Antisense oligonucleotide capable of regulating expression and / or function of RPS25 gene

Abstract

Provided is a single-stranded antisense oligonucleotide capable of regulating the expression and / or function of RPS25 gene or a pharmaceutically acceptable salt thereof. In the single-stranded antisense oligonucleotide, each nucleoside is bound through a phosphate group and / or a modified phosphate group. 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, wherein the gap region comprises a nucleic acid composed of deoxyribose molecules in which a sugar moiety may be modified, the gap region includes at least one 5'-CP nucleic acid, and each of the 3'-wing region and the 5'-wing region comprises a modified nucleic acid having a substituent at position-2'. The single-stranded antisense oligonucleotide has a nucleotide length of 12- to 30-mer, and the nucleotide sequence for the single-stranded antisense oligonucleotide is a nucleotide sequence having sequence identity of 90% to 100% inclusive with respect to a nucleotide sequence complementary to at least one target region included in the nucleotide sequence represented by SEQ ID NO:1 and composed of the same nucleotide length as that of the single-stranded antisense oligonucleotide, or a nucleotide sequence complementary to a nucleotide sequence having a structure that one or several nucleotides are deleted, substituted, inserted or added in the above-mentioned target region, or a nucleotide sequence capable of hybridizing, under stringent conditions, with an oligonucleotide having the above-mentioned target region.

Description

Antisense oligonucleotides that regulate the expression and / or function of the RPS25 gene

[0001] The present invention relates to antisense oligonucleotides that regulate the expression and / or function of the RPS25 gene, and agents that regulate the expression and / or function of the RPS25 gene, including the same.

[0002] The ribosomal protein S25 (RPS25) gene 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 within the ribosomal 40S subunit has also been elucidated (Non-Patent Document 1).

[0003] The RPS25 protein binds to an RNA element that enables cap-independent translation initiation as part of the protein synthesis process, and regulates 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 particularly common in viruses (Non-Patent Document 2).

[0004] Repeat associated non-ATG translation (RAN translation) was first confirmed in patients with spinocerebellar ataxia type 8 in 2011 (Non-Patent Document 3). Ran translation refers to a mechanism by which a specific sequence is repeated to be translated into a protein (e.g., dipeptide repeat (DPR)) in an ATG-independent manner. Subsequently, the involvement of RAN translation has been reported in several repeat diseases (diseases caused by the repetition of specific gene sequences), 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 into 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 Documents 4 and 5).

[0005] In 2019, the RPS25 protein was reported as a molecule that contributes significantly to the production of dipeptide repeats by RAN translation. Knocking down the RPS25 gene inhibited the RAN translation-dependent production of DPRs derived from GGGGCC repeat sequences or CAG repeat sequences. The GGGGCC repeat sequence is known as an abnormal expansion mutation in the C9orf72 gene, which is one of the familial mutations in amyotrophic lateral sclerosis. The CAG repeat sequence is known as an abnormal expansion mutation in the huntingtin gene and the ATXN2 gene. It was revealed that knocking down the RPS25 gene inhibited the production of DPRs derived from GGGGCC repeat sequences and also inhibited motor neuron cell death in induced purullipotent stem cells (iPSCs)-derived motor neurons established from patients with C9orf72 gene mutations. (Non-Patent Document 6)

[0006] The antisense oligonucleotide for 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 is phosphorothioated. The base sequence of the antisense oligonucleotide contains a portion of the base sequence of the sense strand of the RPS25 gene.

[0007] As a result of extensive research, the inventors of the present application have reported a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that binds to the RPS25 gene and effectively regulates the expression of the RPS25 gene (Patent Document 7).

[0008] Antisense oligonucleotides, commonly referred to as gapmers, are compounds that contain a gap region composed of deoxyribose and wing regions containing modified nucleic acids developed for improved nuclease resistance, binding affinity, and specificity to target RNA. They exert their effects by hybridizing to target RNA sequences and suppressing gene expression. Various modified nucleic acids have been developed for clinical applications of gapmers, and numerous compounds (gapmers) utilizing these modified nucleic acids have been developed and marketed. Nucleic acid drugs, including gapmers, are known to accumulate in specific organs, such as the liver, after systemic exposure. During research and development, issues related to nucleic acid drug toxicity, such as hepatotoxicity, have become apparent. Efforts to resolve these issues (e.g., elucidating mechanisms and exploring methods to avoid them) are ongoing. However, the potential toxicity of gapmers to highly exposed sites, such as the eyes, brain, and skin, upon topical administration has not been fully elucidated, and challenges remain, such as elucidating the mechanisms of their expression and developing methods to avoid them.

[0009] The toxicity of antisense oligonucleotides can be categorized into so-called "toxicity due to hybridization with off-target RNA (narrowly defined off-target toxicity)" and "toxicity not due to hybridization with RNA (broadly defined off-target toxicity)," and various approaches have been adopted to avoid these toxicities. Regarding broadly defined off-target toxicity, as mentioned above, many studies have been conducted using toxicity in the liver, which is the organ of accumulation, as an indicator. Meanwhile, in recent years, while the development of antisense oligonucleotides for central nervous system diseases via intrathecal administration has been actively promoted, the issue of toxicity to the central nervous system (hereinafter sometimes referred to as "central toxicity") has emerged.

[0010] Regarding the relationship between antisense oligonucleotides and central nervous system toxicity, it is known that central nervous system symptoms such as convulsions appear within one hour of exposure to the central nervous system, and a correlation between changes in intracellular free calcium concentration in neurons and central toxicity has been disclosed (Patent Document 8, Non-Patent Document 7). Furthermore, a document evaluating the central toxicity of phosphorothioate modifications in antisense oligonucleotides discloses that in gapmers having phosphorothioate modifications, central toxicity is reduced by replacing the phosphorothioate bonds located in the wing portions with phosphodiester bonds (Non-Patent Document 8).

[0011] Patent Document 9 shows that the central toxicity observed immediately after exposure of antisense oligonucleotides to the central nervous system (hereinafter sometimes referred to as "acute central toxicity"; also referred to herein as "in vivo acute neuroTox") can be reduced when modified nucleic acids have a substituent at the 5' carbon atom of the nucleoside sugar moiety. In addition to acute central toxicity, it is also known that there is central toxicity that develops a certain time after administration (hereinafter sometimes referred to as "delayed central toxicity"; also referred to herein as "in vivo delayed neuroTox"), and symptoms observed from the day after exposure to the central nervous system include decreased spontaneous movement, abnormal gait and hind limb function, tremors, hind limb or tail weakness, loss of hind limb reflexes, weight loss, etc. To the inventor's knowledge, no specific solution to this delayed central toxicity has been reported.

[0012] International Publication No. WO 2019 / 084068, International Publication No. WO 2011 / 052436, International Publication No. WO 2014 / 046212, International Publication No. WO 2015 / 125783, International Publication No. WO 2020 / 158910, International Publication No. WO 99 / 14226, International Publication No. WO 2022 / 097727, International Publication No. WO 2016 / 127000, International Publication No. WO 2022 / 234855

[0013] Science (2011) 331(6018):730-736Genes Dev. (2009) 23(23):2753-2764 Proc. Natl. Acad. Sci. USA (2011) 108(1):260-265 Neuron (2015) 88:667-677 Neuron (2020) 105:645-662 Nat. Neurosci. (2019) 22(9):1383-1388 Mol. Ther. Nucleic Acids (2023) 31:182-196BioRxiv. doi:https: / / doi. org / 10.1101 / 2021.02.14.431096 (2021) Mol. Gen. Genet. (1979) 169:1-6Curr. Opin. Struct. Biol. (2014) 24:165-169J. Med. Chem. (2016)59:9645-9667Nature (2015)518(7539):409-412

[0014] Although Patent Document 1 reports that inhibiting the RPS25 gene suppresses DPR production, it is unclear whether a similar effect can be achieved by approaches using nucleic acids such as antisense. Furthermore, the antisense oligonucleotide described in Non-Patent Document 5 suppresses RPS25 gene expression, but the inhibitory effect is partial. Although Patent Document 7 reports a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that effectively regulates RPS25 gene expression, there is a need for the creation of an antisense oligonucleotide against the RPS25 gene with reduced toxicity (e.g., delayed central nervous system toxicity) for use as a pharmaceutical.

[0015] The present invention has been made in view of the above circumstances, and an object of the present invention is 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.

[0016] As a result of intensive research to solve the above-mentioned problems, the present inventors have discovered that delayed central nervous system toxicity can be reduced by positioning a modified nucleic acid having a cyclopropane ring at the 5'-position of the nucleoside sugar moiety in the gap region of a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof (hereinafter sometimes referred to as the "antisense oligonucleotide of the present invention") that binds to the RPS25 gene and effectively regulates expression of the RPS25 gene, and have completed the present invention. That is, the present invention is as follows.

[0017] [1] An antisense oligonucleotide according to a first aspect of the present invention is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that regulates the expression and / or function of the RPS25 gene, wherein each nucleoside in the single-stranded antisense oligonucleotide is linked via a phosphate group and / or a modified phosphate group, the single-stranded antisense oligonucleotide comprises a gap region, a 3' wing region linked to the 3' end of the gap region, and a 5' wing region linked 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 substituent at the 2' position, the base length of the single-stranded antisense oligonucleotide is 12 to 30 mer, and the base sequence of the single-stranded antisense oligonucleotide is The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof is a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to at least one target region in the base sequence set forth in SEQ ID NO: 1, which is composed of the same base length as the single-stranded antisense oligonucleotide; a base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted or added in the target region; or a base sequence which hybridizes under stringent conditions to an oligonucleotide having the target region.

[0018] [2] An antisense oligonucleotide according to a second aspect of the present invention is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that regulates the expression and / or function of the RPS25 gene, wherein each nucleoside in the single-stranded antisense oligonucleotide is linked via a phosphate group and / or a modified phosphate group, the single-stranded antisense oligonucleotide comprises a gap region, a 3' wing region linked to the 3' end of the gap region, and a 5' wing region linked 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 substituent at the 2' position, the base length of the single-stranded antisense oligonucleotide is 12 to 30 mer, and the base sequence of the single-stranded antisense oligonucleotide is A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof (excluding the single-stranded antisense oligonucleotides of Reference Examples 1 to 31) is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, which is a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to at least one target region in the base sequence set forth in SEQ ID NO: 1, which is composed of the same base length as the single-stranded antisense oligonucleotide; a base sequence complementary to a base sequence in the target region in which one or several bases have been deleted, substituted, inserted or added; or a base sequence which hybridizes under stringent conditions to an oligonucleotide having the target region.

[0019] [3] An antisense oligonucleotide according to a third aspect of the present invention is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that regulates the expression and / or function of the RPS25 gene, wherein each nucleoside in the single-stranded antisense oligonucleotide is linked via a phosphate group and / or a modified phosphate group, the single-stranded antisense oligonucleotide comprises a gap region, a 3' wing region linked to the 3' end of the gap region, and a 5' wing region linked 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 substituent at the 2' position, the base length of the single-stranded antisense oligonucleotide is 12 to 30 mer, and the base sequence of the single-stranded antisense oligonucleotide is A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof is a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to at least one target region of the base sequence set forth in SEQ ID NO: 1, which is composed of the same base length as the single-stranded antisense oligonucleotide; a base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted or added in the target region; or a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region (excluding the single-stranded antisense oligonucleotides of Reference Examples 1 to 31 and Design Examples 1 to 18).

[0020] In the antisense oligonucleotide of the present invention, modified nucleic acids are arranged in the gap region, 5' wing region, and 3' wing region. The gap region contains at least one 5'-CP (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") and AmNA (amide-bridged modified nucleic acid, Amido-bridged The antisense oligonucleotides of the present invention are preferably selected from the group consisting of 2'-O,4'-C-spirocyclopropyl-2'-pyridinone (2'-O,4'-C-pyridinone), 2'-O,4'-C ...

[0021] [4] In any of [1] to [3] above, 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 in the base sequence set forth in SEQ ID NO: 1, which has the same base length as the single-stranded antisense oligonucleotide.

[0022] [5] In any of [1] to [4] above, 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.

[0023] [6] In any of the above [1] to [5], the gap region has a length of 5 to 20 nucleotides, the 3' wing region is a 3 to 5 nucleotide modified nucleic acid having a substituent at the 2' position, and the 5' wing region is a 3 to 5 nucleotide modified nucleic acid having a substituent at the 2' position.

[0024] [7] In any one of the above [1] to [6], the 5'-CP nucleic acid is located at least at the second position counting from the 5' side of the gap region.

[0025] [8] In any one of the above [1] to [7], two or more of the 5'-CP nucleic acids are arranged in the gap region.

[0026] [9] In any one of the above [1] to [8], 2 to 5 of the 5'-CP nucleic acids are arranged in the gap region.

[0027]

[10] In any one of the above [1] to [7], the 5'-CP nucleic acid is arranged in one-ninth or more of the gap region.

[0028]

[11] In the above

[10] , the 5'-CP nucleic acid is arranged at one-fifth or more of the gap region.

[0029]

[12] In any one of the above [1] to

[11] , the 5'-CP nucleic acid is arranged in a continuous 2- to 4-mer sequence at least at one position in the gap region.

