  • a naturally occurring nucleotide may be referred to as a "natural nucleotide”.
  • a modified nucleotide that does not exist in nature may be referred to as a "modified nucleotide” or "modified nucleic acid”.
  • modified nucleotide or “modified nucleic acid” include a compound in which a phosphate group is bound to the sugar moiety of the modified nucleoside, a compound in which a modified phosphate group described below is bound to the sugar moiety of the modified nucleoside, and a compound in which a modified phosphate group described below is bound to the sugar moiety of a natural nucleoside.
  • sugar modification means that the sugar moiety of the nucleotide is modified.
  • the modified sugar moiety may be particularly referred to as a "modified sugar”.
  • Modified nucleotides that have been sugar modified can be used as modified nucleic acids, and examples of such modified nucleotides include LNA, AmNA, GuNA, scpBNA, 2'-O-alkyl (e.g., 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, etc.), 2'-F, 5'-methyl-DNA, ENA (2'-O,4'-C-Ethylene-Bridged Nucleic Acid), S-cEt (2',4'-constrained Ethyl Nucleic Acid), 5'-CP nucleic acid (5'-cyclopropyl Nucleic Acid), and the like.
  • LNA examples include those containing structures represented by the symbols “A(L)”, “5(L)”, “G(L)”, and “T(L)” as described below.
  • AmNA examples include those containing structures represented by the symbols “A(Y)”, “5(Y)”, “G(Y)”, and “T(Y)” as described below.
  • GuNA examples include those containing structures represented by the symbols “A(Gx)”, “5(Gx)”, “G(Gx)”, and “T(Gx)” as described below.
  • scpBNA examples include those containing structures represented by the symbols “A(S)”, “5(S)”, “G(S)”, and “T(S)” as described below.
  • Examples of 2'-MOE nucleic acids include those containing structures represented by the symbols “A(m)”, “5(m)”, “G(m)”, and “T(m)” as described below.
  • Examples of 5'-CP nucleic acids include those containing structures represented by the symbols “A(5'-CP)”, “5(5'-CP)”, “G(5'-CP)”, and “T(5'-CP)” as described below.
  • Examples of 2'-OMe nucleic acids include those containing structures represented by the symbols “A(M)”, “5(M)”, “G(M)”, “U(M)", and “T(M)” as described below.
  • Examples of MCE nucleic acids include those containing structures represented by the symbols “A(Mx)”, “5(Mx)”, “G(Mx)”, and "U(Mx)” as described below.
  • modified nucleic acid having a substituent at the 2'-position refers to a modified nucleic acid having a substituent at the 2'-position of the sugar moiety of the above nucleotide.
  • non-bridged modified nucleic acids include 2'-O-alkyl (e.g., 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, MCE nucleic acid, etc.) and 2'-F, while bridged modified nucleic acids include LNA, AmNA, GuNA, scpBNA, ENA, S-cEt, etc.
  • Nucleotide modifications known in the art other than sugar modifications can be used as modified nucleic acids for producing single-stranded antisense oligonucleotides of the present invention.
  • nucleotide modifications phosphate group modifications and nucleic acid base modifications described below are known. Examples of such nucleotide modifications include nucleotide modifications described in W. Brad Wan et. Al. J. Med. Chem. (2016) 59: 9645-9667. (Non-Patent Document 11) and the like. These nucleotide modifications can be carried out based on methods known in the art described in the documents cited in the above documents.
  • phosphate group refers to a nucleotide in which the bond at the phosphate moiety is a naturally occurring phosphodiester bond (a bond indicated by the symbol "-" as described below).
  • phosphate group modification means that the phosphate moiety of the nucleotide is modified.
  • the modified phosphate moiety may be specifically referred to as a "modified phosphate group.”
  • nucleobase modification means that the nucleobase portion of the nucleotide is modified.
  • the modified nucleobase portion may be specifically referred to as a "modified nucleobase.”
  • modified nucleobases include 5-methylcytosine, 5-hydroxymethylcytosine, and 5-propynylcytosine.
  • DNA or RNA analogues refer to molecules having a structure similar to that of DNA or RNA, such as peptide nucleic acid (pNA) and morpholino nucleic acid.
  • pNA peptide nucleic acid
  • morpholino nucleic acid morpholino nucleic acid
  • ncRNA refers to a general term for RNA that is not involved in protein translation.
  • examples of the ncRNA include ribosomal RNA, transfer RNA, miRNA, and Natural Antisense Transcript (NAT).
  • nucleic acid base moiety of the oligonucleotide examples include thyminyl, cytosinyl, adeninyl, guaninyl, 5-methylcytosinyl, uracilyl, 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl, 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl, and 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl groups.
  • examples of the nucleic acid base moiety include thyminyl, cytosinyl, adeninyl, guaninyl, 5-methylcytosinyl, and uracilyl groups.
  • uracil (U) and thymine (T) are interchangeable. Both uracil (U) and thymine (T) can form base pairs with adenine (A) in a complementary strand.
  • Cytosine (C) and 5-methylcytosine (5(x)) are interchangeable and can both form base pairs with guanine (G) in a complementary strand. The same is true for the nucleobase portion of antisense oligonucleotides.
  • target RNA refers to an RNA whose function is inhibited by the binding of the single-stranded antisense oligonucleotide.
  • target RNA refers to the mRNA and pre-mRNA of RPS25.
  • Examples of the target RNA include human RPS25 mRNA having the base sequence set forth in SEQ ID NO: 1 (hereinafter, sometimes referred to as "hRPS25”), monkey RPS25 mRNA having the base sequence set forth in SEQ ID NO: 2 (hereinafter, sometimes referred to as “cRPS25”), mouse RPS25 mRNA having the base sequence set forth in SEQ ID NO: 3 (hereinafter, sometimes referred to as "mRPS25”), rat RPS25 mRNA having the base sequence set forth in SEQ ID NO: 4 (hereinafter, sometimes referred to as "rRPS25”), etc.
  • hRPS25 human RPS25 mRNA having the base sequence set forth in SEQ ID NO: 1
  • cRPS25 monkey RPS25 mRNA having the base sequence set forth in SEQ ID NO: 2
  • mRPS25 mouse RPS25 mRNA having the base sequence set forth in SEQ ID NO: 3
  • rRPS25 rat RPS25 mRNA
  • binding to a target RNA means that the nucleic acid base of the single-stranded antisense oligonucleotide forms a double-stranded nucleic acid together with the nucleic acid base of the target RNA due to complementarity with the target RNA.
  • the double-stranded nucleic acid may be formed in at least a part of the target RNA.
  • the strength of the binding to the target RNA can be measured, for example, by an index of thermal stability.
  • An example of the index of thermal stability is 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 refers to a region in the mRNA and pre-mRNA of RPS25 to which the single-stranded antisense oligonucleotide binds, including the target region consisting of the indicated nucleotide sequence and a region on the pre-mRNA of RPS25.
  • the pre-mRNA refers to a primary transcript of RNA transcribed from DNA. That is, the pre-mRNA is an RNA including an exon region, an intron region, and an untranslated region (UTR).
  • the pre-mRNA can also be understood as an RNA before splicing after transcription. When the pre-mRNA is spliced, it becomes an mRNA.
  • the 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, but can form a double strand with a part of the target region. That is, the single-stranded antisense oligonucleotide of the present invention is preferably one that has complete complementarity with the target region, but can be complementary to at least a part of the target region as long as it binds to the target RNA of RPS25.
  • the part of the target region means a region of the target region having a length of 10 to 15 nucleotide bases.
  • “Complementary to at least a portion of the target region” means complementary to the bases of at least a portion of the target region on the target RNA, including complementary to the bases of a region on an mRNA or pre-mRNA corresponding to the at least a portion of the region.
  • the base sequence of the single-stranded antisense oligonucleotide according to this embodiment is: (A) a base sequence having a sequence identity of 90% to 100% based on a base sequence complementary to a target region consisting of a continuous 14-22 mer from bases located at positions 123, 124, 185, 186, 263, 264, 324, 325, 442, 443, or 447 to 453 from the 5' end in the base sequence set forth in SEQ ID NO: 1; (B) a base sequence complementary to a base sequence in which one or several bases are deleted, substituted, inserted or added in the target region; or (C) A base sequence that hybridizes under stringent conditions to an oligonucleotide having the above-mentioned target region is preferable.
  • each base sequence shown in the sequence listing is used to indicate only the sequence information of the nucleic acid base portion.
  • the structural information of the oligonucleotide including the sugar portion and the phosphate portion in addition to the nucleic acid base portion is shown in the format shown in Tables 2-1 to 2-4, 3-1, 3-2, and 4 described below.
  • sequence identity refers to the percentage of identical bases in the total overlapping base sequence in the optimal alignment when two base sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm can take into account the introduction of gaps into one or both of the sequences for optimal alignment).
  • 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 preferably has a sequence identity of 95% to 100% with the base sequence complementary to the above-mentioned specified target region in the base sequence set forth in SEQ ID NO:1, more preferably has a sequence identity of 98% to 100%, and even more preferably has a sequence identity of 100%.
  • a base sequence in which one or several bases have been deleted, substituted, inserted or added can be, for example, a base sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity due to the deletion, substitution, insertion or addition to the base sequence before the deletion, substitution, insertion or addition.
  • the specific number of "one or several bases” may be one, two, three, four or five of the above-mentioned deletions, substitutions, insertions or additions, each independently, or a combination of multiple bases.
  • stringent conditions refers to conditions in which the sample is incubated for 12 hours at room temperature in a solution containing 6xSSC (1xSSC composition: 0.15M NaCl, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5x Denhardt's solution, 100 ⁇ g/mL denatured salmon sperm DNA, and 50% (v/v) formamide, followed by washing with 0.5xSSC at a temperature of 50°C or higher.
  • more stringent conditions such as incubation for 12 hours at 45°C or 60°C, washing with 0.2xSSC or 0.1xSSC, and washing at a temperature of 60°C or 65°C or higher, are also included.
  • the target region is preferably a 14-22mer base sequence consisting of consecutive bases located at positions 123, 124, 185, 186, 263, 264, 324, 325, 442, 443, or 447-453 from the 5' end in the base sequence set forth in SEQ ID NO:1.
  • the target region is preferably a base sequence consisting of a contiguous 14-22 mer starting from bases located at positions 324, 325, or 448 to 453 from the 5' end in the base sequence set forth in SEQ ID NO:1.
  • the target region is preferably a base sequence consisting of a continuous 18-22 mer starting from bases 448 to 453 counting from the 5' end in the base sequence set forth in SEQ ID NO:1.
  • the target region is preferably a base sequence consisting of 18 to 20 consecutive bases from the 450th to 451st or 453rd base counting from the 5' end in the base sequence set forth in SEQ ID NO:1.