[0030]

[13] In any one of the above [1] to

[12] , the 5'-CP nucleic acid is located on the 5'-end side of the gap region.

[0031]

[14] In any one of the above [1] to

[13] , the 5'-CP nucleic acid is located at the 5'-end and 3'-end of the gap region.

[0032]

[15] In any one of the above [1] to

[14] , the single-stranded antisense oligonucleotide has a base length of 15 to 20 mer.

[0033]

[16] In any one of the above [1] to

[15] , the single-stranded antisense oligonucleotide has a base length of 18 to 20 mer.

[0034]

[17] In any of [1] to

[16] above, 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, and 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.

[0035]

[18] In any of [1] to

[17] above, 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, and the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

[0036]

[19] In any of [1] to

[18] above, 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, and the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA.

[0037]

[20] In any one of the above [1] to

[19] , at least one internucleoside bond of the single-stranded antisense oligonucleotide is a phosphorothioate bond.

[0038]

[21] In any one of the above [1] to

[20] , at least one internucleoside bond of the single-stranded antisense oligonucleotide is a phosphodiester bond.

[0039]

[22] In any one of the above [1] to

[21] , the proportion of phosphorothioate bonds in the internucleoside bonds constituting the single-stranded antisense oligonucleotide is 50% to 80%.

[0040]

[23] In the above

[22] , the proportion of phosphorothioate bonds in the internucleoside bonds constituting the single-stranded antisense oligonucleotide is 50% to 70%.

[0041]

[24] In any of [1] to

[23] above, in 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 positioned at the 3' end of the gap region).

[0042]

[25] In any of [1] to

[24] above, in 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 cases where the 5'-CP nucleic acid is positioned 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).

[0043]

[26] In any of the above [1] to

[25] , in the gap region, 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.

[0044]

[27] In any of [1] to

[26] above, the base sequence of the single-stranded antisense oligonucleotide is: a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to a target region consisting of a consecutive 14-22mer consisting of bases located at positions 123, 124, 185, 186, 263, 264, 324, 325, 442, 443, or 447 to 453, counting 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 have been deleted, substituted, inserted, or added in the target region; or a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region.

[0045]

[28] In any of [1] to

[27] above, the base sequence of the single-stranded antisense oligonucleotide is a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to a target region consisting of a consecutive 14-22mer starting from bases 324, 325, or 448 to 453, counting 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 have been deleted, substituted, inserted, or added in the target region; or a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region.

[0046]

[29] In any of the above [1] to

[28] , the base sequence of the single-stranded antisense oligonucleotide is a base sequence that has 90% to 100% sequence identity with a base sequence complementary to a target region consisting of 18 to 22 consecutive bases from the 448th to 453rd bases counting from the 5' end in the base sequence set forth in SEQ ID NO: 1, the 3' wing region is a 3 to 5 mer, and the 5' wing region is a 3 to 5 mer.

[0047]

[30] In any of the above items [1] to

[29] , 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 a continuous 18- to 20-mer sequence beginning with bases 450 to 451 or 453 counting from the 5'-end in the base sequence set forth in SEQ ID NO: 1.

[0048]

[31] In any of [1] to

[30] above, the base sequence of the single-stranded antisense oligonucleotide is one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 5 to 47 and 49 to 56.

[0049]

[32] In any of [1] to

[31] above, the base sequence of the single-stranded antisense oligonucleotide is one 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.

[0050]

[33] In any of [1] to

[32] above, the base sequence of the single-stranded antisense oligonucleotide is one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 41, 47, 50, and 53 to 55.

[0051]

[34] The antisense oligonucleotide conjugate of the present invention is an antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof, comprising the single-stranded antisense oligonucleotide of any one of [1] to

[33] above or a pharmaceutically acceptable salt thereof, and an additional substance bound to the single-stranded antisense oligonucleotide, wherein the additional substance is selected from the group consisting of polyethylene glycol, a peptide, an alkyl chain, a nucleic acid, a ligand compound, an antibody, a protein, and a sugar chain.

[0052]

[35] A pharmaceutical product according to the present invention comprises, as an active ingredient, the single-stranded antisense oligonucleotide of any one of [1] to

[33] above or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide conjugate of

[34] above or a pharmaceutically acceptable salt thereof.

[0053]

[36] In the above

[35] , the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof is administered so as to be exposed to the central nervous system.

[0054]

[37] In the above

[35] or

[36] , the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof is administered to a subject susceptible to delayed central toxicity.

[0055]

[38] The agent for regulating the expression and / or function of the RPS25 gene according to the present invention comprises, as an active ingredient, any of the single-stranded antisense oligonucleotides [1] to

[33] above or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide conjugate

[34] above or a pharmaceutically acceptable salt thereof.

[0056]

[39] The inhibitor of dipeptide repeat production by RNA translation of the present invention comprises, as an active ingredient, any of the single-stranded antisense oligonucleotides [1] to

[33] above or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex

[34] above or a pharmaceutically acceptable salt thereof.

[0057]

[40] The therapeutic agent for repeat disease according to the present invention comprises, as an active ingredient, any of the single-stranded antisense oligonucleotides [1] to

[33] above or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide conjugate

[34] above or a pharmaceutically acceptable salt thereof.

[0058]

[41] The preventive agent for repeat disease according to the present invention comprises, as an active ingredient, any of the single-stranded antisense oligonucleotides [1] to

[33] above or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide conjugate

[34] above or a pharmaceutically acceptable salt thereof.

[0059]

[42] In the therapeutic agent of

[40] above and the preventive agent of

[41] above, 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.

[0060]

[43] A method for treating or preventing a repeat disease according to the present invention comprises administering to an individual the single-stranded antisense oligonucleotide of any of [1] to

[33] above or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide conjugate of

[34] above or a pharmaceutically acceptable salt thereof.

[0061]

[44] In the method for treating or preventing a repeat disease according to

[43] above, 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.

[0062]

[45] The present invention provides a single-stranded antisense oligonucleotide according to any one of [1] to

[33] above or a pharmaceutically acceptable salt thereof, or an antisense oligonucleotide conjugate according to

[34] above or a pharmaceutically acceptable salt thereof, for use in treating or preventing C9orf72 ALS.

[0063]

[46] The present invention provides a single-stranded antisense oligonucleotide according to any one of [1] to

[33] above or a pharmaceutically acceptable salt thereof, or an antisense oligonucleotide conjugate according to

[34] above or a pharmaceutically acceptable salt thereof, for use in producing an agent for treating or preventing C9orf72 ALS.

[0064] According to the present invention, it is possible to provide a single-stranded antisense oligonucleotide that regulates the expression and / or function of the RPS25 gene, which has reduced delayed central nervous system toxicity, and an agent for regulating the expression and / or function of the RPS25 gene, which comprises the same.

[0065] 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 RPS25 gene expression is suppressed when a single-stranded antisense oligonucleotide according to this embodiment is used.

[0066] An embodiment of the present invention (hereinafter sometimes referred to as "this embodiment") will be described below. However, this embodiment is not limited thereto. In this specification, the notation in the form of "I to J" means the upper and lower limits of a range (i.e., I or more and J or less). When no unit is specified for I and a unit is specified only for J, the unit of I and the unit of J are the same.

[0067] <Single-stranded antisense oligonucleotide that modulates expression and / or function of RPS25 gene> The single-stranded antisense oligonucleotide of this embodiment is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that modulates expression and / or function of RPS25 gene, wherein each nucleoside in the single-stranded antisense oligonucleotide is linked via 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 substituent at the 2' position, the base length of the single-stranded antisense oligonucleotide is 12 to 30 mer, and the base sequence of the single-stranded antisense oligonucleotide is A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof is a single-stranded antisense oligonucleotide having a base sequence that has 90% to 100% sequence identity with respect to a base sequence complementary to at least one target region of the base sequence set forth in SEQ ID NO: 1, which is composed of the same base length as the single-stranded antisense oligonucleotide, a base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted or added in the target region, or a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region. These will be explained in detail below.

[0068] <Definitions of Terms, etc.> First, definitions of terms used in this specification will be explained below.

[0069] (RPS25 gene) In this embodiment, 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.

[0070] (Single-Stranded Antisense Oligonucleotide) In this embodiment, a "single-stranded antisense oligonucleotide" or "antisense oligonucleotide" (hereinafter sometimes referred to as "ASO") refers to an oligonucleotide or a pharmacologically acceptable salt thereof that is 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"). Antisense oligonucleotides are composed of DNA, RNA, and / or analogs thereof. Antisense oligonucleotides form a duplex with the target mRNA, pre-mRNA, or ncRNA, thereby suppressing the activity of the target mRNA, pre-mRNA, or ncRNA. Antisense oligonucleotides include those that have a base sequence that is completely complementary to the base sequence of the target mRNA, pre-mRNA, or ncRNA; those that have a base sequence in which one or more bases have been deleted, substituted, inserted, or added in the complementary base sequence; and those that contain bases that form wobble base pairs in their base sequence. Furthermore, the antisense oligonucleotide of the present invention may further contain modified nucleotides known in the art other than "modified nucleic acids in which the sugar moiety is a modified sugar" (sugar-modified modified nucleotides) described below. Examples of modified nucleotides known in the art include sugar-modified modified nucleotides, as well as phosphate-modified nucleotides and nucleobase-modified nucleotides described below. The structures of both termini of the antisense oligonucleotide in this embodiment are 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 below). The single-stranded antisense oligonucleotide in this embodiment may be in a single-stranded form, or may hybridize with a second-strand oligonucleotide described below to form a double-stranded form.A double-stranded oligonucleotide consisting of the single-stranded antisense oligonucleotide and a second-stranded oligonucleotide hybridized to the single-stranded antisense oligonucleotide may be referred to as a "double-stranded antisense oligonucleotide."

[0071] (Oligonucleotide) In this embodiment, "oligonucleotide" refers to a nucleoside polymer in which 2 to 30 identical or different nucleosides are linked together via phosphodiester bonds or other bonds. The oligonucleotide can also be understood as being composed of a nucleic acid base moiety, a phosphate moiety, and a sugar moiety, as shown in the following structural formula:

[0072]

[0073] The oligonucleotides are broadly classified into natural oligonucleotides and non-natural oligonucleotides. "Natural oligonucleotides" refer to oligonucleotides composed of naturally occurring nucleotides. "Non-natural oligonucleotides" refer 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 moiety is modified; phosphorothioate derivatives in which one non-bridging oxygen atom in the phosphodiester bond is replaced with a sulfur atom; phosphorodithioate derivatives in which two non-bridging oxygen atoms in the phosphodiester bond are replaced with sulfur atoms; ester derivatives in which the phosphodiester bond is triesterized; 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 in 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 nucleobase is modified. More preferably, the unnatural oligonucleotide includes a cross-linked modified sugar derivative in which the sugar moiety is modified; a phosphorothioate derivative in which one non-bridging oxygen atom in 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 moiety is modified with a modified sugar (e.g., a cross-linked sugar) described below and one non-bridging oxygen atom in the phosphodiester bond is replaced with a sulfur atom or the non-bridging oxygen atom in the phosphodiester bond is substituted with an alkyl group.

[0074] (Nucleoside) In this embodiment, "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."

[0075] (Nucleotide) In this embodiment, "nucleotide" refers to a compound in which a phosphate group is bound to the sugar of the above-mentioned nucleoside. A naturally occurring nucleotide may be referred to as a "natural nucleotide." A non-naturally occurring modified nucleotide may be referred to as a "modified nucleotide" or "modified nucleic acid." Examples of "modified nucleotide" or "modified nucleic acid" include a compound in which a phosphate group is bound to the sugar moiety of the above-mentioned modified nucleoside, a compound in which a modified phosphate group described below is bound to the sugar moiety of the above-mentioned modified nucleoside, and a compound in which a modified phosphate group described below is bound to the sugar moiety of a natural nucleoside.

[0076] (Sugar modification, modified sugar) In this embodiment, "sugar modification" means that the sugar moiety of the nucleotide is modified. The modified sugar moiety may be specifically referred to as a "modified sugar." Modified nucleotides that have been sugar modified can be used as modified nucleic acids, and examples thereof 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. Examples of LNAs include those containing structures represented by the symbols "A(L)", "5(L)", "G(L)", and "T(L)" as described below. Examples of AmNAs include those containing structures represented by the symbols "A(Y)", "5(Y)", "G(Y)", and "T(Y)" as described below. Examples of GuNAs include those containing structures represented by the symbols "A(Gx)", "5(Gx)", "G(Gx)", and "T(Gx)" as described below. Examples of scpBNAs 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.

[0077] (Modified Nucleic Acid Having a Substituent at the 2'-Position) In this embodiment, the term "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 nucleotide. For example, non-crosslinked 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 crosslinked modified nucleic acids include LNA, AmNA, GuNA, scpBNA, ENA, S-cEt, etc.

[0078] (Nucleotide Modifications Known in the Art Other Than Sugar Modifications) Nucleotide modifications known in the art other than the sugar modifications described above can be used as modified nucleic acids for producing the single-stranded antisense oligonucleotide of the present invention. Known nucleotide modifications include phosphate group modifications and nucleobase modifications, which will be described later. Examples of such nucleotide modifications include the 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 literature cited in the above literature.

[0079] (Phosphate Group) In this embodiment, the term "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 "-" described below).

[0080] (Phosphate group modification, modified phosphate group) In this embodiment, "phosphate group modification" means that the phosphate moiety of the nucleotide is modified. The modified phosphate moiety may be particularly referred to as a "modified phosphate group." Examples of bond types containing the modified phosphate group include a phosphorothioate bond (a bond indicated by the symbol "∧" described below), a phosphorodithioate bond, a phosphoamidate bond (a bond indicated by the symbol "=" described below), a boranophosphate bond (a bond indicated by the symbol "x" described below), and an alkylphosphonate.

[0081] (Nucleobase modification, modified nucleobase) In this embodiment, "nucleobase modification" means that the nucleobase portion of the nucleotide is modified. The modified nucleobase portion may be particularly referred to as a "modified nucleobase". Examples of modified nucleobases include 5-methylcytosine, 5-hydroxymethylcytosine, and 5-propynylcytosine.

[0082] (DNA or RNA Analogs) The DNA or RNA analogs mentioned above refer to molecules having a structure similar to that of DNA or RNA, such as peptide nucleic acids (pNA) and morpholino nucleic acids.

[0083] (ncRNA) In this embodiment, "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).

[0084] (Nucleobase Moiety of Oligonucleotide) Examples of the nucleobase moiety of the oligonucleotide 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. Preferred examples of the nucleobase moiety include thyminyl, cytosinyl, adeninyl, guaninyl, 5-methylcytosinyl, and uracilyl groups. Among these nucleobases, 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.

[0085] (Target RNA) In this embodiment, the term "target RNA" refers to RNA whose function is inhibited by binding of the single-stranded antisense oligonucleotide. In other words, in this embodiment, the term "target RNA" refers to RPS25 mRNA and pre-mRNA. Examples of the target RNA include human RPS25 mRNA having the nucleotide sequence set forth in SEQ ID NO: 1 (hereinafter, sometimes referred to as "hRPS25"), monkey RPS25 mRNA having the nucleotide sequence set forth in SEQ ID NO: 2 (hereinafter, sometimes referred to as "cRPS25"), mouse RPS25 mRNA having the nucleotide sequence set forth in SEQ ID NO: 3 (hereinafter, sometimes referred to as "mRPS25"), and rat RPS25 mRNA having the nucleotide sequence set forth in SEQ ID NO: 4 (hereinafter, sometimes referred to as "rRPS25").

[0086] (Binding to target RNA) In this embodiment, "binding to target RNA" means that the nucleic acid bases of the single-stranded antisense oligonucleotide form a double-stranded nucleic acid together with the nucleic acid bases of the target RNA due to complementarity with the target RNA. The double-stranded nucleic acid may be formed in at least a portion of the target RNA. The strength of 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.

[0087] (Target region) The target region refers to a region in the RPS25 mRNA or pre-mRNA to which the single-stranded antisense oligonucleotide binds. The target region includes the target region consisting of the indicated nucleotide sequence and a region on the RPS25 pre-mRNA.

[0088] (Pre-mRNA) The pre-mRNA refers to the primary transcript of RNA transcribed from DNA. That is, the pre-mRNA is RNA containing exon regions, intron regions, and untranslated regions (UTRs). The pre-mRNA can also be understood as RNA before splicing after transcription. When the pre-mRNA is spliced, it becomes mRNA.

[0089] (Binding to the target region) 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. However, the single-stranded antisense oligonucleotide of the present invention does not necessarily need to form a double strand with the entire target region, as long as it forms a double strand with a partial region of the target region. In other words, the single-stranded antisense oligonucleotide of the present invention is preferably one that is completely complementary to the target region, but as long as it binds to the target RNA of RPS25, it is sufficient that it is complementary to at least a partial region of the target region.

[0090] (Part of the target region) The part of the target region means a region of the target region having a length of 10 to 15 nucleotide bases.

[0091] (Complementary to at least a portion of the target region) "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, which also includes complementary to the bases of a region on the mRNA or pre-mRNA corresponding to at least a portion of the region.

[0092] <Base Sequence of Single-Stranded Antisense Oligonucleotide> The base sequence of the single-stranded antisense oligonucleotide according to this embodiment is preferably: (A) a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to a target region consisting of a 14-22mer sequence consisting of consecutive bases at positions 123, 124, 185, 186, 263, 264, 324, 325, 442, 443, or 447-453 in the base sequence set forth in SEQ ID NO: 1, counting from the 5' end; (B) a base sequence complementary to a base sequence in the target region in which one or several bases have been deleted, substituted, inserted, or added; or (C) a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region. Furthermore, in this embodiment, the base sequences shown in the sequence listing are used to indicate only the sequence information of the nucleic acid base portion. The structural information of the oligonucleotide, including the sugar moiety and phosphate moiety in addition to the nucleic acid base moiety, is shown in the format shown in Tables 2-1 to 2-4, 3-1, 3-2 and 4 below.

[0093] In this embodiment, "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). The "sequence identity" of a base sequence 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.

[0094] The base sequence of the single-stranded antisense oligonucleotide of this embodiment preferably has a sequence identity of 95% to 100% with the base sequence complementary to the above-mentioned predetermined target region in the base sequence set forth in SEQ ID NO: 1, more preferably 98% to 100%, and even more preferably 100% sequence identity.

[0095] In this embodiment, examples of "a base sequence in which one or several bases have been deleted, substituted, inserted or added" include a base sequence that has, due to the deletion, substitution, insertion or addition, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity 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 deletions, substitutions, insertions or additions.

[0096] In this embodiment, "stringent conditions" refers to conditions in which the sample is incubated for 12 hours at room temperature in a solution containing 6xSSC (1xSSC has the composition: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5x Denhardt's solution, 100 μg / mL denatured salmon sperm DNA, and 50% (v / v) formamide, followed by washing in 0.5xSSC at a temperature of 50°C or higher. More stringent conditions are also encompassed, such as incubation at 45°C or 60°C for 12 hours, washing in 0.2xSSC or 0.1xSSC, or washing at a temperature of 60°C or 65°C or higher.

[0097] In one aspect of this embodiment, the target region is preferably a 14- to 22-mer base sequence consisting of consecutive bases located at positions 123, 124, 185, 186, 263, 264, 324, 325, 442, 443, or 447 to 453 in the base sequence set forth in SEQ ID NO: 1, counting from the 5' end.

[0098] In another aspect of this embodiment, the target region is preferably a 14- to 22-mer base sequence consisting of consecutive 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.

[0099] In another aspect of this embodiment, the target region is preferably a base sequence consisting of a continuous 18- to 22-mer starting from bases 448 to 453, counting from the 5' end, in the base sequence set forth in SEQ ID NO: 1.

[0100] In another aspect of this embodiment, the target region is preferably a base sequence consisting of a continuous 18- to 20-mer base starting from bases located at positions 450 to 451 or 453 from the 5' end in the base sequence set forth in SEQ ID NO: 1.

[0101] The single-stranded antisense oligonucleotide can bind to the target region of RPS25. As used herein, the phrase "binding to the target region of RPS25" of the single-stranded antisense oligonucleotide of the present invention encompasses direct binding of the single-stranded antisense oligonucleotide of the present invention to RPS25 mRNA and direct binding to RPS25 pre-mRNA.

[0102] One embodiment of the single-stranded antisense oligonucleotide of the present invention is a single-stranded oligonucleotide that regulates expression of the RPS25 gene, having any of the nucleotide sequences listed in Table 1, and complementary to a target region in the human RPS25 mRNA listed in Table 1. As long as the single-stranded oligonucleotide contains a nucleotide sequence listed in Table 1, it may extend by 1 to 5 nucleotides on the 3' and / or 5' ends. The target region can be considered to be a region in the human RPS25 mRNA that is particularly involved in regulating expression of the human RPS25 gene (e.g., a region of the mRNA with a secondary structure that facilitates binding of antisense nucleotides). For example, if the 5'-terminal position is "451" and the 3'-terminal position is "465" in Table 1, the nucleotide sequence from 451 to 465, counting from the 5'-end, in the nucleotide sequence set forth in SEQ ID NO: 1 is the target region in the human RPS25 mRNA targeted by the corresponding single-stranded antisense oligonucleotide (sequence name "h451-465").

[0103]

[0104] In Table 1 above, 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). As for the modified sugar nucleoside, the symbol "A'" is selected from A(M), A(m), A(L), A(Y), A(Gx), A(5'-CP), A(Mx), or A(S), as 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), as 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), as described below; and the symbol "T'" is selected from U(M), T(m), T(L), T(Y), T(Gx), T(5'-CP), U(Mx), or T(S), as described below.

[0105] In one aspect of this embodiment, 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.

[0106] In one aspect of this embodiment, the base sequence of the single-stranded antisense oligonucleotide is more preferably one base sequence selected from the group consisting 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.

[0107] In one aspect of this embodiment, it is further preferable that the base sequence of the single-stranded antisense oligonucleotide is one base sequence 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.

[0108] In one aspect of this embodiment, it is even more preferable that the base sequence of the single-stranded antisense oligonucleotide is one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 41, 47, 50, and 53-55.

[0109] <Pharmacologically acceptable salt> The single-stranded antisense oligonucleotide according to this embodiment may be in the form of a pharmacologically acceptable salt. Here, "pharmacologically acceptable salt" refers to a salt of the single-stranded antisense oligonucleotide of the present invention, which is a physiologically acceptable salt of the single-stranded antisense oligonucleotide of the present invention, that is, a salt that retains the desired biological activity of the single-stranded antisense oligonucleotide and does not retain undesired toxicological effects. The same applies to the double-stranded antisense oligonucleotide and antisense oligonucleotide complex described below.

[0110] Pharmaceutically Acceptable Salts In one aspect of this embodiment, the single-stranded antisense oligonucleotide may be in the form of a pharmaceutically acceptable salt. Here, "pharmaceutically acceptable salts" refers to the pharmacologically acceptable salts described above that are acid addition salts or base addition salts. Examples of acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate, as well as organic acid salts such as citrate, oxalate, phthalate, fumarate, maleate, succinate, malate, acetate, formate, propionate, benzoate, trifluoroacetate, methanesulfonate, benzenesulfonate, para-toluenesulfonate, and camphorsulfonate. Examples of 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. Further examples include salts with basic or acidic amino acids such as arginine, lysine, ornithine, aspartic acid, and glutamic acid (amino acid salts). The same applies to the double-stranded antisense oligonucleotides and antisense oligonucleotide complexes described below.

[0111] <Structure of Single-Stranded Antisense Oligonucleotide> The single-stranded antisense oligonucleotide according to this embodiment 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 (see, for example, FIG. 1). The single-stranded antisense oligonucleotide is preferably in a single-stranded form. In one aspect of this embodiment, the single-stranded antisense oligonucleotide may hybridize with a second-strand oligonucleotide described below to form a double-stranded form (double-stranded antisense oligonucleotide). The base sequence of the second-strand oligonucleotide preferably has a sequence identity of 90% to 100% based on a base sequence complementary to the base sequence of the single-stranded antisense oligonucleotide.

[0112] The single-stranded antisense oligonucleotide is a so-called gapmer-type single-stranded antisense oligonucleotide. Gapmer-type single-stranded antisense oligonucleotides inhibit the function of target RNA through the following mechanism. First, the single-stranded antisense oligonucleotide binds to the target region of the target RNA (top to center of Figure 2). Next, RNase H, an RNase, recognizes and binds to the complex of the single-stranded antisense oligonucleotide and the target RNA (center of Figure 2). The target RNA is then cleaved and degraded by an enzymatic degradation reaction caused by RNase H. At this time, the single-stranded antisense oligonucleotide is not affected by the enzymatic degradation by RNase H (bottom of Figure 2). Therefore, the single-stranded antisense oligonucleotide can bind to another target RNA and cleave and degrade that RNA. In this way, gapmer-type single-stranded antisense oligonucleotides function as a catalyst in the enzymatic degradation reaction caused by RNase H, and are therefore thought to have a sustained effect even when administered in small amounts.

[0113] In this embodiment, the single-stranded antisense oligonucleotide can be suitably used to regulate RPS25 gene expression through the mechanism described above (including when it acts via regulating maturation of RPS25 pre-mRNA). Furthermore, according to this embodiment, the effect of regulating RPS25 gene expression by the single-stranded antisense oligonucleotide can be exerted even through intrathecal administration, which is an administration route commonly used in clinical applications. Here, "regulating RPS25 gene expression" at least means suppressing RPS25 gene expression, and as a result, at least means suppressing RPS25 protein function (e.g., RNA translation).

[0114] (Gap Region) The gap region contains at least one 5'-CP nucleic acid. The gap region is preferably a 5- to 20-mer nucleic acid composed of deoxyribose, which may contain a nucleic acid whose sugar moiety is modified. In other words, the gap region can also be understood as a 5- to 20-mer nucleic acid containing deoxyribose, which may contain a nucleic acid whose sugar moiety is modified. The gap region can also be understood as being composed of 5- to 20-mer natural nucleotides, unnatural nucleotides, or both, whose sugar moiety is deoxyribose. By having deoxyribose or modified deoxyribose as the sugar moiety, the gap region can form a complex recognizable by RNase H together with the target RNA, such as the mRNA of RPS25. Here, an example of a nucleic acid containing modified deoxyribose is a 5'-CP nucleic acid.