  • binding to the target region of RPS25 includes direct binding of the single-stranded antisense oligonucleotide of the present invention to the mRNA of RPS25 and direct binding to the mRNA precursor of RPS25.
  • One embodiment of the single-stranded antisense oligonucleotide of the present invention is a single-stranded oligonucleotide that regulates the expression of the RPS25 gene, which has any of the base sequences shown in Table 1, and which is complementary to the target region in the mRNA of human RPS25 shown in Table 1.
  • the single-stranded oligonucleotide may extend 1 to 5 bases on the 3' side and/or 5' side, respectively, as long as it contains the base sequence shown in Table 1.
  • the target region can be said to be a region in the mRNA of human RPS25 that is particularly related to the regulation of the expression of the human RPS25 gene (for example, a region having a secondary structure of the mRNA to which antisense nucleotides are easily bound) in the mRNA of human RPS25.
  • the 5'-end position is "451" and the 3'-end position is "465" in Table 1
  • the base sequence from the 451st to the 465th bases counting from the 5'-end in the base sequence shown in SEQ ID NO: 1 is the target region in the mRNA of human RPS25 targeted by the corresponding single-stranded antisense oligonucleotide (sequence name "h451-465").
  • the symbols "A'", “C'”, “G'” and “T'” are each selected from natural nucleosides (a, A, c, C, g, G, t, and U, as described below) or modified nucleosides (including modified sugar nucleosides).
  • the symbol “A'” is selected from A(M), A(m), A(L), A(Y), A(Gx), A(5'-CP), A(Mx) or A(S) described below
  • the symbol “C'” is selected from 5(x), C(M), 5(m), 5(L), 5(Y), 5(Gx), 5(5'-CP), C(Mx) or 5(S) described below
  • the symbol “G'” is selected from G(M), G(m), G(L), G(Y), G(Gx), G(5'-CP), G(Mx) or G(S) described below
  • the symbol “T'” is selected from U(M), T(m), T(L), T(Y), T(Gx), T(5'-CP), U(Mx) or T(S) described below.
  • the base sequence of the single-stranded antisense oligonucleotide is preferably one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 5 to 47 and 49 to 56.
  • the base sequence of the single-stranded antisense oligonucleotide is preferably one selected from the group consisting of SEQ ID NOs: 6, 9, 12, 15-18, 22, 24, 27-29, 31-36, 38, 40-42, 47, and 49-54.
  • the base sequence of the single-stranded antisense oligonucleotide is one selected from the group consisting of the base sequences of SEQ ID NOs: 6, 9, 12, 15-18, 22, 24, 27-29, 31-32, 34-36, 38, 40-42, 47, and 49-54.
  • the base sequence of the single-stranded antisense oligonucleotide is one selected from the group consisting of the base sequences of SEQ ID NOs: 41, 47, 50, and 53 to 55.
  • the single-stranded antisense oligonucleotide according to this embodiment may be in the form of a pharmacologically acceptable salt.
  • pharmaceutically acceptable salt refers to the salt of the single-stranded antisense oligonucleotide of the present invention, which is the physiologically acceptable salt of the single-stranded antisense oligonucleotide of the present invention, that is, the salt that retains the desired biological activity of the single-stranded antisense oligonucleotide and does not retain undesired toxicological effects.
  • the double-stranded antisense oligonucleotide and antisense oligonucleotide complex described below.
  • the single-stranded antisense oligonucleotide may be in the form of a pharma- ceutically acceptable salt.
  • pharmaceutically acceptable salt refers to a salt that is the above-mentioned pharmacologically acceptable salt and is an acid addition salt or a base addition salt.
  • acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate, and organic acid salts such as citrate, oxalate, phthalate, fumarate, maleate, succinate, malate, acetate, formate, propionate, benzoate, trifluoroacetate, methanesulfonate, benzenesulfonate, para-toluenesulfonate, and camphorsulfonate.
  • inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate
  • organic acid salts such as citrate, oxalate, phthalate, fumarate, maleate, succinate, malate, acetate, formate, propionate, benzoate, trifluoroacetate, methanesulfonate, benzenesulf
  • base addition salts include inorganic base salts such as sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, and aluminum salts, as well as organic base salts such as trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, dicyclohexylamine, and N,N-dibenzylethylamine.
  • inorganic base salts such as sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, and aluminum salts
  • organic base salts such as trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexy
  • salts with basic or acidic amino acids such as arginine, lysine, ornithine, aspartic acid, or glutamic acid (amino acid salts).
  • basic or acidic amino acids such as arginine, lysine, ornithine, aspartic acid, or glutamic acid (amino acid salts).
  • the single-stranded antisense oligonucleotide includes a gap region, a 3' wing region bound to the 3' end of the gap region, and a 5' wing region bound to the 5' end of the gap region (see, for example, FIG. 1).
  • the single-stranded antisense oligonucleotide is preferably in a single-stranded form.
  • the single-stranded antisense oligonucleotide may hybridize with a second-stranded oligonucleotide described below to take a double-stranded form (double-stranded antisense oligonucleotide).
  • the base sequence of the second-stranded oligonucleotide is preferably a base sequence having a sequence identity of 90% or more and 100% or less based on a base sequence complementary to the base sequence of the single-stranded antisense oligonucleotide.
  • the single-stranded antisense oligonucleotide is a so-called gapmer type single-stranded antisense oligonucleotide.
  • the gapmer type single-stranded antisense oligonucleotide inhibits the function of the target RNA by the following mechanism. First, the single-stranded antisense oligonucleotide binds to the target region of the target RNA (top to center of FIG. 2). Next, RNase H, an RNA degrading enzyme, recognizes and binds to the complex of the single-stranded antisense oligonucleotide and the target RNA (center of FIG. 2).
  • the target RNA is then cleaved and degraded by an enzymatic degradation reaction by RNase H.
  • the single-stranded antisense oligonucleotide is not affected by the enzymatic degradation by RNase H (lower part of FIG. 2). Therefore, the single-stranded antisense oligonucleotide can bind to another target RNA and cleave and degrade the RNA.
  • the gapmer type single-stranded antisense oligonucleotide functions as a catalyst in the enzymatic degradation reaction by RNase H described above, and is therefore considered to have a predetermined effect even when administered in small amounts.
  • the single-stranded antisense oligonucleotide can be suitably used to regulate the expression of the RPS25 gene by the mechanism described above (including the case where it acts through regulating the maturation of RPS25 mRNA precursors). Furthermore, according to this embodiment, the effect of regulating the expression of the RPS25 gene by the single-stranded antisense oligonucleotide can be exerted even by intrathecal administration, which is an administration route commonly used in clinical applications.
  • regulating the expression of the RPS25 gene means at least suppressing the expression of the RPS25 gene, and as a result, at least suppressing the function of the RPS25 protein (such as RNA translation).
  • the gap region includes at least one 5'-CP nucleic acid.
  • the gap region is preferably a 5-20 mer nucleic acid composed of deoxyribose which may include a nucleic acid whose sugar portion is modified.
  • the gap region can also be understood as a 5-20 mer nucleic acid containing deoxyribose which may include a nucleic acid whose sugar portion is modified.
  • the gap region can also be understood as being composed of 5-20 mer natural nucleotides, non-natural nucleotides, or both, whose sugar portion is deoxyribose.
  • the gap region can form a complex recognizable by RNase H together with the target RNA, such as the mRNA of RPS25, by having the sugar portion be deoxyribose or modified deoxyribose.
  • the target RNA such as the mRNA of RPS25
  • the sugar portion be deoxyribose or modified deoxyribose.
  • an example of a nucleic acid containing modified deoxyribose is 5'-CP nucleic acid.
  • the number of bases in the gap region is preferably 5-20 mer, more preferably 6-17 mer, even more preferably 7-13 mer, and even more preferably 9-13 mer.
  • Examples of natural nucleotides in which the sugar moiety is deoxyribose include deoxyadenosine monophosphate, deoxyguanosine monophosphate, thymidine monophosphate, deoxycytidine monophosphate, and deoxy-5-methylcytidine monophosphate (also called 5-methyldeoxycytidine).
  • examples of natural nucleotides that make up the gap region include those that contain structural formulas corresponding to the symbols a, g, t, and c, which will be described later.
  • non-natural nucleotides in which the sugar moiety is deoxyribose or modified deoxyribose include 5'-CP nucleic acid, 2-thio-thymidine monophosphate, 2-aminoadenosine monophosphate, and 7-deazaguanosine monophosphate.
  • the above gap region may be a nucleic acid in which some of the sugar moieties of a natural nucleotide, the sugar moiety of which is deoxyribose, are modified sugars, as long as the effects of the present invention are achieved. That is, in one aspect of this embodiment, the above gap region may be a nucleic acid in which some of the sugar moieties are deoxyribose and other sugar moieties are modified sugars (e.g., modified deoxyribose).
  • the 5'-CP nucleic acid is located at least at the second position counting from the 5' side of the gap region.
  • two or more of the 5'-CP nucleic acid are arranged in the gap region, and it is more preferable that two to five of the 5'-CP nucleic acid are arranged.
  • "two or more are arranged” is a concept that includes an embodiment in which two or more of the target 5'-CP nucleic acid are arranged consecutively, and an embodiment in which two or more are arranged dispersedly in the cap region.
  • the 5'-CP nucleic acid is arranged in a continuous 2-4 mer sequence at least at one location in the gap region.
  • the 5'-CP nucleic acid is preferably located on the 5'-end side of the gap region. Also, the 5'-CP nucleic acid is preferably located on the 5'-end side and the 3'-end side of the gap region.
  • located on the 5'-end side of the gap region means located on the 5'-end side of the center of the gap region.
  • located on the 3'-end side of the gap region means located on the 3'-end side of the center of the gap region.
  • the 5'-CP nucleic acid is preferably arranged in a ninth or more portion of the gap region, and more preferably in a fifth or more portion of the gap region.
  • the upper limit may be, for example, half or less.
  • the bond between the 5'-CP nucleic acid and the nucleoside adjacent to the 3' side of the 5'-CP nucleic acid is preferably a phosphorothioate bond (excluding the case where the 5'-CP nucleic acid is disposed at the 3' end of the gap region).
  • the 5'-CP nucleic acid is disposed at the 3' end of the gap region
  • the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is preferably a phosphorothioate bond (excluding the cases where the 5'-CP nucleic acid is positioned at the 3' end of the gap region and where the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is a 5'-CP nucleic acid).
  • the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 5' side of the 5'-CP nucleic acid is preferably a phosphodiester bond.
  • the 3' wing region is a modified nucleic acid having a substituent at the 2' position.
  • the 3' wing region can be understood to be composed of modified nucleotides having a substituent at the 2' position.