[0115] The number of bases in the gap region is preferably 5 to 20 mer, more preferably 6 to 17 mer, even more preferably 7 to 13 mer, and even more preferably 9 to 13 mer.

[0116] Examples of natural nucleotides in which the sugar moiety is deoxyribose include deoxyadenosine monophosphate, deoxyguanosine monophosphate, thymidine monophosphate, deoxycytidine monophosphate, deoxy-5-methylcytidine monophosphate (also referred to as 5-methyldeoxycytidine), etc. In other words, natural nucleotides constituting the gap region include those containing structural formulas corresponding to the symbols a, g, t, and c described below.

[0117] Examples of 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.

[0118] In addition, the gap region may be a nucleic acid in which some of the sugar moieties of a natural nucleotide whose sugar moiety 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 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).

[0119] In this embodiment, the 5'-CP nucleic acid is preferably located at least at the second position counting from the 5' side of the gap region.

[0120] In this embodiment, it is preferable that two or more 5'-CP nucleic acids are arranged in the gap region, and more preferably 2 to 5. Here, the term "two or more 5'-CP nucleic acids" refers to a concept including an embodiment in which two or more 5'-CP nucleic acids of interest are arranged contiguously, and an embodiment in which two or more 5'-CP nucleic acids are arranged dispersedly in the cap region.

[0121] In this embodiment, it is preferable that the 5'-CP nucleic acid is arranged in a continuous 2- to 4-mer sequence at least at one position in the gap region.

[0122] In this embodiment, 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. Here, "located on the 5'-end side of the gap region" means located closer to the 5'-end than the center of the gap region. "located on the 3'-end side of the gap region" means located closer to the 3'-end than the center of the gap region.

[0123] In this embodiment, the 5'-CP nucleic acid is preferably arranged in an amount of at least one-ninth of the gap region, more preferably at least one-fifth of the gap region, and the upper limit may be, for example, one-half or less.

[0124] In this embodiment, the bond between the 5'-CP nucleic acid and the nucleoside adjacent to the 3' side of the 5'-CP nucleic acid in the gap region is preferably a phosphorothioate bond (excluding the case where the 5'-CP nucleic acid is positioned at the 3' end of the gap region). Here, "the 5'-CP nucleic acid is positioned at the 3' end of the gap region" can also be understood as "the 5'-CP nucleic acid is bonded to a nucleoside in the 3' wing region." In one aspect of this embodiment, the bond between the 5'-CP nucleic acid and the nucleoside adjacent to the 3' side of the 5'-CP nucleic acid in the gap region is preferably a phosphorothioate bond (excluding the case where the 5'-CP nucleic acid is positioned at the 3' end of the gap region or the case where the nucleoside adjacent to the 3' side of the 5'-CP nucleic acid is a 5'-CP nucleic acid).

[0125] In this embodiment, in the gap region, 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.

[0126] (3' Wing Region) The 3' wing region is a modified nucleic acid having a substituent at the 2' position. In other words, the 3' wing region can also be understood as being 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, as non-crosslinked 2'-modified nucleic acids, 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, and MCE nucleic acid, and as crosslinked modified nucleic acids, at least one selected from the group consisting of LNA, AmNA, GuNA, and scpBNA. By comprising the 3' wing region and the 5' wing region described below with the specified modified nucleotides, high binding affinity to the target RNA can be expected, and ultimately, the function of the target RNA can be effectively inhibited. In one aspect of this embodiment, the 3' wing region may be a modified nucleic acid in which the sugar moiety is a modified sugar. Examples of modified nucleic acids in which the sugar moiety is a modified sugar include those listed above (Sugar modification, modified sugar). In one aspect of this embodiment, the modified nucleic acid having a substituent at the 2'-position in the 3'-wing region may consist solely of 2'-MOE nucleic acids. Note that multiple types of modified nucleic acids in the 3'-wing region may be contained in one single-stranded antisense oligonucleotide.

[0127] In this embodiment, the modified nucleic acid having a substituent at the 2'-position in the 3'-wing region preferably comprises 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. In another aspect of this embodiment, 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.

[0128] The number of bases in the 3' wing region is preferably 3 to 5 mer, more preferably 3 to 4 mer.

[0129] (5' Wing Region) The 5' wing region is a modified nucleic acid having a substituent at the 2' position. In other words, the 5' wing region can also 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 non-crosslinked 2'-modified nucleic acids, 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, and MCE nucleic acid, and as crosslinked modified nucleic acids, at least one selected from the group consisting of LNA, AmNA, GuNA, and scpBNA. In one aspect of this embodiment, the 5' wing region may be a modified nucleic acid in which the sugar moiety is a modified sugar. Examples of modified nucleic acids in which the sugar moiety is a modified sugar include those listed above (Sugar modification, modified sugar). In one aspect of this embodiment, the modified nucleic acid in the 5' wing region having a substituent at the 2' position may be composed solely of 2'-MOE nucleic acid. Note that multiple types of modified nucleic acids in the 5' wing region may be contained in a single single-stranded antisense oligonucleotide. In another aspect of this embodiment, the modified nucleic acids in the 3' wing region and the 5' wing region may be composed solely of 2'-MOE nucleic acids, or may be composed solely of cross-linked modified nucleic acids.

[0130] In this embodiment, 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, 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. In another aspect of this embodiment, 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.

[0131] In one aspect of this embodiment, it is preferred that 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, and that 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. It is also more preferred that 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, and that the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

[0132] In another aspect of this embodiment, it is more preferred that the modified nucleic acid having a substituent at the 2'-position in the 3'-wing region is a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA, and that the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region is a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA.

[0133] The number of bases in the 5' wing region is preferably 3 to 5 mer, more preferably 3 to 4 mer.

[0134] In one aspect of this embodiment, it is preferred that the gap region has a base length of 5 to 20 mer, the 3' wing region has a base length of 3 to 5 mer, and the 5' wing region has a base length of 3 to 5 mer.

[0135] In one aspect of this embodiment, it is more preferred that the gap region has a base length of 7 to 13 mer, the 3' wing region has a base length of 3 to 5 mer, and the 5' wing region has a base length of 3 to 5 mer.

[0136] In one aspect of this embodiment, it is more preferred that the gap region has a base length of 9 to 13 mer, the 3' wing region has a base length of 3 to 4 mer, and the 5' wing region has a base length of 3 to 4 mer.

[0137] (Method of Representing Gapmer-Type Structures) The single-stranded antisense oligonucleotides of the present invention are gapmer-type. When representing gapmer-type structures, the notation method of "X-Y-Z" may be used. In the above notation method, "X" represents the number of bases in the 5' wing region, "Y" represents the number of bases in the gap region, and "Z" represents the number of bases in the 3' wing region.

[0138] "X-Y-Z" includes 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, 3-13 -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, 5-10-5, 5-11-2, 5-11-3, 5-11-4, 5-11-5, etc. For example, "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.

[0139] The single-stranded antisense oligonucleotide of the present invention has a base length of 12 to 30 mer, preferably 15 to 20 mer, and more preferably 18 to 20 mer. When the single-stranded antisense oligonucleotide of the present invention has a base length of 15 to 20 mer or 18 to 20 mer, it binds particularly strongly to RPS25 mRNA and can more effectively regulate RPS25 gene expression.

[0140] In this embodiment, 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.

[0141] In one aspect of this embodiment, at least one internucleoside linkage of the single-stranded antisense oligonucleotide is preferably a phosphorothioate linkage. In another aspect of this embodiment, at least one internucleoside linkage of the single-stranded antisense oligonucleotide is preferably a phosphodiester linkage.

[0142] In one aspect of this embodiment, the proportion of phosphorothioate bonds in the internucleoside linkages constituting the single-stranded antisense oligonucleotide is preferably 50% to 80%, more preferably 50% to 70%.

[0143] 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 mers, a 5' wing region consisting of 3 to 5 mers, and a 3' wing region consisting of 3 to 5 mers. 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. Preferably, 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. As the 2'-O-alkylated nucleotide, a 2'-O-alkylated (e.g., 2'-O-methylated) D-ribofuranose nucleotide may be used. The gapmer-type single-stranded antisense oligonucleotide may also form a double strand by hybridizing with a second strand oligonucleotide.

[0144] In one aspect of this embodiment, the single-stranded antisense oligonucleotide is preferably at least one selected from the group consisting of Example 2, Example 5, Example 8, Examples 11 to 14, Example 18, Example 20, Examples 23 to 25, Examples 27 to 32, Example 34, Examples 36 to 38, Example 43, and Examples 45 to 50 in Tables 2-1 to 2-4 described below. Furthermore, 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.

[0145] <Double-stranded antisense oligonucleotide> The double-stranded antisense oligonucleotide of this embodiment is a double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, comprising the single-stranded antisense oligonucleotide and a second-strand oligonucleotide hybridized to the single-stranded antisense oligonucleotide. The base sequence of the second-strand oligonucleotide preferably has a sequence identity of 90% to 100% with respect to a base sequence complementary to the base sequence of the single-stranded antisense oligonucleotide.

[0146] 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.In addition, among the oligonucleotides constituting the double-stranded antisense 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".

[0147] <Method for producing single-stranded antisense oligonucleotides> The single-stranded antisense oligonucleotides 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 support using a commercially available automated nucleic acid synthesizer. Next, the synthesized single-stranded oligonucleotide is cleaved from the solid support using a basic substance or the like, and deprotected to obtain a crude single-stranded oligonucleotide. The crude single-stranded oligonucleotide obtained is then purified using HPLC or the like. In addition to the above-mentioned production method, the single-stranded antisense oligonucleotides of the present invention can be produced by appropriately modifying the base sequence, modification site, etc. of the nucleic acid according to methods known to those skilled in the art. In addition, 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 using commercially available amidites as reagents. 5'-CP nucleic acids can be produced by the method described in WO 2020 / 158910 (Patent Document 5). LNAs can be produced by the method described in WO 99 / 14226 (Patent Document 6).

[0148] <<Method for Producing Double-Stranded Antisense Oligonucleotide>> 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 a base sequence complementary to the single-stranded antisense oligonucleotide using a production method similar to that of the single-stranded antisense oligonucleotide, and then hybridizing the single-stranded antisense oligonucleotide and the second strand oligonucleotide.

[0149] <<Antisense Oligonucleotide Conjugate>> The antisense oligonucleotide conjugate of this embodiment comprises the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, and an additional substance bound to the single-stranded antisense oligonucleotide or the second strand 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.).

[0150] In one aspect of this embodiment, the antisense oligonucleotide complex comprises the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, and an additional substance bound to the single-stranded antisense oligonucleotide, wherein 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.).

[0151] In another aspect of this embodiment, the antisense oligonucleotide complex comprises the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, and an additional substance bound to the single-stranded antisense oligonucleotide or the second strand oligonucleotide, wherein 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.).

[0152] In this embodiment, the term "additional substance" refers to a substance bound to the single-stranded antisense oligonucleotide or the second-strand oligonucleotide and used to impart a predetermined effect. The additional 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 additional substance may be bound to the 5'-end, the 3'-end, or both the 5'-end and the 3'-end of the second-strand oligonucleotide. In one aspect of this embodiment, the additional substance is preferably bound to either the 5'-end or the 3'-end of the single-stranded antisense oligonucleotide or the second-strand oligonucleotide. The additional substance may be directly bound to the single-stranded antisense oligonucleotide or the second-strand oligonucleotide via a covalent bond. The additional substance may be bound to the single-stranded antisense oligonucleotide or the second-strand oligonucleotide via a linker substance. Examples of the linker substance include linkers composed of alkyl, polyethylene glycol, peptide, disulfide, nucleic acid, etc., and / or combinations thereof. Examples of methods for binding the additional substance to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide include the methods described in the Examples below.

[0153] Examples of peptides that can be used as the additional substance include, but are not limited to, 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-translocating peptides.

[0154] Examples of the ligand compound used as the additional substance include, but are not limited to, 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, and monoamine receptor ligands (indatraline, etc.).

[0155] Examples of 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, and anti-HER2 antibody.

[0156] Examples of proteins that can be used as the additional substance include, but are not limited to, the following: albumin

[0157] Examples of 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.

[0158] <<RPS25 Gene Expression Regulator>> The RPS25 gene expression regulator of this embodiment comprises the single-stranded antisense oligonucleotide, the double-stranded antisense oligonucleotide, or the antisense oligonucleotide conjugate of the present invention as an active ingredient. In one aspect of this embodiment, the expression regulator can also be understood as an expression inhibitor for the RPS25 gene. In another aspect of this embodiment, the expression regulator can also be understood as an inhibitor of RAN translation. In another aspect of this embodiment, the expression regulator can also be understood as an inhibitor of dipeptide repeat expression via inhibition of RAN translation. The single-stranded antisense oligonucleotide of the present invention inhibits RPS25 gene expression by binding to RPS25 mRNA or pre-mRNA, thereby inhibiting RAN translation by its translation product. Any administration method and formulation known in the art can be used for the RPS25 gene expression regulator of the present invention.