  • the modified nucleic acid having a substituent at the 2' position in the 3' wing region preferably includes at least one selected from the group consisting of 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, and MCE nucleic acid as non-bridged 2'-position modified nucleic acids, and LNA, AmNA, GuNA, and scpBNA as bridged modified nucleic acids.
  • the 3' wing region and the 5' wing region described below being composed of the predetermined modified nucleotides, high binding affinity to the target RNA can be expected, and the function of the target RNA can be effectively suppressed.
  • the 3' wing region may be a modified nucleic acid in which the sugar moiety is a modified sugar.
  • modified nucleic acids in which the sugar moiety is a modified sugar include those listed above (sugar modification, modified sugar).
  • the modified nucleic acid having a substituent at the 2'-position in the 3'-wing region may be composed of only 2'-MOE nucleic acid. Note that, the modified nucleic acid in the 3'-wing region may be of multiple types contained in one single-stranded antisense oligonucleotide.
  • the modified nucleic acid having a substituent at the 2' position in the 3' wing region preferably includes at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA, and more preferably is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.
  • the modified nucleic acid having a substituent at the 2' position in the 3' wing region preferably is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA.
  • the number of bases in the 3' wing region is preferably 3-5 mer, and more preferably 3-4 mer.
  • the 5' wing region is a modified nucleic acid having a substituent at the 2' position.
  • the 5' wing region can be understood to be composed of modified nucleotides having a substituent at the 2' position.
  • the modified nucleic acid in the 5' wing region preferably includes, as a non-bridged 2'-position modified nucleic acid, 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, and MCE nucleic acid, and as a bridged modified nucleic acid, at least one selected from the group consisting of LNA, AmNA, GuNA, and scpBNA.
  • the 5' wing region may be a modified nucleic acid in which the sugar moiety is a modified sugar.
  • modified nucleic acids in which the sugar moiety is a modified sugar include those listed above (sugar modification, modified sugar).
  • the modified nucleic acid in the 5' wing region having a substituent at the 2' position may be composed only of 2'-MOE nucleic acid. Note that multiple types of modified nucleic acids in the 5' wing region may be included in one single-stranded antisense oligonucleotide.
  • the modified nucleic acids in the 3' wing region and the 5' wing region may consist solely of 2'-MOE nucleic acids, or may consist solely of cross-linked modified nucleic acids.
  • the modified nucleic acid having a substituent at the 2' position in the 5' wing region preferably includes at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA, and more preferably is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.
  • the modified nucleic acid having a substituent at the 2' position in the 5' wing region preferably is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA.
  • the modified nucleic acid having a substituent at the 2' position in the 3' wing region comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA;
  • the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region preferably comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.
  • the modified nucleic acid having a substituent at the 2'-position in the 3'-wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA, More preferably, the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region is comprised of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.
  • the modified nucleic acid having a substituent at the 2' position in the 3' wing region is comprised of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA; More preferably, the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region is comprised of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA.
  • the number of bases in the 5' wing region is preferably 3-5mer, and more preferably 3-4mer.
  • the number of bases in the gap region is 5-20 mer
  • the number of bases in the 3' wing region is 3-5 mer
  • the number of bases in the 5' wing region is 3-5 mer.
  • the number of bases in the gap region is 7-13 mer
  • the number of bases in the 3' wing region is 3-5 mer
  • the number of bases in the 5' wing region is 3-5 mer.
  • the number of bases in the gap region is 9-13 mer
  • the number of bases in the 3' wing region is 3-4 mer
  • the number of bases in the 5' wing region is 3-4 mer.
  • the single-stranded antisense oligonucleotide of the present invention is a gapmer type.
  • the notation method of "X-Y-Z” may be used. In the above notation method, "X” indicates the number of bases in the 5' wing region, “Y” indicates the number of bases in the gap region, and “Z” indicates the number of bases in the 3' wing region.
  • X-Y-Z is 2-8-4, 2-8-3, 2-8-5, 2-9-2, 2-9-3, 2-9-4, 2-9-5, 2-10-3, 2-10-4, 2-10-5, 2-11-3, 2-11-4, 2-11-5, 2-12-3, 2-12-4, 2-12-5, 3-8-2, 3-8-3, 3-8-4, 3-8-5, 3-9-3, 3-9-4, 3-9-5, 3-10-3, 3-10-4, 3-10-5, 3-11-3, 3-11-4, 3-11-5, 3-12-3, 3-12-4, 3-12-5, 3-1 Examples include 3-3, 3-13-4, 4-8-2, 4-8-3, 4-8-4, 4-8-5, 4-9-3, 4-9-4, 4-9-5, 4-10-3, 4-10-4, 4-10-5, 4-11-2, 4-11-3, 4-11-4, 4-11-5, 4-12-4, 4-13-3, 5-8-2, 5-8-3, 5-8-4, 5-8-5, 5-9-2, 5-9-3, 5-9-4, 5-9-5, 5-10-2, 5-10-3, 5-10-4,
  • “2-8-4" means that the 5' wing region is a 2-mer oligonucleotide, the gap region is an 8-mer oligonucleotide, and the 3' wing region is a 4-mer oligonucleotide.
  • the base length of the single-stranded antisense oligonucleotide of the present invention is 12-30 mer, preferably 15-20 mer, and more preferably 18-20 mer.
  • the base length of the single-stranded antisense oligonucleotide of the present invention is 15-20 mer or 18-20 mer, the binding to the mRNA of RPS25 is particularly strong, and the expression of the RPS25 gene can be regulated more effectively.
  • the single-stranded antisense oligonucleotide has each nucleoside linked via a phosphate group and/or a modified phosphate group, and is preferably linked via a phosphodiester bond or a phosphorothioate bond.
  • At least one internucleoside bond in the single-stranded antisense oligonucleotide is a phosphorothioate bond. In another aspect of this embodiment, it is preferred that at least one internucleoside bond in the single-stranded antisense oligonucleotide is a phosphodiester bond.
  • the proportion of phosphorothioate bonds in the internucleoside bonds constituting the single-stranded antisense oligonucleotide is preferably 50% to 80%, and more preferably 50% to 70%.
  • One embodiment of the single-stranded antisense oligonucleotide of the present invention is a gapmer-type single-stranded antisense oligonucleotide having a gap region consisting of 5 to 20 mer, a 5' wing region consisting of 3 to 5 mer, and a 3' wing region consisting of 3 to 5 mer.
  • the gap region is positioned between the 5' wing region and the 3' wing region.
  • the gap region contains at least one 5'-CP nucleic acid. It is preferable that the 5' wing region and the 3' wing region each contain at least one 2'-MOE nucleic acid, LNA, AmNA, GuNA, or scpBNA.
  • the 5' wing region and the 3' wing region may contain 2'-O-alkylated or 2'-F-substituted nucleotides.
  • a 2'-O-alkylated nucleotide e.g., 2'-O-methylated, etc.
  • the above-mentioned gapmer-type single-stranded antisense oligonucleotide may form a double strand by hybridizing with a second strand oligonucleotide.
  • the single-stranded antisense oligonucleotide is preferably at least one selected from the group consisting of Example 2, Example 5, Example 8, Example 11 to Example 14, Example 18, Example 20, Example 23 to Example 25, Example 27 to Example 32, Example 34, Example 36 to Example 38, Example 43, and Example 45 to Example 50 in Tables 2-1 to 2-4 described below.
  • the single-stranded antisense oligonucleotide is more preferably at least one selected from the group consisting of Example 37, Example 43, Example 46, and Examples 49 to 51 in Tables 2-1 to 2-4 described below.
  • the double-stranded antisense oligonucleotide comprises the single-stranded antisense oligonucleotide and and a second strand oligonucleotide hybridized to the single-stranded antisense oligonucleotide, or a pharma- ceutically acceptable salt thereof.
  • the base sequence of the second strand oligonucleotide is preferably a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to the base sequence of the single-stranded antisense oligonucleotide.
  • the double-stranded antisense oligonucleotide can be dissociated in solution and separated into the single-stranded antisense oligonucleotide and the second-stranded oligonucleotide.
  • the separated single-stranded antisense oligonucleotide can bind to the target RNA.
  • the single-stranded antisense oligonucleotide can also be understood as a "first-stranded oligonucleotide" in relation to the second-stranded oligonucleotide.
  • the first-stranded oligonucleotide has an antisense strand to the target RNA, but for convenience, the double-stranded oligonucleotide consisting of the first-stranded oligonucleotide and the second-stranded oligonucleotide will be referred to as a "double-stranded antisense oligonucleotide.”
  • the single-stranded antisense oligonucleotide of the present invention can be produced by solid-phase synthesis using the phosphoramidite method. For example, a single-stranded oligonucleotide having a predetermined base sequence is first synthesized on a solid-phase support using a commercially available automatic nucleic acid synthesizer. Next, the synthesized single-stranded oligonucleotide is cut out from the solid-phase support 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 by appropriately changing the base sequence, modification site, etc. of the nucleic acid according to a method known to those skilled in the art.
  • AmNA, GuNA, and scpBNA can be produced by the methods described in WO 2011/052436 (Patent Document 2), WO 2014/046212 (Patent Document 3), and WO 2015/125783 (Patent Document 4), respectively.
  • 2'-MOE nucleic acids can be produced by using amidites that are commercially available as reagents.
  • 5'-CP nucleic acids can be produced by the method described in International Publication No. 2020/158910 (Patent Document 5).
  • LNA can be produced by the method described in International Publication No. 99/14226 (Patent Document 6).
  • the double-stranded antisense oligonucleotide of the present invention can be produced by first producing an oligonucleotide (second strand oligonucleotide) having a predetermined sequence identity based on the base sequence complementary to the single-stranded antisense oligonucleotide using the same production method as the single-stranded antisense oligonucleotide, and then hybridizing the single-stranded antisense oligonucleotide and the second strand oligonucleotide.
  • the antisense oligonucleotide complex comprises: the single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof, or the double-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof; an additional substance bound to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide;
  • the additional substance is selected from the group consisting of polyethylene glycol, peptides, alkyl chains (e.g., saturated aliphatic hydrocarbons, etc.), nucleic acids, ligand compounds, antibodies, proteins, and sugar chains (e.g., carbohydrates, polysaccharides, etc.).
  • the antisense oligonucleotide conjugate comprises: The single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof; and an additional substance bound to the single-stranded antisense oligonucleotide, the additional substance being selected from the group consisting of polyethylene glycol, peptides, alkyl chains (e.g., saturated aliphatic hydrocarbons, etc.), nucleic acids, ligand compounds, antibodies, proteins, and sugar chains (e.g., carbohydrates, polysaccharides, etc.).
  • the antisense oligonucleotide conjugate comprises: The double-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof; and an additional substance bound to the single-stranded antisense oligonucleotide or the second strand oligonucleotide, the additional substance being selected from the group consisting of polyethylene glycol, peptides, alkyl chains (e.g., saturated aliphatic hydrocarbons, etc.), nucleic acids, ligand compounds, antibodies, proteins, and sugar chains (e.g., carbohydrates, polysaccharides, etc.).
  • additive substance refers to a substance bound to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide, and used to impart a predetermined action.
  • the additive substance may be bound to the 5' end, the 3' end, or both the 5' end and the 3' end of the single-stranded antisense oligonucleotide.
  • the additive substance may be bound to the 5' end, the 3' end, or both the 5' end and the 3' end of the second-stranded oligonucleotide.
  • the additive substance is preferably bound to either the 5' end or the 3' end of the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide.
  • the additive substance may be directly bound to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide by a covalent bond.
  • the additive substance may be bound to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide via a linker substance.
  • linker substance examples include linkers composed of alkyl, polyethylene glycol, peptide, disulfide, nucleic acid, etc., and/or combinations thereof.
  • the method for binding the additional substance to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide can be, for example, the method described in the Examples below.
  • Peptides used as the above-mentioned additional substances include, but are not limited to, the following: CPPs (Cell Penetrating Peptides), nuclear transport peptides, TAT (Trans-Activator of Transcription protein), polyarginine, glucagon-like peptide-1 analogue peptides, synthetic cyclic RGD peptides, and brain transport peptides.
  • ligand compounds used as the above-mentioned additional substances include, but are not limited to, the following: N-acetylgalactosamine (GalNAc), sugars (glucose, mannose, etc.), lipids (cholesterol, palmitic acid, docosahexaenoic acid, etc.), vitamins (folic acid, vitamin A, vitamin E (tocopherol), etc.), amino acids, monoamine receptor ligands (indatraline, etc.)
  • antibodies that can be used as the additional substance 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 that can be used as the above-mentioned additional substances include, but are not limited to, the following: Albumin
  • nucleic acids used as the additional substance include, but are not limited to, the following: natural nucleotides, aptamers.
  • the nucleic acids used as the additional substance are not counted in the base length of the antisense oligonucleotide.
  • the expression regulator of the RPS25 gene according to this embodiment contains the single-stranded antisense oligonucleotide of the present invention, the double-stranded antisense oligonucleotide, or the antisense oligonucleotide complex as an active ingredient.
  • the expression regulator can also be understood as an expression inhibitor for the RPS25 gene.
  • the expression regulator can also be understood as an inhibitor for RNA translation.
  • the expression regulator can also be understood as an expression inhibitor for dipeptide repeats via inhibition of RNA translation.
  • the single-stranded antisense oligonucleotide of the present invention inhibits the expression of the RPS25 gene by binding to the mRNA or pre-mRNA of RPS25, and inhibits the translation of RNA by the translation product.
  • the administration method and formulation of the expression regulator of the RPS25 gene according to the present invention can be any administration method and formulation known in the art.
  • the pharmaceutical composition according to this embodiment contains the single-stranded antisense oligonucleotide of the present invention or a pharma- ceutical acceptable salt thereof, the above-mentioned double-stranded antisense oligonucleotide or a pharma-ceutical acceptable salt thereof, or the above-mentioned antisense oligonucleotide complex or a pharma-ceutical acceptable salt thereof as an active ingredient.
  • the administration method and formulation of the pharmaceutical composition according to this embodiment can be any administration method and formulation known in the art.
  • the above-mentioned pharmaceutical composition may be referred to as "pharmaceutical composition of antisense oligonucleotide, etc.”
  • the pharmaceutical composition is used for treating or preventing diseases related to the RPS25 gene, i.e., diseases that can be caused by dipeptide repeats produced by RNA translation.
  • diseases related to the RPS25 gene i.e., diseases that can be caused by dipeptide repeats produced by RNA translation.
  • the pharmaceutical composition can be used for treating or preventing diseases whose symptoms can be expected to improve by suppressing the expression of the RPS25 gene.
  • repeat diseases include various psychiatric and neurological diseases and muscular diseases, including C9orf72 ALS, C9orf72 FTLD, Huntington's disease, spinocerebellar ataxia (types 1, 2, 3, 6, 7, 8, 12, and 17), dentatorubral-pallidoluysian atrophy, spinal-bulbar muscular atrophy, Friedreich ataxia, fragile X-associated tremor ataxia syndrome, and myotonic dystrophy.
  • C9orf72 ALS C9orf72 FTLD
  • Huntington's disease Huntington's disease
  • spinocerebellar ataxia types 1, 2, 3, 6, 7, 8, 12, and 17
  • dentatorubral-pallidoluysian atrophy spinal-bulbar muscular atrophy
  • Friedreich ataxia fragile X-associated tremor ataxia syndrome
  • myotonic dystrophy myotonic dystrophy.
  • the therapeutic agent for repeat disease contains the above single-stranded antisense oligonucleotide or its pharma- ceutical acceptable salt, the above double-stranded antisense oligonucleotide or its pharma-ceutical acceptable salt, or the above antisense oligonucleotide complex or its pharma-ceutical acceptable salt as an active ingredient.
  • the prophylactic agent for repeat disease contains the above single-stranded antisense oligonucleotide or its pharma-ceutical acceptable salt, the above double-stranded antisense oligonucleotide or its pharma-ceutical acceptable salt, or the above antisense oligonucleotide complex or its pharma-ceutical acceptable salt as an active ingredient.
  • the above repeat disease is preferably at least one selected from the group consisting of C9orf72 ALS, C9orf72 FTLD, Huntington's disease, spinocerebellar ataxia, dentatorubral-pallidoluysian atrophy, spinal-bulbar muscular atrophy, Friedreich ataxia, fragile X-associated tremor ataxia syndrome, and myotonic dystrophy.
  • the C9orf72 ALS refers to ALS having a mutation in which the GGGGCC sequence present in the intron region between the exon 1a and exon 1b regions of the C9orf72 gene is abnormally repeated and expanded.
  • the C9orf72 gene is a causative gene for ALS.
  • ALS is the most common type, accounting for approximately 6% of sporadic ALS cases and approximately 40% of familial ALS cases.
  • ALS is a neurodegenerative disease in which motor neurons selectively die, causing muscle atrophy. It is diagnosed by combining clinical and/or electrophysiologic features of upper and lower motor neuropathy.
  • a laboratory-based diagnosis is made. If it is in the second area, it is diagnosed as supported probable, if it is in the third area, it is diagnosed as probable, and if it is in the third area, it is diagnosed as definite.
  • the C9orf72 FTLD refers to FTLD having a mutation in which the GGGGCC sequence present in the intron region between the exon 1a and exon 1b regions of the C9orf72 gene is abnormally repeated and expanded.
  • the FTLD is characterized by progressive abnormal behavior.
  • patients are observed to have three or more symptoms, such as disinhibited behavior, apathy, lethargy, fixation, stereotypy, or lip-smacking, and changes in eating habits.
  • FTLD neurodegenerative disease
  • the Huntington's disease refers to a hereditary neurodegenerative disease that is inherited in an autosomal dominant manner and is caused by the abnormal repetitive expansion of the CAG sequence present in the exon 1 region of the Huntington's gene.
  • the Huntington's disease presents movement disorders characterized by involuntary movements, psychiatric symptoms, and cognitive symptoms. Huntington's disease is diagnosed when specific neurological findings are observed and abnormal expansion mutations of the CAG sequence are observed in genetic diagnosis, or when a progressive course is observed, a family history of autosomal dominant inheritance, specific neurological findings, and clinical test findings are observed, and similar diseases are denied in differential diagnosis.
  • spinocerebellar ataxia types 1, 2, 3, 6, 7, 8, 12, and 17
  • dentatorubral-pallidoluysian atrophy refers to hereditary neurodegenerative diseases that are inherited in an autosomal dominant manner and are caused by the abnormal repetition and expansion of a specific three-base sequence (CAG or CTG) present on the responsible gene in each disease.
  • CAG or CTG three-base sequence
  • spinocerebellar ataxia type 8 a repeat sequence of CTG is observed.
  • the above-mentioned spinocerebellar ataxia and dentatorubral-pallidoluysian atrophy are diagnosed by combining genetic diagnosis, neuropathological diagnosis, etc., with cerebellar or posterior column ataxia or spastic paraplegia as the main symptoms and being slowly progressive.
  • the above-mentioned spinal and bulbar muscular atrophy refers to a hereditary disease caused by abnormal repeated expansion of the CAG sequence present in the exon region of the androgen receptor gene.
  • the above-mentioned spinal and bulbar muscular atrophy is diagnosed by combining neurological findings (bulbar symptoms, lower motor nerve symptoms, finger tremor, decreased tendon reflexes), clinical findings, laboratory findings, genetic diagnosis, etc.
  • the Friedreich ataxia refers to a hereditary neurodegenerative disease that is autosomal recessive and occurs due to a mutation in the frataxin gene. In most cases of Friedreich ataxia, the GAA sequence in the first intron is abnormally repeated and expanded. By doing so.
  • the fragile X-associated tremor and ataxia syndrome refers to a hereditary neurodegenerative disease caused by abnormal repeated expansion of the CGG sequence present in the 5'UTR of the FMP1 gene.
  • the fragile X-associated tremor and ataxia syndrome is diagnosed by combining clinical symptoms (cerebellar ataxia, motor tremor, Parkinsonism, dementia, intellectual disability), middle cerebellar peduncle signs by MRI examination, genetic diagnosis, etc.
  • the myotonic dystrophy refers to a hereditary muscle disease that is inherited in an autosomal dominant manner and is caused by an abnormal repeated expansion of the CUG sequence present in the 3'UTR of the DMPK gene.
  • the above-mentioned individual means a mammal.
  • the above-mentioned individual is preferably a human, a monkey, a marmoset, a dog, a pig, a rabbit, a guinea pig, a rat, or a mouse.
  • the above-mentioned individual is more preferably a human.
  • the single-stranded antisense oligonucleotide or pharmaceutical composition thereof may be administered to a subject (individual) sensitive to delayed central toxicity by a suitable administration route.
  • delayed central toxicity refers to central toxicity that appears after a period during which acute central toxicity may appear has passed and recovery has occurred. Examples of symptoms observed due to delayed central toxicity include decreased spontaneous movement, abnormal gait and abnormal function of the hind limbs, tremors, weakness of the hind limbs or tail, loss of hind limb reflexes, weight loss, etc. This toxicity is scored based on the general condition findings observed on the observation day according to the following criteria, and the Clinical sign score is calculated by adding up the scores throughout the observation period.
  • the score is added depending on the intensity of the toxicity findings, a single-stranded antisense oligonucleotide with a low Clinical sign score can be judged to be highly safe.