[0159] <<Pharmaceutical Compositions Comprising Single-Stranded Antisense Oligonucleotides, etc., as Active Ingredients>> The pharmaceutical compositions of this embodiment comprise, as active ingredients, the single-stranded antisense oligonucleotides of the present invention or pharmaceutically acceptable salts thereof, the double-stranded antisense oligonucleotides or pharmaceutically acceptable salts thereof, or the antisense oligonucleotide complexes or pharmaceutically acceptable salts thereof. Any administration method and formulation known in the art can be used for the pharmaceutical compositions of this embodiment. Hereinafter, the pharmaceutical compositions may be referred to as "pharmaceutical compositions of antisense oligonucleotides, etc."

[0160] The pharmaceutical composition is used for the treatment or prevention of diseases associated with the RPS25 gene, i.e., diseases that can be caused by dipeptide repeats produced by RNA translation. In other words, the pharmaceutical composition can be used for the treatment or prevention of diseases whose symptoms can be expected to be improved by suppressing expression of the RPS25 gene. Such diseases are sometimes referred to as "repeat diseases." Specific examples of repeat diseases include various neuropsychiatric and muscular disorders, 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.

[0161] <Therapeutic and Preventive Agents for Repeat Diseases> The therapeutic agent for repeat disease according to this embodiment comprises, as an active ingredient, the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof. The preventive agent for repeat disease according to this embodiment comprises, as an active ingredient, the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide conjugate or a pharmaceutically 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.

[0162] <C9orf72 ALS> The above-mentioned C9orf72 ALS refers to ALS with 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 the most common causative gene for ALS, accounting for approximately 6% of sporadic ALS cases and approximately 40% of familial ALS cases. ALS is a neurodegenerative disease in which muscles atrophy due to the selective death of motor neurons. ALS is diagnosed by combining clinical or electrophysiological features of upper and lower motor neuropathy. Specifically, if there are findings indicating upper and lower motor nerve disorders in one of the four areas of the brainstem, cervical spinal cord, thoracic spinal cord, and lumbosacral spinal cord, and electromyographic findings are observed in the other areas, the diagnosis is laboratory-supported probable; if there are findings in two areas, probable; and if there are findings in three areas, definite.

[0163] <C9orf72 FTLD> The term "C9orf72 FTLD" refers to FTLD with an abnormal repeat expansion of the GGGGCC sequence present in the intron region between exon 1a and exon 1b of the C9orf72 gene. FTLD is characterized by progressive abnormal behavior and cognitive impairment that interfere with daily life, as well as three or more symptoms, including disinhibited behavior, apathy, lethargy, perseveration, stereotypy, or lip-smacking, and changes in eating habits. FTLD is diagnosed as behavioral abnormality-type FTLD when imaging findings show atrophy of the frontal lobe or anterior temporal lobe or decreased metabolism or blood flow, and the condition can be differentiated from other specific disorders. The above-mentioned FTLD is diagnosed as semantic dementia FTLD when, in addition to impairment in object naming and impairment in word comprehension, there is impairment in knowledge of objects or symptoms such as superficial dyslexia and agraphia, atrophy is observed in the anteriorly dominant temporal lobe, and it can be differentiated from specific diseases.

[0164] <Huntington's disease> Huntington's disease refers to a hereditary neurodegenerative disease with autosomal dominant inheritance that develops due to an abnormal repeat expansion of the CAG sequence present in exon 1 of the huntingtin gene. Huntington's disease presents with movement disorders characterized by involuntary movements, psychiatric symptoms, and cognitive symptoms. Huntington's disease is diagnosed when specific neurological findings are observed and an abnormal expansion mutation of the CAG sequence is found 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 ruled out in differential diagnosis.

[0165] Spinocerebellar ataxia (types 1, 2, 3, 6, 7, 8, 12, and 17) and dentatorubral-pallidoluysian atrophy are hereditary neurodegenerative diseases that are inherited in an autosomal dominant manner and are caused by an abnormal repeat expansion of a specific triplet sequence (CAG or CTG) present in the responsible gene for each disease. In spinocerebellar ataxia types 1, 2, 3, 6, 7, 12, and 17 and dentatorubral-pallidoluysian atrophy, a CAG repeat sequence is observed. In spinocerebellar ataxia type 8, a CTG repeat sequence is observed. The above-mentioned spinocerebellar ataxia and dentatorubral-pallidoluysian atrophy are characterized by cerebellar or posterior column ataxia or spastic paraplegia as their main symptoms, and are generally slowly progressive. They are diagnosed by combining genetic diagnosis, neuropathological diagnosis, etc.

[0166] Spinal-bulbar muscular atrophy is a genetic disease caused by an abnormal repeat expansion of a CAG sequence present in the exon region of the androgen receptor gene. Spinal-bulbar muscular atrophy is diagnosed based on a combination of neurological findings (bulbar symptoms, lower motor neuron signs, finger tremor, decreased tendon reflexes), clinical findings, laboratory findings, genetic diagnosis, etc.

[0167] Friedreich ataxia is an autosomal recessive hereditary neurodegenerative disease caused by a mutation in the frataxin gene. Most cases of Friedreich ataxia are caused by an abnormal repeat expansion of the GAA sequence in the first intron.

[0168] <Fragile X-associated ataxia syndrome> The fragile X-associated ataxia syndrome refers to a hereditary neurodegenerative disease caused by an abnormal repeat expansion of a CGG sequence present in the 5'UTR of the FMP1 gene. The fragile X-associated ataxia syndrome is diagnosed by combining clinical symptoms (cerebellar ataxia, motor tremor, Parkinsonism, dementia, intellectual disability), middle cerebellar peduncle signs by MRI, genetic diagnosis, etc.

[0169] <Myotonic Dystrophy> The above-mentioned 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.

[0170] <Individual> The individual refers to a mammal. The individual is preferably a human, monkey, marmoset, dog, pig, rabbit, guinea pig, rat, or mouse. The individual is more preferably a human.

[0171] In one aspect of this embodiment, the single-stranded antisense oligonucleotide or pharmaceutical composition thereof can be administered via a suitable administration route to a subject (individual) sensitive to delayed central toxicity. Here, "delayed central toxicity" refers to central toxicity that appears after recovery following a period during which acute central toxicity may appear. Symptoms observed due to delayed central toxicity include, for example, decreased spontaneous activity, abnormal gait and hindlimb function, tremor, hindlimb or tail weakness, loss of hindlimb reflexes, and weight loss. To evaluate this toxicity, the general condition findings observed on the observation day are scored according to the following criteria, and the scores are summed throughout the observation period to calculate a clinical sign score. Since scores are added depending on the intensity of the toxicity findings, single-stranded antisense oligonucleotides with low clinical sign scores can be determined to be highly safe. Furthermore, the pathological score is calculated by scoring cases in which abnormal findings were observed and cases in which no abnormal findings were observed in the brain pathology examination. For each score, the average value for each group is used. Furthermore, "subjects (individuals) sensitive to delayed central toxicity" refers to subjects selected using biomarkers, etc. Clinical sign score; 0 points: no abnormalities 1 point: abnormal hind limb function, tremors, decreased spontaneous movement 2 points: dragging of hind limbs, weakness of tail or hind limbs 3 points: complete hind limb dysfunction, paralysis of hind limbs, recumbency, prone position 4 points: euthanasia Pathological score; 0 points: no abnormalities 1 point: abnormalities (single cell necrosis, vacuolation, etc.)

[0172] It is known that 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, thereby affecting the off-target candidate gene and thereby exhibiting toxicity (sometimes referred to as "narrow-sense off-target toxicity"). To avoid unexpected narrow-sense off-target toxicity, it is important to design the target RNA in advance in a region that does not hybridize to non-target RNA. In one aspect of this embodiment, as a method for evaluating the effect of the single-stranded antisense oligonucleotide or a pharmaceutical composition thereof on an off-target candidate gene, a search for similar base sequences can be performed using "high-speed base sequence search: GGGenome (https: / / gggenome.dbcls.jp / ja / )" or the like, and the narrow-sense off-target candidate gene can be estimated. If there are few off-target candidate genes, it can be determined that the risk of narrow-sense off-target toxicity is low.

[0173] When administering the single-stranded antisense oligonucleotide of the present invention or a pharmaceutical composition thereof (including therapeutic and preventive agents for C9orf72 ALS), the administration method and dosage form are not particularly limited. That is, any administration method and formulation known in the art can be used as the administration method and formulation of the antisense oligonucleotide of the present invention. Examples of administration methods include oral administration and parenteral administration. Examples of parenteral administration include ophthalmic administration, intravaginal administration, rectal administration, intranasal administration, transdermal administration, intravenous injection, infusion, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration by aspiration or inhalation, intrathecal administration, and intraventricular administration. In one aspect of this embodiment, the single-stranded antisense oligonucleotide or a pharmaceutical composition thereof is preferably administered so as to be exposed to the central nervous system. Examples of administration methods that result in exposure to the central nervous system include intrathecal administration and intraventricular administration.

[0174] Various pharmaceutical additives such as excipients, binders, wetting agents, disintegrants, lubricants, diluents, flavoring agents, fragrances, solubilizers, suspending agents, emulsifiers, stabilizers, preservatives, and isotonic agents may be mixed into the formulations of the antisense oligonucleotides of the present invention as needed.

[0175] When pharmaceutical compositions such as the antisense oligonucleotides of the present invention are administered locally, formulations such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders can be used.

[0176] When pharmaceutical compositions of the present invention, such as antisense oligonucleotides, are orally administered, formulations such as powders, granules, suspensions or solutions dissolved in water or non-aqueous media, capsules, powders, tablets, etc. can be used.

[0177] When pharmaceutical compositions such as antisense oligonucleotides of the present invention are administered parenterally, intrathecally, or intracerebroventricularly, formulations such as sterile aqueous solutions can be used.

[0178] The effective dose 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 dose may be 0.01 to 100 mg / kg, preferably 0.1 to 50 mg / kg, and more preferably 0.1 to 10 mg / kg.

[0179] <<Method for regulating expression of the RPS25 gene>> In this embodiment, 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 pharmaceutically acceptable salt thereof, or the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof to a cell, tissue, or individual expressing the RPS25 gene.

[0180] In this embodiment, the single-stranded antisense oligonucleotide or the like may be administered to a cell, tissue, or individual in vitro or in vivo. When administered in vivo, the administration route is the same as that described above.

[0181] In this embodiment, examples of "cells expressing the RPS25 gene" include nerve cells that constitute the central nervous system, nerve cells that constitute the peripheral nervous system, and other cells that constitute skin tissue.

[0182] The method for treating or preventing a repeat disease in this embodiment comprises administering the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof, as an active ingredient to an individual suffering from the repeat disease. The individual is preferably a subject (individual) sensitive to delayed central nervous system toxicity.

[0183] The repeat diseases include the above-mentioned neuropsychiatric disorders, muscular disorders, etc. The dosage form, administration route and dosage when administered to an individual can be appropriately selected from those described above.

[0184] The antisense oligonucleotide according to this embodiment has been described above. The single-stranded antisense oligonucleotide having the above-described configuration is capable of regulating the expression of the RPS25 gene. Here, the inhibitory activity (knockdown activity) on the expression of the RPS25 gene can be measured by known methods. Examples of methods for measuring knockdown activity include those described in Nature (2015) 518(7539):409-12 (Non-Patent Document 12) and WO 2022 / 097727 (Patent Document 7). Furthermore, knockdown activity can also be measured by transfecting HEK293T cells with the antisense oligonucleotide, as described below. Furthermore, the single-stranded antisense oligonucleotide contains at least one 5'-CP nucleic acid in the gap region, thereby reducing delayed central nervous system toxicity.

[0185] <<Method for Evaluating Inhibitory Activity Against RPS25 Gene Expression>> <Introduction of Antisense Oligonucleotide into Cells> "RPS25 gene-expressing cells" are treated with an antisense oligonucleotide for 6 hours to 3 days using methods such as lipofection, electroporation, or direct addition. Any cells expressing the RPS25 gene may be used, such as HEK293T cells, more preferably neurons, and even more preferably human-derived neurons. The cells treated with the antisense oligonucleotide may be harvested immediately after treatment, or the antisense oligonucleotide may be removed and the cells continued to be cultured.

[0186] <Assessing RPS25 mRNA Levels> Total RNA extracted from the recovered cells is reverse-transcribed, and the resulting complementary DNA is subjected to real-time PCR or other methods using an RPS25 gene-specific probe to measure the level of RPS25 mRNA. Examples of probes used in real-time PCR include Taqman probes. Reaction methods include, for example, a three-step process (cDNA denaturation, annealing, and extension) or a two-step process (cDNA denaturation, annealing, and extension) repeated any number of times. The two or three steps are repeated, 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 temperature (for extension reaction) is, for example, 65°C to 75°C, preferably the optimum temperature for the polymerase used in the reaction. The temperature (for annealing and extension reaction) is, for example, 55°C to 70°C.

[0187] <Assessing RPS25 Protein Amount> The collected cells are lysed to obtain an extract. The amount of RPS25 protein contained in the extract is assessed using immunochemical techniques such as Western blotting or ELISA (Enzyme-Linked Immunosorbent Assay). In Western blotting, any equipment can be used for the electrophoresis, transfer, and detection steps. The reaction time and temperature of the membrane with the primary or secondary antibody can be set as desired, for example, overnight at 4°C or 1 to 3 hours at room temperature.