  • the Pathological score is calculated by scoring the cases where abnormal findings were observed and those where they were not observed in the pathological examination of the brain. The average score for each group will be used.
  • subjects (individuals) sensitive to delayed central nervous system toxicity refers to subjects selected using biomarkers, etc.
  • Clinical sign score 0 points: no abnormality 1 point: abnormal hind leg function, tremor, decreased spontaneous movement 2 points: dragging of hind legs, weakness of tail or hind legs 3 points: complete hind leg dysfunction, paralysis of hind legs, recumbency, prone position 4 points: euthanasia Pathological score; 0 points: no abnormality 1 point: abnormality (single cell necrosis, vacuolation, etc.)
  • nucleic acid drugs may hybridize to RNA (off-target candidate gene or off-target candidate RNA) having the same or similar base sequence as the target RNA, and may manifest toxicity due to affecting the off-target candidate gene (sometimes referred to as "narrow-sense off-target toxicity").
  • RNA off-target candidate gene or off-target candidate RNA
  • a search for similar base sequences using "high-speed base sequence search: GGGenome (https://gggenome.dbcls.jp/ja/)" or the like can be performed to estimate the narrow-sense off-target candidate gene.
  • GGGenome https://gggenome.dbcls.jp/ja/
  • the administration method and dosage form are not particularly limited.
  • any administration method and formulation known in the art can be used as the administration method and formulation of the antisense oligonucleotide of the present invention.
  • the administration method include oral administration and parenteral administration.
  • parenteral administration include eye drop administration, intravaginal administration, intrarectal administration, intranasal administration, transdermal administration, intravenous injection, drip infusion, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration by aspiration or inhalation, intrathecal administration, and intraventricular administration.
  • the single-stranded antisense oligonucleotide or pharmaceutical composition thereof is preferably administered so as to be exposed to the central nervous system.
  • the administration method that exposes the central nervous system include intrathecal administration and intraventricular administration.
  • the antisense oligonucleotides and other formulations of the present invention may be mixed with various pharmaceutical additives, such as excipients, binders, wetting agents, disintegrants, lubricants, diluents, flavoring agents, fragrances, solubilizing agents, suspending agents, emulsifiers, stabilizers, preservatives, and isotonicity agents, as necessary.
  • various pharmaceutical additives such as excipients, binders, wetting agents, disintegrants, lubricants, diluents, flavoring agents, fragrances, solubilizing agents, suspending agents, emulsifiers, stabilizers, preservatives, and isotonicity agents, as necessary.
  • formulations such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, etc. can be used.
  • the pharmaceutical composition of the present invention such as the antisense oligonucleotide
  • it can be in the form of, for example, a powder, granules, a suspension or solution dissolved in water or a non-aqueous medium, capsules, powders, tablets, or other formulations.
  • compositions such as the antisense oligonucleotides of the present invention parenterally, intrathecally, or intraventricularly
  • formulations such as sterile aqueous solutions can be used.
  • the effective dosage of the single-stranded antisense oligonucleotide of the present invention can be determined arbitrarily depending on the sex, age, weight, symptoms, etc. of the individual to be administered. Furthermore, it can also be determined arbitrarily depending on the method, route, frequency, etc. of administration. For example, the dosage can be 0.01 to 100 mg/kg, etc. Preferably, it is 0.1 to 50 mg/kg, and more preferably, it is 0.1 to 10 mg/kg.
  • the method for regulating expression of the RPS25 gene comprises the step of administering, as an active ingredient, the single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof, or the antisense oligonucleotide complex or a pharma- ceutical acceptable salt thereof to a cell, tissue, or individual expressing the RPS25 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 is the administration route described above.
  • examples of "cells expressing the RPS25 gene” include nerve cells that make up the central nervous system, nerve cells that make up the peripheral nervous system, and other cells that make up skin tissue.
  • the method for treating or preventing a repeat disease in this embodiment includes a step of administering the above-mentioned single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof, or the above-mentioned antisense oligonucleotide complex or a pharma-ceutical acceptable salt thereof, as an active ingredient, to an individual suffering from the repeat disease.
  • the above-mentioned individual is preferably a subject (individual) sensitive to delayed central nervous system toxicity.
  • the repeat disease may be the above-mentioned psychiatric and neurological disorders, muscular disorders, etc.
  • the dosage form, administration route, and dosage when administered to an individual may be appropriately selected from those described above.
  • the above describes the antisense oligonucleotide according to this embodiment.
  • the single-stranded antisense oligonucleotide having the above-mentioned configuration can regulate the expression of the RPS25 gene.
  • the inhibitory activity (knockdown activity) against the expression of the RPS25 gene can be measured by a known method. Examples of the method for measuring the knockdown activity include the methods described in Nature (2015) 518 (7539): 409-12 (Non-Patent Document 12) and WO 2022/097727 (Patent Document 7).
  • the knockdown activity can also be measured by transfection of the antisense oligonucleotide into HEK293T cells, which will be described later.
  • the single-stranded antisense oligonucleotide contains at least one 5'-CP nucleic acid in the gap region, thereby reducing delayed central nervous toxicity.
  • the "cells expressing the RPS25 gene” are treated with the antisense oligonucleotide for 6 hours to 3 days using a method such as lipofection, electroporation, or introduction by direct addition.
  • the cells used may be any cells expressing the RPS25 gene, such as HEK293T cells, more preferably nerve cells, and even more preferably human-derived nerve cells.
  • the cells treated with the antisense oligonucleotide may be collected immediately after treatment, or the antisense oligonucleotide may be removed and the cells may be continuously cultured.
  • RNA extracted from the collected cells is subjected to a reverse transcription reaction, and the amount of RPS25 mRNA is measured by carrying out real-time PCR or the like on the obtained complementary DNA using an RPS25 gene-specific probe.
  • An example of a probe used in real-time PCR is a Taqman probe.
  • reaction methods include a method in which the three steps of "(cDNA denaturation)-(annealing)-(extension reaction)" or the two steps of "(cDNA denaturation)-(annealing and extension reaction)" are repeated any number of times. The number of times the two or three steps are repeated is, for example, 25 to 45 times, preferably 35 to 40 times.
  • the (cDNA denaturation) temperature is, for example, 90°C to 98°C, preferably 92°C to 95°C.
  • the (annealing) temperature is, for example, 40°C to 70°C, preferably 50°C to 60°C.
  • the (extension reaction) temperature is, for example, 65°C to 75°C, preferably the optimal temperature for the polymerase used in the reaction.
  • the temperature (annealing and extension reaction) is, for example, 55°C to 70°C.
  • the collected cells are lysed to obtain an extract.
  • the amount of RPS25 protein contained in the extract is evaluated using immunochemical techniques such as Western blotting and ELISA (Enzyme-Linked Immunosorbent Assay).
  • Western blotting any device can be used for each step of electrophoresis, transfer, and detection.
  • the reaction time and reaction temperature of the membrane with the primary or secondary antibody can be set arbitrarily, for example, overnight at 4°C or 1 to 3 hours at room temperature.
  • the present invention is not limited to the above-mentioned embodiments.
  • the above-mentioned single-stranded antisense oligonucleotide includes the following embodiments.
  • the antisense oligonucleotide according to the first aspect of the present invention comprises: A single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof that regulates the expression and/or function of the RPS25 gene,
  • the single-stranded antisense oligonucleotide has each nucleoside linked by a phosphate group and/or a modified phosphate group;
  • the single-stranded antisense oligonucleotide comprises a gap region, a 3' wing region attached to the 3' end of the gap region, and a 5' wing region attached to the 5' end of the gap region;
  • the gap region is a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the gap region comprises at least one 5'-CP nucleic acid;
  • the 3' wing region and the 5' wing region are modified nucleic acids having a substitution at
  • the antisense oligonucleotide according to the second aspect of the present invention is A single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof that regulates the expression and/or function of the RPS25 gene,
  • the single-stranded antisense oligonucleotide has each nucleoside linked by a phosphate group and/or a modified phosphate group;
  • the single-stranded antisense oligonucleotide comprises a gap region, a 3' wing region attached to the 3' end of the gap region, and a 5' wing region attached to the 5' end of the gap region;
  • the gap region is a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the gap region comprises at least one 5'-CP nucleic acid;
  • the 3' wing region and the 5' wing region are modified nucleic acids having a substitution at the
  • the antisense oligonucleotide according to the third aspect of the present invention is A single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof that regulates the expression and/or function of the RPS25 gene,
  • the single-stranded antisense oligonucleotide has each nucleoside linked by a phosphate group and/or a modified phosphate group;
  • the single-stranded antisense oligonucleotide comprises a gap region, a 3' wing region attached to the 3' end of the gap region, and a 5' wing region attached to the 5' end of the gap region;
  • the gap region is a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the gap region comprises at least one 5'-CP nucleic acid;
  • the 3' wing region and the 5' wing region are modified nucleic acids having a substitution at the
  • the base sequence of the single-stranded antisense oligonucleotide has a sequence identity of 95% to 100% based on a base sequence complementary to at least one target region of the base sequence set forth in SEQ ID NO: 1, the base sequence being composed of the same base length as the single-stranded antisense oligonucleotide.
  • the base sequence of the single-stranded antisense oligonucleotide is a base sequence complementary to at least one target region in the base sequence set forth in SEQ ID NO: 1, the target region having the same base length as the single-stranded antisense oligonucleotide.
  • the number of bases in the gap region is 5 to 20 mer
  • the 3' wing region is a 3-5 mer modified nucleic acid having a substituent at the 2' position
  • the 5' wing region is preferably a 3- to 5-mer modified nucleic acid having a substituent at the 2' position.
  • the 5'-CP nucleic acid is located at least at the second position counting from the 5' side of the gap region.
  • two or more of the 5'-CP nucleic acid are arranged in the gap region.
  • the 5'-CP nucleic acids it is preferable that 2 to 5 of the 5'-CP nucleic acids are arranged in the gap region.
  • the 5'-CP nucleic acid is arranged in an amount of at least 1/9 of the gap region.
  • the 5'-CP nucleic acid is arranged in a position that is at least one-fifth of the gap region.
  • the 5'-CP nucleic acid is arranged in a continuous 2-4 mer sequence at least at one location in the gap region.
  • the 5'-CP nucleic acid is located on the 5' end side of the gap region.
  • the 5'-CP nucleic acid is preferably located at the 5' end and 3' end of the gap region.
  • the base length of the single-stranded antisense oligonucleotide is preferably 15 to 20 mer.
  • the base length of the single-stranded antisense oligonucleotide is preferably 18 to 20 mer.
  • the modified nucleic acid having a substituent at the 2'-position in the 3'-wing region comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA;
  • the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region preferably comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.
  • the modified nucleic acid having a substituent at the 2' position in the 3' wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA
  • the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region is preferably comprised of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.
  • the modified nucleic acid having a substituent at the 2' position in the 3' wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA
  • the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region is preferably composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA.
  • At least one internucleoside bond of the single-stranded antisense oligonucleotide is a phosphorothioate bond.
  • At least one internucleoside bond in the single-stranded antisense oligonucleotide is a phosphodiester bond.
  • the ratio of phosphorothioate bonds in the internucleoside bonds constituting the single-stranded antisense oligonucleotide is 50% to 80%.
  • the ratio of phosphorothioate bonds in the internucleoside bonds constituting the single-stranded antisense oligonucleotide is 50% to 70%.
  • the bond between the 5'-CP nucleic acid and the nucleoside adjacent to the 3' side of the 5'-CP nucleic acid is preferably a phosphorothioate bond (except when the 5'-CP nucleic acid is located at the 3' end of the gap region).
  • the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is preferably a phosphorothioate bond (excluding cases where the 5'-CP nucleic acid is located at the 3' end of the gap region and where the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is a 5'-CP nucleic acid).
  • the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 5' side of the 5'-CP nucleic acid is preferably a phosphodiester bond.
  • the base sequence of the single-stranded antisense oligonucleotide is A base sequence having a sequence identity of 90% to 100% based on a base sequence complementary to a target region consisting of a continuous 14-22 mer from bases located at positions 123, 124, 185, 186, 263, 264, 324, 325, 442, 443, or 447 to 453 from the 5' end in the base sequence set forth in SEQ ID NO: 1; A base sequence complementary to a base sequence in which one or several bases are deleted, substituted, inserted or added in the target region; or It is preferable that the base sequence hybridizes under stringent conditions to the oligonucleotide having the above-mentioned target region.
  • the base sequence of the single-stranded antisense oligonucleotide is A base sequence having a sequence identity of 90% to 100% based on a base sequence complementary to a target region consisting of a continuous 14-22mer from the bases located at positions 324, 325, or 448 to 453 from the 5' end in the base sequence set forth in SEQ ID NO: 1; A base sequence complementary to a base sequence in which one or several bases are deleted, substituted, inserted or added in the target region; or It is preferable that the base sequence hybridizes under stringent conditions to the oligonucleotide having the above-mentioned target region.
  • the base sequence of the single-stranded antisense oligonucleotide has a sequence identity of 90% to 100% based on a base sequence complementary to a target region consisting of a continuous 18-22mer from bases 448 to 453 counting from the 5' end in the base sequence set forth in SEQ ID NO: 1, the 3' wing region is a 3-5 mer, The 5' wing region is preferably a 3-5 mer.
  • the base sequence of the single-stranded antisense oligonucleotide is preferably a base sequence that has 90% to 100% sequence identity with respect to a base sequence complementary to a target region consisting of 18 to 20 consecutive bases from the 450th to 451st or 453rd bases counting from the 5' end in the base sequence set forth in SEQ ID NO: 1.
  • the base sequence of the single-stranded antisense oligonucleotide is preferably one selected from the group consisting of the base sequences of SEQ ID NOs: 5 to 47 and 49 to 56.
  • the base sequence of the single-stranded antisense oligonucleotide is preferably one selected from the group consisting of the base sequences of SEQ ID NOs: 6, 9, 12, 15-18, 22, 24, 27-29, 31-36, 38, 40-42, 47, and 49-54.
  • the base sequence of the single-stranded antisense oligonucleotide is preferably one selected from the group consisting of the base sequences of SEQ ID NOs: 41, 47, 50, and 53 to 55.
  • An antisense oligonucleotide complex according to one embodiment of the present invention comprises the single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof, and an additional substance bound to the single-stranded antisense oligonucleotide, or a pharma- ceutically acceptable salt thereof,
  • the additional substance is selected from the group consisting of polyethylene glycol, peptides, alkyl chains, nucleic acids, ligand compounds, antibodies, proteins, and sugar chains.
  • a pharmaceutical product according to one aspect of the present invention contains the above-mentioned single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof, or the above-mentioned antisense oligonucleotide complex or a pharma-ceutical acceptable salt thereof, as an active ingredient.
  • the single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof, or the antisense oligonucleotide complex or a pharma- ceutically acceptable salt thereof is preferably administered so as to be exposed to the central nervous system.
  • the single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof, or the antisense oligonucleotide complex or a pharma- ceutically acceptable salt thereof is preferably administered to a subject susceptible to delayed central nervous system toxicity.
  • an agent for regulating the expression and/or function of the RPS25 gene contains the above-mentioned single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof, or the above-mentioned antisense oligonucleotide complex or a pharma-ceutical acceptable salt thereof, as an active ingredient.
  • An inhibitor of dipeptide repeat production by RNA translation contains the above-mentioned single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof, or the above-mentioned antisense oligonucleotide complex or a pharma-ceutical acceptable salt thereof, as an active ingredient.
  • a therapeutic agent for repeat disease contains the above-mentioned single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof, or the above-mentioned antisense oligonucleotide complex or a pharma-ceutical acceptable salt thereof, as an active ingredient.
  • a preventive agent for repeat disease contains the above-mentioned single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof, or the above-mentioned antisense oligonucleotide complex or a pharma-ceutical acceptable salt thereof, as an active ingredient.
  • the repeat disease is preferably at least one selected from the group consisting of C9orf72 ALS, C9orf72 FTLD, Huntington's disease, spinocerebellar ataxia, dentatorubral-pallidoluysian atrophy, spinal-bulbar muscular atrophy, Friedreich ataxia, fragile X-associated tremor ataxia syndrome, and myotonic dystrophy.
  • a method for treating or preventing a repeat disease comprises administering to an individual the single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof, or the antisense oligonucleotide complex or a pharma-ceutical acceptable salt thereof.
  • the repeat disease is preferably at least one selected from the group consisting of C9orf72 ALS, C9orf72 FTLD, Huntington's disease, spinocerebellar ataxia, dentatorubral-pallidoluysian atrophy, spinal-bulbar muscular atrophy, Friedreich ataxia, fragile X-associated tremor ataxia syndrome, and myotonic dystrophy.
  • One aspect of the present invention provides the above-mentioned single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof, or the above-mentioned antisense oligonucleotide complex or a pharma-ceutical acceptable salt thereof, for use in the treatment or prevention of C9orf72 ALS.
  • One aspect of the present invention provides the above-mentioned single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof, or the above-mentioned antisense oligonucleotide complex or a pharma-ceutical acceptable salt thereof, for use in producing a therapeutic or preventive agent for C9orf72 ALS.
  • Single-stranded antisense oligonucleotides containing modified nucleic acids such as 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, AmNA, scpBNA, 5'-CP nucleic acid, and/or GuNA, and/or nucleic acid whose nucleic acid base is 5-methylcytosine, were synthesized on a 0.2 ⁇ mol scale using an automatic nucleic acid synthesizer (nS-8 type, manufactured by Gene Design Co., Ltd.). Chain length was extended using a standard phosphoramidite protocol.
  • modified nucleic acids such as 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, AmNA, scpBNA, 5'-CP nucleic acid, and/or GuNA
  • nS-8 type automatic nucleic acid synthesizer
  • the solid support was deprotected by treating it with 0.5 M hydrazine hydrate in pyridine-acetic acid (1:1 v/v) at room temperature for 1 hour outside the synthesizer, and then the solid support was attached to the synthesizer and synthesis was resumed.
  • CPG resin was used as the solid support.
  • DDTT ((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazaoline-3-thione) or the like was used.
  • Single-stranded antisense oligonucleotides containing 2'-MOE nucleic acid, AmNA and/or scpBNA were obtained in which the hydroxyl group at the terminal 5' position was not protected with a DMTr (4,4'-dimethoxytrityl) group and the 3' position was supported on a solid phase.
  • the single-stranded antisense oligonucleotide was subsequently cut out from the solid phase support by alkali treatment and recovered in the form of a solution. Thereafter, the solvent was distilled off from the recovered solution to obtain a crude product.
  • the crude product obtained was purified by reverse phase HPLC to obtain a purified single-stranded antisense oligonucleotide. The purity and structure of each single-stranded antisense oligonucleotide obtained were confirmed by LC-MS (Waters).
  • Tables 2-1 to 2-4, 3-1, and 3-2 below list the single-stranded antisense oligonucleotides produced by the above-mentioned method.
  • the single-stranded antisense oligonucleotides shown in Tables 2-1 to 2-4, 3-1, and 3-2 are single-stranded antisense oligonucleotides against human RPS25 mRNA (SEQ ID NO: 1).
  • R 1 and R 2 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 3 carbon atoms.
  • R 3 , R 4 , and R 5 each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 7 carbon atoms, or a cycloalkyl group having 3 to 7 carbon atoms.
  • R 3 and R 5 in the GuNA shown by “Gx” above are both hydrogen atoms and R 4 is a methyl group, it is represented as “Gm”
  • R 3 is a hydrogen atom and R 4 and R 5 are both methyl groups
  • Gdm when R 3 and R 5 are hydrogen atoms and R 4 is a tert-butyl group, it is represented as "GtB”.
  • the expression evaluation of the RPS25 gene was performed by expression evaluation using human fetal kidney cells according to the single-stranded antisense oligonucleotide produced.
  • the expression evaluation of the RPS25 gene can also be performed using human iPS cell-derived nerve cells.
  • gene expression evaluation means evaluating the amount of mRNA by measuring the amount of complementary DNA (cDNA) obtained by reverse transcription reaction. The specific procedures for each expression evaluation are described below.
  • Human embryonic kidney cells HEK293T (ATCC® CRL-3216TM) were cultured in a culture medium at 37° C. and 5% CO 2.
  • the culture medium for HEK293T cells had the following composition:
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • S1820 100-fold diluted penicillin-streptomycin mixed solution: Nacalai Tesque Cat#09367-34 (penicillin 10,000 units/ml, streptomycin 10,000 ⁇ g/ml, stabilizer included)
  • HEK293T cells (12,000 cells/well) were seeded in a 96-well plate and cultured overnight at 37°C and 5% CO2 . Then, each single-stranded antisense oligonucleotide (final concentration 0.5 nM, 5 nM, 15 nM, or 50 nM) diluted with phosphate-buffered saline (PBS) was transfected into the above-mentioned cells by lipofection. As a negative control, cells transfected with PBS in which the single-stranded antisense oligonucleotide was not dissolved were used.
  • PBS phosphate-buffered saline
  • the transfected cells were cultured in growth medium at 37°C and 5% CO2 for 48 hours.
  • the growth medium was then removed, and the extracted total RNA was subjected to reverse transcription using the Taqman Fast Cells-to-CT Kit (Thermo Fisher Scientific, Cat#4399003).