[0188] The present invention is not limited to the above-described embodiments. For example, the single-stranded antisense oligonucleotide includes the following embodiments.

[0189] An antisense oligonucleotide according to a first aspect of the present invention is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that regulates the expression and / or function of the RPS25 gene, wherein each nucleoside in the single-stranded antisense oligonucleotide is linked via 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 substituent at the 2' position, the base length of the single-stranded antisense oligonucleotide is 12 to 30 mer, and the base sequence of the single-stranded antisense oligonucleotide is The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof is a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to at least one target region in the base sequence set forth in SEQ ID NO: 1, which is composed of the same base length as the single-stranded antisense oligonucleotide; a base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted or added in the target region; or a base sequence which hybridizes under stringent conditions to an oligonucleotide having the target region.

[0190] An antisense oligonucleotide according to a second aspect of the present invention is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that regulates the expression and / or function of the RPS25 gene, wherein each nucleoside in the single-stranded antisense oligonucleotide is linked via a phosphate group and / or a modified phosphate group, the single-stranded antisense oligonucleotide comprises a gap region, a 3' wing region linked to the 3' end of the gap region, and a 5' wing region linked 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 substituent at the 2' position, the base length of the single-stranded antisense oligonucleotide is 12 to 30 mer, and the base sequence of the single-stranded antisense oligonucleotide is A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof (excluding the single-stranded antisense oligonucleotides of Reference Examples 1 to 31) is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, which is a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to at least one target region in the base sequence set forth in SEQ ID NO: 1, which is composed of the same base length as the single-stranded antisense oligonucleotide; a base sequence complementary to a base sequence in the target region in which one or several bases have been deleted, substituted, inserted or added; or a base sequence which hybridizes under stringent conditions to an oligonucleotide having the target region.

[0191] An antisense oligonucleotide according to a third aspect of the present invention is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that regulates the expression and / or function of the RPS25 gene, wherein each nucleoside in the single-stranded antisense oligonucleotide is linked via 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 substituent at the 2' position, the base length of the single-stranded antisense oligonucleotide is 12 to 30 mer, and the base sequence of the single-stranded antisense oligonucleotide is A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof is a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to at least one target region of the base sequence set forth in SEQ ID NO: 1, which is composed of the same base length as the single-stranded antisense oligonucleotide; a base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted or added in the target region; or a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region (excluding the single-stranded antisense oligonucleotides of Reference Examples 1 to 31 and Design Examples 1 to 18).

[0192] In one aspect of the present invention, it is preferable that the base sequence of the single-stranded antisense oligonucleotide has a sequence identity of 95% or more and 100% or less with respect to a base sequence complementary to at least one target region in the base sequence set forth in SEQ ID NO: 1, the base sequence having the same base length as the single-stranded antisense oligonucleotide.

[0193] In one aspect of the present invention, it is preferable that 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.

[0194] In one aspect of the present invention, it is preferred that the gap region has a length of 5 to 20 nucleotides, the 3' wing region is a 3 to 5 nucleotide modified nucleic acid having a substituent at the 2' position, and the 5' wing region is a 3 to 5 nucleotide modified nucleic acid having a substituent at the 2' position.

[0195] In one aspect of the present invention, the 5'-CP nucleic acid is preferably located at least at the second position counting from the 5' side of the gap region.

[0196] In one aspect of the present invention, it is preferable that two or more of the 5'-CP nucleic acid be arranged in the gap region.

[0197] In one aspect of the present invention, it is preferable that 2 to 5 of the 5'-CP nucleic acids are arranged in the gap region.

[0198] In one aspect of the present invention, it is preferable that the 5'-CP nucleic acid is arranged in an amount of one-ninth or more relative to the gap region.

[0199] In one aspect of the present invention, it is preferable that the 5'-CP nucleic acid is arranged in an amount of one-fifth or more relative to the gap region.

[0200] In one aspect of the present invention, it is preferable that the 5'-CP nucleic acid is arranged in a continuous 2- to 4-mer sequence at least at one position in the gap region.

[0201] In one aspect of the present invention, the 5'-CP nucleic acid is preferably located on the 5'-end side of the gap region.

[0202] In one aspect of the present invention, the 5'-CP nucleic acid is preferably located on the 5'-end side and the 3'-end side of the gap region.

[0203] In one aspect of the present invention, the single-stranded antisense oligonucleotide preferably has a base length of 15 to 20 mer.

[0204] In one aspect of the present invention, the single-stranded antisense oligonucleotide preferably has a base length of 18 to 20 mer.

[0205] In one aspect of the present invention, it is preferred that 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, and that 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.

[0206] In one aspect of the present invention, it is preferred that 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, and that the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

[0207] In one aspect of the present invention, it is preferred that 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, and that the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA.

[0208] In one embodiment of the present invention, at least one internucleoside bond of the single-stranded antisense oligonucleotide is preferably a phosphorothioate bond.

[0209] In one embodiment of the present invention, at least one internucleoside bond of the single-stranded antisense oligonucleotide is preferably a phosphodiester bond.

[0210] In one embodiment of the present invention, the proportion of phosphorothioate bonds in the internucleoside bonds constituting the single-stranded antisense oligonucleotide is preferably 50% to 80%.

[0211] In one embodiment of the present invention, the proportion of phosphorothioate bonds in the internucleoside linkages constituting the single-stranded antisense oligonucleotide is preferably 50% to 70%.

[0212] In one aspect of the present invention, in 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 preferably a phosphorothioate bond (excluding the case where the 5'-CP nucleic acid is positioned at the 3' end of the gap region).

[0213] In one aspect of the present invention, in 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 the case where the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is a 5'-CP nucleic acid).

[0214] In one aspect of the present invention, in the gap region, 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.

[0215] In one aspect of the present invention, 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 a consecutive 14-22mer starting from bases located at positions 123, 124, 185, 186, 263, 264, 324, 325, 442, 443, or 447 to 453 in the base sequence set forth in SEQ ID NO: 1, counting from the 5' end; a base sequence complementary to a base sequence in the target region in which one or several bases have been deleted, substituted, inserted, or added; or a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region.

[0216] In one aspect of the present invention, 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 a consecutive 14-22mer starting from bases 324, 325, or 448 to 453, counting from the 5' end, in the base sequence set forth in SEQ ID NO: 1; a base sequence complementary to a base sequence in the target region in which one or several bases have been deleted, substituted, inserted, or added; or a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region.

[0217] In one aspect of the present invention, 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 18 to 22 consecutive bases from the 448th to 453rd bases counting from the 5' end in the base sequence set forth in SEQ ID NO: 1, and it is preferable that the 3' wing region is a 3 to 5 mer and the 5' wing region is a 3 to 5 mer.

[0218] In one aspect of the present invention, 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 a continuous 18- to 20-mer sequence beginning with bases 450 to 451 or 453 counting from the 5' end in the base sequence set forth in SEQ ID NO: 1.

[0219] In one aspect of the present invention, 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.

[0220] In one aspect of the present invention, 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: 6, 9, 12, 15 to 18, 22, 24, 27 to 29, 31 to 36, 38, 40 to 42, 47, and 49 to 54.

[0221] In one aspect of the present invention, 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: 41, 47, 50, and 53 to 55.

[0222] An antisense oligonucleotide conjugate according to one embodiment of the present invention is an antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof, comprising: the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof; and an additional substance bound to the single-stranded antisense oligonucleotide, wherein the additional substance is selected from the group consisting of polyethylene glycol, a peptide, an alkyl chain, a nucleic acid, a ligand compound, an antibody, a protein, and a sugar chain.

[0223] A pharmaceutical product according to one aspect of the present invention comprises, as an active ingredient, the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof.

[0224] In one embodiment of the pharmaceutical, the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof is preferably administered so as to be exposed to the central nervous system.

[0225] In one embodiment of the pharmaceutical, the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof is preferably administered to a subject susceptible to delayed central nervous system toxicity.

[0226] An agent for regulating the expression and / or function of the RPS25 gene according to one embodiment of the present invention comprises, as an active ingredient, the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof.

[0227] An inhibitor of dipeptide repeat production by RNA translation according to one embodiment of the present invention comprises the above-mentioned single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the above-mentioned antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof as an active ingredient.

[0228] A therapeutic agent for repeat disease according to one embodiment of the present invention comprises, as an active ingredient, the above-mentioned single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the above-mentioned antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof.

[0229] A preventive agent for repeat disease according to one embodiment of the present invention comprises the above-mentioned single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the above-mentioned antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof as an active ingredient.

[0230] In one embodiment of the therapeutic agent and the preventive agent, 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.

[0231] A method for treating or preventing a repeat disease according to one embodiment of the present invention comprises administering to an individual the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof.

[0232] In one embodiment of the method for treating or preventing a repeat disease, 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.

[0233] One aspect of the present invention provides the above-mentioned single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the above-mentioned antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of C9orf72 ALS.

[0234] One aspect of the present invention provides the above-mentioned single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the above-mentioned antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof, for use in producing an agent for treating or preventing C9orf72 ALS.

[0235] Examples of the present invention will be described below, but the present invention is not limited to these examples.

[0236] <<Preparation of Single-Stranded Antisense Oligonucleotides Against the RPS25 Gene>> First, the single-stranded antisense oligonucleotides shown in Tables 2-1 to 2-4, 3-1, 3-2, and 4 were designed. For some of the designed oligonucleotides, single-stranded antisense oligonucleotides against the RPS25 gene were prepared by the following procedure.

[0237] Single-stranded antisense oligonucleotides containing modified nucleic acids, such as 2'-O-methyl nucleic acids, 2'-MOE nucleic acids, AmNA, scpBNA, 5'-CP nucleic acids, and / or GuNA, and / or nucleic acids containing 5-methylcytosine as the nucleobase moiety, were synthesized on a 0.2 μmol scale using an automated nucleic acid synthesizer (nS-8 model, Gene Design Co., Ltd.). Chain elongation was carried out using a standard phosphoramidite protocol. However, when adding a monomer bearing a levunilyl protecting group at the 5' position, 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 outside the synthesizer for 1 hour, after which the solid support was attached to the synthesizer and synthesis resumed. CPG resin was used as the solid support. Furthermore, DDTT (((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazaoline-3-thione) or the like was used for sulfurization to form the phosphorothioate (PS) backbone. Single-stranded antisense oligonucleotides containing 2'-MOE nucleic acid, AmNA, and / or scpBNA were obtained in such a way that the terminal 5'-position hydroxyl group was not protected with a DMTr (4,4'-dimethoxytrityl) group and the 3'-position was supported on a solid phase. Subsequently, the single-stranded antisense oligonucleotide was cleaved from the solid phase support by alkali treatment and recovered in solution. Thereafter, the solvent was evaporated from the recovered solution to obtain a crude product. The resulting crude product was purified by reverse-phase HPLC to obtain a purified single-stranded antisense oligonucleotide. The purity and structure of each of the resulting single-stranded antisense oligonucleotides were confirmed by LC-MS (Waters).

[0238] The single-stranded antisense oligonucleotides prepared by the above-described method are listed in Tables 2-1 to 2-4, 3-1, and 3-2 below. The single-stranded antisense oligonucleotides shown in Tables 2-1 to 2-4, 3-1, and 3-2 are single-stranded antisense oligonucleotides directed against human RPS25 mRNA (SEQ ID NO: 1).

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246] In this specification, the following symbols or notations may be used to represent corresponding structures:

[0247]

[0248]

[0249] In the structural formula shown above, R １ and R ２ are each independently a hydrogen atom or a linear or branched alkyl group having 1 to 3 carbon atoms. １ and R ２ Both represent a methyl group. ３ , R ４ , and R ５ are each independently 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. ３ and R ５ are both hydrogen atoms, and R ４ When R is a methyl group, it is represented as "Gm" and R ３ is a hydrogen atom, and R４ and R ５ When both are methyl groups, it is represented as "Gdm", and R ３ and R ５ is a hydrogen atom, and R ４ When is a tert-butyl group, it is represented as "GtB".

[0250] <<Evaluation of RPS25 Gene Expression>> Evaluation of RPS25 gene expression was performed using human fetal kidney cells according to the single-stranded antisense oligonucleotides produced. The evaluation of RPS25 gene expression can also be performed using human iPS cell-derived neurons. In this example, gene expression evaluation refers to assessing the amount of mRNA by measuring the amount of complementary DNA (cDNA) obtained by reverse transcription. The specific procedures for each expression evaluation are described below.