  • the complementary DNA (cDNA) obtained from this reverse transcription reaction was used to perform real-time PCR using pre-designed gene-specific probes (see below) in Taqman gene expression assays (Applied Biosystems) (40 cycles of 95°C for 3 seconds and 60°C for 30 seconds).
  • the expression ratio of human RPS25 mRNA in each single-stranded antisense oligonucleotide relative to human RPS25 mRNA, determined by the above-mentioned method, is shown in Tables 5-1 to 5-3 and Tables 6-1 to 6-2 (column "hRPS25 expression ratio"). At this time, the expression ratio of human RPS25 mRNA determined in the negative control group was set to 1.00. Those with an expression ratio of 0.80 or less were determined to be single-stranded antisense oligonucleotides capable of suppressing the expression of human RPS25 mRNA. In the tables, "-" indicates that no measurement was performed.
  • those with the above expression ratio of 0.80 or less can be determined to be single-stranded antisense oligonucleotides capable of regulating the function of the human RPS25 gene.
  • Mouse primary cultured nerve cells are cultured in a culture medium at 37° C. and 5% CO 2.
  • the culture medium for mouse primary cultured nerve cells has the following composition.
  • mouse primary cultured nerve cells (derived from mouse fetal cerebrum) are seeded in a 96-well plate with each cell (40,000 cells/well) and cultured for 5 days under conditions of 37 ° C and 5% CO 2. Then, each single-stranded antisense oligonucleotide (final concentration 0.01 ⁇ M, 0.1 ⁇ M, or 1 ⁇ M) diluted with phosphate-buffered saline (PBS) is added to the culture medium. As a negative control group, PBS in which the single-stranded antisense oligonucleotide is not dissolved is added to the culture medium. The cells are cultured in the culture medium for 48 hours under conditions of 37 ° C and 5% CO 2.
  • PBS phosphate-buffered saline
  • RNA is subjected to reverse transcription reaction using Taqman Fast Cells-to-CT Kit (manufactured by Thermo Fisher Scientific, Cat # 4399003).
  • the complementary DNA (cDNA) obtained from this reverse transcription reaction is used to perform real-time PCR using predesigned gene-specific probes (see below) in Taqman gene expression assays (Applied Biosystems) (40 cycles of 95°C for 3 seconds and 60°C for 30 seconds).
  • Motor neuron cells are differentiated from human iPS cells and used for evaluation. Cell maintenance and differentiation induction are performed in the medium described below at 37°C and 5% CO2 .
  • Mitomycin treatment of feeder cells As feeder cells for seeding human iPS cells, mitomycin-treated SNL cells (Cell Bio Labs, Cat# CBA-316) are prepared. Mitomycin treatment of SNL cells is performed as follows. First, 0.1% gelatin (FUJIFILM Wako Pure Chemical Industries, Cat# 190-15805) is added to a 10 cm petri dish (Iwaki, Cat# 3020-100), and the petri dish is left to stand for 1 hour or more in an incubator under conditions of 37°C and 5% CO2 (hereinafter, this operation may be referred to as "gelatin treatment").
  • 0.1% gelatin (FUJIFILM Wako Pure Chemical Industries, Cat# 190-15805) is added to a 10 cm petri dish (Iwaki, Cat# 3020-100), and the petri dish is left to stand for 1 hour or more in an incubator under conditions of 37°C and 5% CO2 (hereinafter, this operation may be referred to as "gelatin treatment").
  • SNL cells are seeded using SNL cell medium so that 1 to 2 x 106 cells are seeded per petri dish.
  • the cells are diluted 8 to 16 times every 3 to 4 days and passaged to grow to the required number of cells.
  • 2-4 x 106 SNL cells are seeded per dish in a 15 cm dish (Iwaki, Cat#3030-150) that has been treated with 0.1% gelatin.
  • mitomycin C (Kyowa Kirin, YJ code 4231400D1031) diluted to 0.4 mg/mL with SNL cell medium is added to the dish to a final concentration of 6.2 ⁇ g/mL.
  • the dish is left to stand for 2 hours and 15 minutes in an incubator under conditions of 37°C and 5% CO2 . Thereafter, the medium is removed from the dish, and the SNL cells are washed once with PBS. 2.5% trypsin/EDTA (ThermoFisher Scientific, Cat#15090-046) was diluted with PBS (final concentration 0.25%) and added to the SNL cells and allowed to stand at room temperature for 1 minute.
  • the SNL cells were then collected in a tube and centrifuged, suspended in Cellbanker® (ZENOAQ Resources, Cat#CB011), and frozen for storage.
  • human iPS cells (201B7 strain, obtained from iPS Academia Japan, Inc., AJ-H1-01) suspended in iPS cell medium containing 1/1000 amount of Y-27632 (manufactured by Tocris, Cat#1254) are seeded in the petri dish. The medium is replaced every day from the day after seeding until differentiation induction begins.
  • ⁇ Induction of differentiation of human iPS cells into motor neurons The iPS cells are exposed to the Y-27632 for 1 hour or more by adding Y-27632 (final concentration 10 ⁇ M) to the cell culture solution of human iPS cells. After removing the culture supernatant and washing the cells with PBS, Cell dissociation solution (CTK solution) (REPROCELL, Cat# RCHETP002) is added and reacted at room temperature for 1 minute. After removing the CTK solution and washing the cells twice with PBS, 1 mL of iPS cell medium is added.
  • CTK solution Cell dissociation solution
  • the cells are detached with a cell scraper, and the cell clumps are dispersed through a cell strainer (Becton Dickinson, Cat# 352350) to obtain a cell suspension.
  • the obtained suspension is transferred to a 6-well plate (Corning, Cat# 3471).
  • Mixed medium A was replaced with a medium supplemented with LDN193189 (Stemgent, Cat#04-0074) (final concentration 0.3 ⁇ M), SB431542 (Tocris, Cat#1614) (final concentration 2 ⁇ M), CHIR-99021 (Stemgent, Cat#04-0004-10) (final concentration 3 ⁇ M), and Y-27632 (final concentration 10 ⁇ M), and the cells were cultured in an incubator at 37° C. and 5% CO 2 (culture day 0).
  • the culture medium was removed with a pipette and replaced with fresh medium containing mixed medium A supplemented with LDN193189 (final concentration 0.3 ⁇ M), SB431542 (final concentration 2 ⁇ M), and CHIR-99021 (final concentration 3 ⁇ M).
  • the culture medium was removed with a pipette and replaced with fresh medium containing mixed medium A supplemented with LDN193189 (final concentration 0.3 ⁇ M), SB431542 (final concentration 2 ⁇ M), CHIR-99021 (final concentration 3 ⁇ M), Purmorphamine (FUJIFILM Wako Pure Chemical Industries, Ltd., Cat#166-23991) (final concentration 0.5 ⁇ M), and Retinoic Acid (Sigma-Aldrich, Cat#R2625) (final concentration 0.1 ⁇ M).
  • the culture medium was removed with a pipette and replaced with a fresh medium containing mixed medium A supplemented with Purmorphamine (final concentration: 0.5 ⁇ M), Retinoic Acid (final concentration: 0.1 ⁇ M), Human BDNF (final concentration: 10 ng/mL), and Ascorbic Acid (Sigma-Aldrich, Cat#A5960) (final concentration: 200 ⁇ M).
  • Purmorphamine final concentration: 0.5 ⁇ M
  • Retinoic Acid final concentration: 0.1 ⁇ M
  • Human BDNF final concentration: 10 ng/mL
  • Ascorbic Acid Sigma-Aldrich, Cat#A5960
  • mixed medium B was replaced with a fresh medium supplemented with Purmorphamine (final concentration 0.5 ⁇ M), Retinoic Acid (final concentration 0.1 ⁇ M), and Compound E (Calbiochem, Cat#565790) (final concentration 0.1 ⁇ M).
  • Purmorphamine final concentration 0.5 ⁇ M
  • Retinoic Acid final concentration 0.1 ⁇ M
  • Compound E Calbiochem, Cat#565790
  • the cell masses are washed with PBS, centrifuged, and the supernatant is removed.
  • Accutase Innovative Cell Technologies, Cat#AT104
  • Y27632 final concentration 10 ⁇ M
  • the human iPS cell-derived motor neurons frozen and stored in the previous section are thawed and suspended in a neuronal medium.
  • the supernatant is then removed by centrifugation, and the cells are resuspended in a neuronal medium containing 1/100 of the amount of Culture One Supplement (ThermoFisher Scientific, A3320201) and Compound E (final concentration 0.1 ⁇ M).
  • the cells are seeded on a coated 96-well plate at 30,000 cells/well and cultured for 28 days in an incubator at 37°C and 5% CO2 .
  • Half of the neuronal medium is replaced every 2 to 3 days. From the start of culture until the 7th day, a medium containing Culture One Supplement and Compound E is used as the neuronal medium.
  • each single-stranded antisense oligonucleotide diluted with PBS (final concentrations 0.01 ⁇ M, 0.1 ⁇ M, and 1 ⁇ M) is added to the culture medium.
  • PBS final concentrations 0.01 ⁇ M, 0.1 ⁇ M, and 1 ⁇ M
  • cells in which PBS in which the single-stranded antisense oligonucleotide is not dissolved is added to the culture medium are used. After culturing the cells in the culture medium at 37° C.
  • the medium containing the single-stranded antisense nucleotide is removed, and the cells are continuously cultured in a neuronal medium (half the medium is replaced once every 2 to 3 days). Thereafter, the culture medium is removed, and the extracted total RNA is subjected to a reverse transcription reaction using Taqman Fast Cells-to-CT Kit (Thermo Fisher Scientific, Cat#4399003).
  • the complementary DNA (cDNA) obtained from this reverse transcription reaction is used to perform real-time PCR using a predesigned gene-specific probe (see below) in Taqman gene expression assays (Applied Biosystems) (95°C; 3 seconds, 60°C; 30 seconds, 40 cycles).
  • the expression evaluation of RPS25 protein is performed using human fetal kidney cells according to the single-stranded antisense oligonucleotide produced.
  • the protein expression level evaluation means evaluating the amount of protein translated from mRNA. The specific procedure for expression evaluation is described below.
  • Human embryonic kidney cells HEK293T (ATCC® CRL-3216TM) are cultured in a culture medium at 37° C. and 5% CO 2.
  • the culture medium for HEK293T cells has the following composition.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • S1820 100-fold diluted penicillin-streptomycin mixed solution: Nacalai Tesque Cat#09367-34 (penicillin 10,000 units/ml, streptomycin 10,000 ⁇ g/ml, stabilizer included)
  • HEK293T cells (500,000 cells/well) are seeded in a 6-well plate and cultured overnight at 37°C and 5% CO2 . Then, each single-stranded antisense oligonucleotide (final concentration 50 nM) diluted with phosphate-buffered saline (PBS) is transfected into the above cells using the lipofection method. As a negative control group, cells transfected with PBS in which the single-stranded antisense oligonucleotide is not dissolved are used. The transfected cells are cultured in a growth medium at 37°C and 5% CO2 for 48 hours.
  • PBS phosphate-buffered saline
  • the growth medium is removed, washed with PBS, and the cells are collected with a cell scraper.
  • the collected solution is centrifuged at 2700 x g, 5 minutes, and 4°C to precipitate the cells.
  • 1 mL of RIPA Lysis and Extraction buffer (ThermoFisher Scientific, Cat # 89900) containing 1/100 of Protease Inhibitor (ThermoFisher Scientific, Cat # 1860932) is added, and the cells are disrupted by an ultrasonic disrupter. Then, the cells are centrifuged under conditions of 15000 ⁇ g, 10 minutes, and 4 ° C., and the supernatant is used as a sample.
  • the collected sample is subjected to protein quantification using Pierce (trademark) BCA Protein Assay kit (ThermoScientific, Cat # 23225). After adjusting the concentration of each sample to a constant level, PierceTM Lane Marker Reducing Sample Buffer (ThermoFisher Scientifier, Cat#39000) is added and heated at 95°C for 5 minutes. The prepared samples are layered so that the protein amount is 10 ⁇ g to 20 ⁇ g/lane, and electrophoresis is performed. Electrophoresis is performed for 30 minutes at a constant voltage of 200V using CriterionTM TDXTM Precast Gel 4-15% (BIO-RAD, Cat#5671085J10). The electrophoresis buffer used is Running Buffer Solution (10x) for SDS-PAGE (Nacalai Tesque, Cat#30329-61) diluted to 1x concentration.
  • a Trans-Blot Turbo Transfer Pack (manufactured by BIO-RAD, Cat#1704157) is used as the membrane, and the Standard protocol (30 minutes) of the BIO-RAD Trans-Blot Turbo Transfer System is used as the transfer device.
  • the membrane is washed with TBST.
  • the composition of TBST is Tris-buffered saline (pH 7.4) (manufactured by Nacalai Tesque, Cat#35438-81) diluted to 1x concentration containing 0.06% polyoxyethylene sorbitan monolaurate (Tween-20) (manufactured by Nacalai Tesque, Cat#28353-85).
  • ⁇ RPS25 Antibody Anti-RPS25 antibody (Abcam, Cat# ab102940)
  • Solvent Canget signal Solution 1 (manufactured by Toyobo Co., Ltd., Cat#NKB-101)
  • ⁇ -actin Antibody ⁇ -Actin (13E5)
  • Rabbit mAb HRP conjugate
  • Solvent Blocking One
  • the secondary antibody and dilution solvent were as follows: ⁇ RPS25 Antibody: Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody (manufactured by Invitrogen, Cat#A24537) Solvent: Canget Signal Solution 2 (manufactured by Toyobo Co., Ltd., Cat#NKB-101) After shaking with the secondary antibody, the plate is washed with TBST and then detected using ECL prime (Amersham, Cat#RPN2232). Amersham Imager 680 is used for detection and analysis.
  • the expression ratio of human RPS25 protein for each single-stranded antisense oligonucleotide determined by the above method is calculated. At this time, the expression ratio of human RPS25 protein determined in the negative control group is set to 1.00. When the protein expression ratio is below 0.80, it can be determined that the single-stranded antisense oligonucleotide is capable of regulating the function of the RPS25 gene.
  • HeLa-S3 cells which are human cervical cancer cells, were cultured in a growth medium at 37° C. and 5% CO 2.
  • the growth medium used had the following composition:
  • composition of growth medium used for cytotoxicity evaluation 10% fetal bovine serum (FBS): GIBCO, CAT# 10437028 1%
  • the above cells (1.0 x 10 4 cells/well) were seeded on a 96-well plate the day before the experiment. After culturing the seeded cells overnight at 37°C and 5% CO 2 , each single-stranded antisense oligonucleotide (final concentration: 1 to 200 nM) complexed with Lipofectamine 3000 (Thermo Fisher Scientific, Cat#L3000-015) in Opti-Minimum Essential Medium (Thermo Fisher Scientific, Cat#31985070) was added, and the above cells were cultured for 24 hours at 37°C and 5% CO 2 .
  • Lipofectamine 3000 Thermo Fisher Scientific, Cat#L3000-015
  • Opti-Minimum Essential Medium Thermo Fisher Scientific, Cat#31985070
  • the solution After adding ultrapure water (10 ⁇ L), the solution is separated by centrifugation into an aqueous layer containing the single-stranded antisense oligonucleotide and a mineral oil layer, and the aqueous layer is analyzed by LC-MS (Waters), and the remaining single-stranded antisense oligonucleotide is calculated from the area intensity of the UV-chromatogram of the obtained single-stranded antisense oligonucleotide.
  • LC-MS Waters
  • Remaining oligonucleotide (%) indicates the remaining rate of undegraded single-stranded antisense oligonucleotide after 72 hours relative to the undegraded single-stranded antisense oligonucleotide at the time of analysis immediately after mixing with serum.
  • Those that have a remaining rate of 50% or more after 72 hours are determined to be stable single-stranded antisense oligonucleotides.
  • the expression evaluation of the RPS25 gene was performed by administering the gene intracerebroventricularly to mice and measuring the amount of mRNA in each area of the prefrontal cortex.
  • the gene expression evaluation means evaluating the amount of mRNA by measuring the amount of complementary DNA (cDNA) obtained by reverse transcription reaction. The specific procedures for each expression evaluation are described below.
  • FVB mice (CLEA Japan) were anesthetized with isoflurane (Pfizer, Cat#114133403).
  • antisense oligonucleotides dissolved in artificial cerebrospinal fluid (Tocris Bioscience, Cat#3525/25mL) were administered to the anesthetized FVB mice at 10 ⁇ L/individual using a two-stage needle (Top, Medical Device Approval Number 15800BZZ01460000) attached to a 50 ⁇ L Hamilton syringe (Hamilton, Cat#705LT).
  • Mice in the negative control group were administered only artificial cerebrospinal fluid at 10 ⁇ L/individual.
  • RNA extraction from the stored tissue samples was performed using RNeasy Mini Kit (QIAGEN, Cat# 74106).
  • Reverse transcription reaction from the extracted mRNA was performed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystem, Cat# 4368814). For the reverse transcription reaction, 1 ⁇ g of mRNA was diluted to 20 ⁇ L and used.
  • cDNA complementary DNA obtained from this reverse transcription reaction
  • real-time PCR was performed using pre-designed gene-specific probes (see below) in Taqman expression assays (Applied Biosystems) (95°C; 3 seconds, 60°C; 30 seconds, 40 cycles).
  • the expression ratio of mouse RPS25 mRNA for each single-stranded antisense oligonucleotide determined by the above method is shown in Tables 8-1 to 8-2.
  • the expression ratio of mouse RPS25 RNA determined in the negative control group was set to 1.00. Since it is generally believed that when mRNA expression is suppressed, subsequent translation into protein is also suppressed, when the expression ratio is 0.80 or less, it can be determined that the single-stranded antisense oligonucleotide is capable of regulating the function of the mouse RPS25 gene.
  • the target regions to which the single-stranded antisense oligonucleotides listed in Tables 8-1 to 8-2 bind are regions whose sequences are conserved between the human RPS25 gene and the mouse RPS25 gene.
  • Ptosis eye closure, decreased locomotor activity (disappearance) (1 point) 3.
  • Staggering (1 point), ataxic gait (2 points) 4.
  • Abnormal posture/irregular breathing (1 point), lying on the side/prone (2 points), slow/abnormal breathing (4 points) 5.
  • the “score” column in "in vivo delayed neuroTox” was calculated based on the following evaluation criteria. Each score was the average value for each group.
  • ICR means Crl:CD1 mice
  • FVB means FVB/NJcl mice.
  • the evaluation items were 1) posture, 2) abnormal appearance, 3) stereotypic behavior, 4) reactivity to stimuli, 5) grip strength, 6) respiration, and 7) trembling/convulsions, and each was scored as 0 for normal, 1 for slightly abnormal, and 2 for extremely abnormal.
  • the total score obtained for each item was entered in the "score" column of "in vivo acute neuroTox” in Tables 9-1 and 9-2.
  • the evaluation items were 1) posture, 2) external abnormality, 3) stereotypic behavior, 4) reactivity to stimuli, 5) grip strength, 6) respiration, and 7) tremors/convulsions, and were scored as 0 for normal, 1 for slightly abnormal, and 2 for extremely abnormal.
  • the total score obtained for each item was entered in the "Clinical Sign Score” column in "in vivo delayed neuroTox” in Tables 9-1 and 9-2.
  • GGGenome high-speed base sequence search system
  • the sequence information of each single-stranded antisense oligonucleotide was entered into GGGenome, and the number of narrow off-target RNAs was counted from the number of hybridization mismatches for human spliced RNA and human pre-spliced RNA registered in the database. If there was a mismatch, deletion, or insertion during hybridization, the number of mismatches was calculated by summing the number of corresponding bases.
  • the base sequence of the target RNA from the base sequence of the single-stranded antisense oligonucleotide is "AGCTGTAC”
  • the RNA having the base sequence of " ATCTGTAC” has a mismatch (underlined base) of "1”
  • the number of mismatches is calculated as "1”.
  • the base sequence of the target RNA is "AGCTGTAC”
  • the RNA having the base sequence " ATCTG ⁇ G>TAC” has a mismatch (underlined base) of "1” and a deletion (base surrounded by angle brackets) of "1”
  • the number of mismatches is calculated as "2".
  • the base sequence of the target RNA is "AGCTGTAC”
  • the RNA having the base sequence " ATCTG *AC” has a mismatch (underlined base) of "1” and an insertion (a portion marked with *) of "1”
  • the number of mismatches is calculated as "2".
  • the number of mismatches during hybridization to RNA other than the target (off-target RNA) is counted, and the number of RNAs that perfectly match, the number of RNAs with a number of mismatches of 1 or less, and the number of RNAs with a number of mismatches of 2 or less are calculated for each single-stranded antisense oligonucleotide.
  • RNAs with a mismatch number of 0, 1 or less, or 2 or less The fewer the number of RNAs with a mismatch number of 0, 1 or less, or 2 or less, the lower the narrowly defined off-target toxicity risk; conversely, the greater the number of RNAs with a mismatch number of 0, 1 or less, or 2 or less, the higher the narrowly defined off-target toxicity risk.
  • Tables 10-1 to 10-4 When the base length is 18 or more, the number of RNAs that hybridize with 1 or less mismatches is 10 or less, which is considered to be a low narrowly defined off-target risk, and is a more preferred single-stranded antisense oligonucleotide.

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