[0251] <Expression Evaluation Using Human Embryonic Kidney Cells> Human embryonic kidney cells HEK293T (ATCC® CRL-3216™) were cultured in a culture medium at 37°C and 5% CO ２ The HEK293T cells were cultured under the following conditions. The culture medium used had the following composition:

[0252] Composition of culture medium for HEK293T cells Dulbecco's modified Eagle's medium (DMEM): Sigma, Cat# D6429 10% Fetal bovine serum (FBS): BioWest, Cat# S1820 100-fold diluted penicillin-streptomycin mixed solution: Nacalai Tesque, Cat# 09367-34 (penicillin 10,000 units / ml, streptomycin 10,000 μg / ml, stabilizer included)

[0253] First, on the day before measuring the expression level of the human RPS25 gene, HEK293T cells (12,000 cells / well) were seeded in a 96-well plate and incubated at 37°C, 5% CO ２The cells were cultured overnight under the conditions of 0.5 nM, 5 nM, 15 nM, or 50 nM. Then, each single-stranded antisense oligonucleotide diluted in phosphate-buffered saline (PBS) (final concentration: 0.5 nM, 5 nM, 15 nM, or 50 nM) was transfected into the above cells by lipofection. As a negative control, cells transfected with PBS containing no single-stranded antisense oligonucleotide were used. The transfected cells were incubated in growth medium at 37°C and 5% CO. ２ The cells were cultured for 48 hours under the conditions of 95°C for 3 seconds and 60°C for 30 seconds. The growth medium was then removed, and the extracted total RNA was reverse-transcribed 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, 60°C for 30 seconds).

[0254] List of gene-specific probes used in the evaluation of human RPS25 gene expression: human RPS25: Hs01568661_g1 human GAPDH: 4326317E (internal control)

[0255] The expression ratio of human RPS25 mRNA for each single-stranded antisense oligonucleotide relative to human RPS25 mRNA, determined by the above-described method, is shown in Tables 5-1 to 5-3 and Tables 6-1 to 6-2 (column "hRPS25 Expression Ratio"). The expression ratio of human RPS25 mRNA determined in the negative control group was set to 1.00. Single-stranded antisense oligonucleotides with an expression ratio of 0.80 or less were determined to be capable of suppressing human RPS25 mRNA expression. In the tables, "-" indicates that no measurement was performed. It is generally believed that suppression of mRNA expression also suppresses subsequent protein translation. Therefore, single-stranded antisense oligonucleotides with an expression ratio of 0.80 or less can be determined to be capable of modulating the function of the human RPS25 gene.

[0256]

[0257]

[0258]

[0259]

[0260]

[0261] <Expression evaluation using mouse primary cultured neurons> Mouse primary cultured neurons were cultured in a culture medium at 37°C and 5% CO ２ The culture medium for primary cultured mouse neurons has the following composition:

[0262] Culture medium composition for mouse primary cultured neurons: B-27 Electrophysiology Kit: Gibco, Cat. #A1413701; 100-fold diluted 200 mM L-glutamine solution: Nacalai Tesque, Cat. #16948-04; 100-fold diluted penicillin-streptomycin mixed solution: Nacalai Tesque, Cat. #09367-34 (penicillin 10,000 units / ml, streptomycin 10,000 μg / ml, stabilizer included)

[0263] First, primary cultured mouse neurons (derived from mouse fetal cerebrum) were seeded in a 96-well plate at 40,000 cells / well and incubated at 37°C, 5% CO ２ The cells are cultured for 5 days under these conditions. Then, each single-stranded antisense oligonucleotide diluted with phosphate-buffered saline (PBS) (final concentration: 0.01 μM, 0.1 μM, or 1 μM) is added to the culture medium. As a negative control, PBS containing no single-stranded antisense oligonucleotide is added to the culture medium. The cells are then cultured in the culture medium at 37°C and 5% CO. ２ The cells were cultured for 48 hours under the conditions of 95°C for 3 seconds, 60°C for 30 seconds, and 40 cycles of 95°C for 3 seconds and 60°C for 30 seconds were performed on the extracted total RNA using a 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).

[0264] List of gene-specific probes used in evaluating the expression of the mouse RPS25 gene: Mouse Rps25: Mm02342783_g1 Mouse GAPDH: 4352339E (internal control)

[0265] Evaluation using human iPS cell-derived motor neurons: Motor neurons are differentiated from human iPS cells and used for evaluation. Cells are maintained and differentiated in the following medium at 37°C and 5% CO ２ This is done under the following conditions.

[0266] List of medium compositions (Medium for SNL cells) DMEM (Sigma-Aldrich, Cat# D6429), 100-fold diluted penicillin-streptomycin mixed solution (ThermoFisher Scientific, Cat# 15140-122), 10-fold diluted fetal bovine serum (ThermoFisher Scientific, Cat# 10437-028) (Medium for iPS cells) Medium for primate ES / iPS cells (REPROCELL, Cat# RCHEMD001B), 100-fold diluted penicillin-streptomycin mixed solution (ThermoFisher Scientific, Cat# 15140-122) (Mixed medium A) DMEM / Ham's F12 GlutaMAX (ThermoFisher Scientific, Cat# 10565-018) 2 mM L-glutamine (ThermoFisher Scientific, Cat# 25030-081) Non-Essential Amino Acid (NEAA) (ThermoFisher Scientific, Cat# 11140-050) 100-fold diluted penicillin-streptomycin mixed solution (ThermoFisher Scientific,(Mixed medium B) Neurobasal medium (ThermoFisher Scientific, cat#21103-049) 2 mM L-glutamine (ThermoFisher Scientific, cat#25030-081) Non-Essential Amino Acid (NEAA) (ThermoFisher Scientific, cat#15140-122) 2 μg / mL Heparin (Sigma-Aldrich, H-4784) N2 supplement (ThermoFisher Scientific, cat#17502-048) Antibiotic-Antimycotic (ThermoFisher Scientific, Cat# 11140-050) 2 μg / mL Heparin (Sigma-Aldrich, H-4784) N2 supplement (ThermoFisher Scientific, Cat# 17502-048) 10 ng / mL IGF-1 (PeproTech, Cat# 100-11) 10 ng / mL Human CNTF (PeproTech, Cat# 450-13) 10 ng / mL Human GDNF (R&D Systems,(Cat #212-GD-050) B27 supplement (ThermoFisher Scientific, cat #12587010) 200 μM Ascorbic acid (Sigma-Aldrich, cat #A5960) 10 ng / mL Human BDNF (Peprotech, cat #450-02) (Culture medium for neurons) Neurobasal medium Electro (ThermoFisher Scientific, cat #A14098-01) 2 mM L-glutamine (ThermoFisher Non-Essential Amino Acid (NEAA) (ThermoFisher Scientific, Cat# 25030-081) Non-Essential Amino Acid (NEAA) (ThermoFisher Scientific, Cat# 11140-050) Antibiotic-Antimycotic (ThermoFisher Scientific, Cat# 15240-062) 2 μg / mL Heparin (Sigma-Aldrich, H-4784) N2 supplement (ThermoFisher Scientific, Cat# 17502-048) 10 ng / mL IGF-1 (PeproTech, cat#100-11) 10 ng / mL Human CNTF (PeproTech, cat#450-13) 10 ng / mL Human GDNF (R&D Systems, cat#212-GD-050) B27 supplement, Electro (ThermoFisher Scientific, cat#A14097-01) 200 μM Ascorbic acid (Sigma-Aldrich, cat#A5960) 10 ng / mL Human BDNF (Peprotech, cat#450-02) 25 μM 2-mercaptoethanol (ThermoFisher Scientific, cat#21985-0123) 0.1% bovine serum albumin (Sigma-Aldrich, cat#A9576),

[0267] <Mitomycin treatment of feeder cells> Mitomycin-treated SNL cells (Cell BioLabs, Cat# CBA-316) are prepared as feeder cells for seeding human iPS cells. Mitomycin treatment of SNL cells is performed as follows: First, 0.1% gelatin (Fujifilm Wako Pure Chemical Industries, Ltd., Cat# 190-15805) is added to a 10 cm Petri dish (Iwaki, Cat# 3020-100), and the cells are incubated at 37°C and 5% CO ２ The petri dish was left to stand in an incubator under the conditions for at least 1 hour (hereinafter, this operation may be referred to as "gelatin treatment"). After that, excess gelatin was removed from the petri dish by suction, and thawed SNL cells were cultured in a SNL cell medium at 1 to 2 × 10 per petri dish. ６ The cells are then diluted 8-16 times every 3-4 days and passaged to grow to the required cell number. Then, 2-4 x 10 cells are seeded in 15 cm dishes (Iwaki, Cat# 3030-150) treated with 0.1% gelatin at 2-4 x 10 cells per dish. ６ SNL cells are seeded onto the dish. After culturing the cells until they are 80-90% confluent, mitomycin C (Kyowa Kirin Co., Ltd., 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 μg / mL. The culture medium is then incubated at 37°C, 5% CO ２ The dish was left in an incubator under these conditions for 2 hours and 15 minutes. The medium was then removed from the dish, and the SNL cells were washed once with PBS. 2.5% trypsin / EDTA (ThermoFisher Scientific, Cat# 15090-046) was diluted with PBS (final concentration 0.25%), added to the SNL cells, and left to stand at room temperature for 1 minute. The SNL cells were then collected in a tube, centrifuged, suspended in Cellbanker® (ZENOAQ Resources, Cat# CB011), and cryopreserved.

[0268] <Maintenance of human iPS cells> 0.1% gelatin was added to a 10 cm petri dish and incubated at 37°C, 5% CO ２The mitomycin-treated SNL cells were suspended in SNL cell medium. Then, 1.5 x 10 ６ The SNL cells are seeded in a 10 cm dish and cultured for 2 to 3 days. The SNL cell medium is then removed from the dish, and the SNL cells are washed with PBS. Human iPS cells (201B7 strain, obtained from iPS Academia Japan, Inc., AJ-H1-01) suspended in iPS cell medium containing 1 / 1000 of the amount of Y-27632 (manufactured by Tocris, Cat. #1254) are then seeded in the dish. The medium is replaced daily from the day after seeding until differentiation induction begins.

[0269] <Induction of Differentiation from Human iPS Cells to Motor Neurons> Y-27632 (final concentration 10 μM) was added to the cell culture medium of human iPS cells, and the iPS cells were exposed to the Y-27632 for at least 1 hour. After removing the culture supernatant and washing the cells with PBS, cell dissociation solution (CTK solution) (REPROCELL, Cat# RCHETP002) was added and allowed to react at room temperature for 1 minute. The CTK solution was removed, and the cells were washed twice with PBS, after which 1 mL of iPS cell medium was added. The cells were detached with a cell scraper, and cell clumps were dispersed through a cell strainer (Becton Dickinson, Cat# 352350) to obtain a cell suspension. The resulting suspension was 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 μM), SB431542 (Tocris, Cat#1614) (final concentration 2 μM), CHIR-99021 (Stemgent, Cat#04-0004-10) (final concentration 3 μM), and Y-27632 (final concentration 10 μM), and the medium was incubated at 37°C and 5% CO ２ The cells are cultured in an incubator under the conditions (culture day 0).

[0270] On days 2 and 4 of culture, the culture medium was removed with a pipette and replaced with fresh mixed medium A supplemented with LDN193189 (final concentration 0.3 μM), SB431542 (final concentration 2 μM), and CHIR-99021 (final concentration 3 μM). On days 7, 9, and 11 of culture, the culture medium was removed with a pipette and replaced with fresh mixed medium A supplemented with LDN193189 (final concentration 0.3 μM), SB431542 (final concentration 2 μM), CHIR-99021 (final concentration 3 μM), Purmorphamine (Fujifilm Wako Pure Chemical Industries, Ltd., Cat# 166-23991) (final concentration 0.5 μM), and Retinoic Acid (Sigma-Aldrich, Cat# R2625) (final concentration 0.1 μM). On days 14 and 16 of culture, 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 μM), retinoic acid (final concentration 0.1 μM), human BDNF (final concentration 10 ng / mL), and ascorbic acid (Sigma-Aldrich, Cat# A5960) (final concentration 200 μM).

[0271] On day 18 of culture, mixed medium B was replaced with fresh medium supplemented with Purmorphamine (final concentration 0.5 μM), Retinoic Acid (final concentration 0.1 μM), and Compound E (Calbiochem, Cat# 565790) (final concentration 0.1 μM). On day 21 of culture, the cell masses were washed with PBS and centrifuged to remove the supernatant. Accutase (Innovative Cell Technologies, Cat# AT104) and Y27632 (final concentration 10 μM) were added to the cell masses and incubated at 37°C for 10 minutes. The cell masses were then cooled on ice and dispersed by pipetting. After centrifugation (300 × g, 5 minutes, 4°C), the precipitated cells were collected and suspended in mixed medium B. This procedure was repeated twice. Through the above procedures, motor neurons derived from iPS cells were obtained. The obtained motor neurons were suspended in Cellbanker®, dispensed, and cryopreserved.

[0272] <Culturing of human iPS cell-derived motor neurons and evaluation of single-stranded antisense nucleotides> Poly-L-Ornitine Solution (PLO) solution (Sigma-Aldrich, Cat. #P4957) diluted 6.66-fold with PBS was added to a 96-well Optical Btm Plt Polybase Black with Lid Cell Culture Sterile PS (ThermoFisher Scientific, Cat. #165305) and allowed to stand at room temperature for at least 2 hours. After washing three times with PBS, 0.5 μg / cm iMatrix diluted with PBS was added. ２ The cells are added to the plate so that the concentration is 100% and allowed to stand overnight at 4°C. Next, the human iPS cell-derived motor neurons cryopreserved in the previous section are thawed and suspended in neuronal medium. The supernatant is then removed by centrifugation, and the cells are resuspended in neuronal medium containing 1 / 100 of the volume of Culture One Supplement (ThermoFisher Scientific, A3320201) and Compound E (final concentration 0.1 μM). The cells are seeded onto a coated 96-well plate at 30,000 cells / well and incubated at 37°C and 5% CO. ２ The cells are cultured in an incubator under the specified conditions for 28 days. Half of the neuronal culture medium is replaced every 2-3 days. From the start of culture until the 7th day, a neuronal culture medium containing Culture One Supplement and Compound E is used.

[0273] After seeding, on days 1, 11, 18, and 26 (referred to as D1, D11, D18, and D26, respectively), each single-stranded antisense oligonucleotide diluted with PBS (final concentrations of 0.01 μM, 0.1 μM, and 1 μM) was added to the culture medium. As a negative control, cells were cultured in PBS containing no single-stranded antisense oligonucleotide. The cells were cultured in the culture medium at 37°C and 5% CO. ２After culturing for 48 or 72 hours under these conditions, the medium containing the single-stranded antisense nucleotides was removed, and the cells were continued to be cultured in neuronal culture medium (half of the medium was replaced every 2-3 days). The culture 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) (95°C for 3 seconds, 60°C for 30 seconds, 40 cycles). The "expression inhibition rate" was calculated using the following formula (1): (Expression inhibition rate)=1−(expression ratio) (Equation 1) The larger the expression inhibition rate, the more the single-stranded antisense oligonucleotide is judged to be able to inhibit the expression of human RPS25 mRNA.

[0274] List of gene-specific probes used in the evaluation of human RPS25 gene expression: human RPS25: Hs01568661_g1 human GAPDH: 4326317E (internal control)

[0275] <<Evaluation of RPS25 Protein Expression>> RPS25 protein expression was evaluated using human fetal kidney cells according to the single-stranded antisense oligonucleotide produced. In this example, evaluation of protein expression level refers to evaluation of the amount of protein translated from mRNA. Specific procedures for expression evaluation are described below.

[0276] <Expression Evaluation Using Human Embryonic Kidney Cells> Human embryonic kidney cells HEK293T (ATCC® CRL-3216™) were cultured in a culture medium at 37°C and 5% CO ２ The culture medium for HEK293T cells is one with the following composition:

[0277] Composition of culture medium for HEK293T cells Dulbecco's modified Eagle's medium (DMEM): Sigma, Cat# D6429 10% Fetal bovine serum (FBS): BioWest, Cat# S1820 100-fold diluted penicillin-streptomycin mixed solution: Nacalai Tesque, Cat# 09367-34 (penicillin 10,000 units / ml, streptomycin 10,000 μg / ml, stabilizer included)

[0278] <Quantitative Analysis of Proteins by Western Blotting> First, HEK293T cells (500,000 cells / well) were seeded in a 6-well plate and incubated at 37°C, 5% CO ２ The cells were then cultured overnight under the conditions of 0.1% ssDNA and 0.2% ssDNA. Then, each single-stranded antisense oligonucleotide (final concentration 50 nM) diluted in phosphate-buffered saline (PBS) was transfected into the cells using the lipofection method. As a negative control, cells transfected with PBS containing no single-stranded antisense oligonucleotide were used. The transfected cells were incubated in growth medium at 37°C and 5% CO. ２The cells were cultured for 48 hours under these conditions. The growth medium was then removed, washed with PBS, and collected using a cell scraper. The collected solution was centrifuged at 2700 x g for 5 minutes at 4°C to precipitate the cells. After removing the supernatant, 1 mL of RIPA Lysis and Extraction buffer (ThermoFisher Scientific, Cat# 89900) containing 1 / 100 of the amount of protease inhibitor (ThermoFisher Scientific, Cat# 1860932) was added, and the cells were disrupted using an ultrasonic disrupter. The cells were then centrifuged at 15,000 x g for 10 minutes at 4°C, and the supernatant was used as a sample. The collected samples were subjected to protein quantification using a Pierce™ BCA Protein Assay kit (ThermoScientific, Cat# 23225). After adjusting the concentration of each sample to a constant level, Pierce™ Lane Marker Reducing Sample Buffer (ThermoFisher Scientifier, Cat# 39000) was added and heated at 95°C for 5 minutes. The prepared samples were layered so that the protein amount was 10 μg to 20 μg per lane, and electrophoresis was performed. Electrophoresis was performed using Criterion™ TDX™ Precast Gel 4-15% (BIO-RAD, Cat# 5671085J10) for 30 minutes at a constant voltage of 200 V. The running buffer used was Running Buffer Solution (10x) for SDS-PAGE (Nacalai Tesque, Cat# 30329-61) diluted to 1x.

[0279] After electrophoresis, transfer was performed using a semi-dry method. A Trans-Blot Turbo Transfer Pack (BIO-RAD, Cat. #1704157) was used as the membrane, and the transfer device used was the BIO-RAD Trans-Blot Turbo Transfer System Standard Protocol (30 minutes). After transfer, the membrane was washed with TBST. TBST consisted of 1x Tris-buffered saline (pH 7.4) (Nacalai Tesque, Cat. #35438-81) diluted to 1x with 0.06% polyoxyethylene sorbitan monolaurate (Tween-20) (Nacalai Tesque, Cat. #28353-85). After washing, the plate is blocked with Blocking One (Nacalai Tesque, Cat. #03953-95) or PVDF Blocking Reagent (Toyobo, Cat. #NYPBR01) by shaking at room temperature for 1 hour. After blocking, the plate is washed with TBST, diluted primary antibody is added, and the plate is shaken overnight at 4°C. The primary antibody and dilution solvent are as follows: RPS25 antibody: Anti-RPS25 antibody (Abcam, Cat# ab102940) Solvent: Canget signal Solution 1 (Toyobo, Cat# NKB-101) β-actin antibody: β-Actin (13E5) Rabbit mAb (HRP conjugate) (Cell Signaling, Cat# 5125) Solvent: Blocking One

[0280] After shaking the primary antibody, wash with TBST, add diluted secondary antibody, and shake at room temperature for 1 hour. The secondary antibody and dilution solvent are as follows: RPS25 antibody: Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody (Invitrogen, Cat# A24537) Solvent: Canget Signal Solution 2 (Toyobo Co., Ltd., Cat# NKB-101) After shaking the secondary antibody, wash with TBST and perform detection using ECL prime (Amersham, Cat# RPN2232). An Amersham Imager 680 is used for detection and analysis.

[0281] The human RPS25 protein expression ratio for each single-stranded antisense oligonucleotide determined by the above method is calculated. The human RPS25 protein expression ratio determined in the negative control group is set to 1.00. A protein expression ratio below 0.80 is determined to be a single-stranded antisense oligonucleotide capable of regulating the function of the RPS25 gene.

[0282] <Evaluation of Cytotoxicity of Single-Stranded Antisense Oligonucleotides> HeLa-S3 cells, which are human cervical cancer cells, were cultured in growth medium at 37°C and 5% CO. ２ The growth medium used was one with the following composition:

[0283] Composition of growth medium used for cytotoxicity evaluation: 10% fetal bovine serum (FBS): manufactured by GIBCO, Cat. No. 10437028; 1% non-essential amino acids (NEAA): manufactured by GIBCO, Cat. No. 11140050; Dulbecco's Modified Eagle Medium low glucose (containing L-glutamine and phenol red) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Cat. No. 041-29775).

[0284] The day before the experiment, the above cells (1.0 × 10 ４ The seeded cells were incubated at 37°C and 5% CO ２After overnight culture under the above conditions, 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 cells were incubated at 37°C and 5% CO ２ The cells were cultured for 24 hours under the conditions of

[0045] . Subsequently, the Caspase-Glo 3 / 7 Assay System (Promega, Cat# G8093) or CellTiter-Blue Cell Viability Assay (Promega, Cat# G8081) was added to the growth medium to evaluate caspase activity and cell viability. The cytotoxicity evaluation results (cell viability) for each single-stranded antisense oligonucleotide, determined by the above-described method, are shown in Tables 7-1 to 7-3.

[0285]

[0286]

[0287]

[0288] <<Evaluation of serum stability of single-stranded antisense oligonucleotides>> Mouse serum (20 μL) or human serum (20 μL) was mixed with 4 μL of a Tris-EDTA buffer (pH 8.0) solution containing 400 pmol of single-stranded antisense oligonucleotide, followed by the addition of mineral oil (15 μL). This solution was incubated at 37°C and then mixed with 10 μL of 8 mol / L urea solution to inactivate the nucleases in the serum. Ultrapure water (10 μL) was added, and the solution was centrifuged to separate the aqueous layer containing the single-stranded antisense oligonucleotide from the mineral oil layer. The aqueous layer was analyzed by LC-MS (Waters), and the remaining single-stranded antisense oligonucleotide was calculated from the area intensity of the UV-chromatogram of the resulting single-stranded antisense oligonucleotide. "Remaining oligonucleotide (%)" indicates the percentage of undegraded single-stranded antisense oligonucleotide remaining after 72 hours relative to the undegraded single-stranded antisense oligonucleotide at the time of analysis immediately after mixing with serum.

[0289] Those with a survival rate of 50% or more after 72 hours are judged to be stable single-stranded antisense oligonucleotides.

[0290] <<In vivo expression evaluation of RPS25 gene>> Expression evaluation of the RPS25 gene was performed by intracerebroventricular administration to mice and measuring the amount of mRNA in each region of the prefrontal cortex. In this example, gene expression evaluation refers to evaluating the amount of mRNA by measuring the amount of complementary DNA (cDNA) obtained by reverse transcription. Specific procedures for each expression evaluation are described below.

[0291] FVB mice (CLEA Japan) were anesthetized with isoflurane (Pfizer, Cat# 114133403). Next, antisense oligonucleotides dissolved in artificial cerebrospinal fluid (Tocris Bioscience, Cat# 3525 / 25 mL) were administered to the anesthetized FVB mice at 10 μL / individual using a two-stage needle (Top, Medical Device Approval Number 15800BZZ01460000) attached to a 50 μL Hamilton syringe (Hamilton, Cat# 705LT). Negative control mice received only artificial cerebrospinal fluid at 10 μL / individual.

[0292] After a certain period of rearing following administration, the FVB mice were euthanized and three sites were collected: the brain, cervical spinal cord, and lumbar spinal cord. The collected tissues were immersed in RNA Later (Applied Biosystems, Cat# AM7024) overnight and then stored at -80°C. RNA was extracted from the stored tissue samples using an RNeasy Mini Kit (QIAGEN, Cat# 74106). Reverse transcription of the extracted mRNA was performed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cat# 4368814). 1 μg of mRNA was diluted to 20 μL and used for the reverse transcription reaction. The complementary DNA (cDNA) obtained from this reverse transcription reaction was used to perform real-time PCR (40 cycles of 95°C for 3 seconds, 60°C for 30 seconds) using predesigned gene-specific probes (see below) in TaqManExpression Assays (Applied Biosystems).

[0293] List of gene-specific probes used in evaluating the expression of the mouse RPS25 gene: Mouse Rps25: Mm02342783_g1 Mouse GAPDH: 4352339E (internal control)

[0294] The expression ratio of mouse RPS25 mRNA for each single-stranded antisense oligonucleotide, determined by the above-described method, is shown in Tables 8-1 and 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 inhibition of mRNA expression will also result in inhibition of subsequent protein translation, an expression ratio of 0.80 or less can be determined to be a single-stranded antisense oligonucleotide 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 and 8-2 bind are regions whose sequences are conserved between the human RPS25 gene and the mouse RPS25 gene.

[0295]

[0296]

[0297] <<In Vivo Evaluation of Central Toxicity of Single-Stranded Antisense Oligonucleotides>> The central toxicity of each single-stranded antisense oligonucleotide was evaluated by intracerebroventricular administration to mice. The mice were maintained immediately to 1 hour after administration and for up to 28 days after administration. The mice were then observed for general condition and body weight, and pathological examination (hematoxylin-eosin staining) of tissue samples was performed at the time of collection. The results are shown in Tables 9-1 and 9-2. The "score" column in the "in vivo acute neuroTox" section of Tables 9-1 and 9-2 was calculated based on the following evaluation criteria. For items 1-5, the highest score among the findings observed was recorded for each individual, and the scores for each item were summed. For example, if ptosis, lateral recumbency, bradypnea, and convulsions were observed in one individual, the total score would be 1 + 4 + 4 = 9. Higher scores indicate more severe findings, so the higher the total score, the more severe the acute central toxicity findings can be judged to be. The average score for each group was used. In the table, "-" indicates that no measurement was performed. 1. Hyperactivity (1 point), stereotypic behavior (grooming, circling) (1 point), rearing behavior (1 point), vocalization (1 point), hypersensitivity / tail lift (1 point) 2. Ptosis, eye closure, decreased (absent) spontaneous movement (1 point) 3. Staggering (1 point), ataxic gait (2 points) 4. Abnormal posture / irregular breathing (1 point), lateral / prone position (2 points), bradypnea / abnormal (4 points) 5. Jumping, tremors, muscle spasms (2 points), convulsions (4 points)

[0298] The "score" column in "in vivo delayed neuroTox" was calculated based on the following evaluation criteria. Each score was calculated as the average value for each group. "ICR" means Crl:CD1 mice, and "FVB" means FVB / NJcl mice. Clinical sign score: 0 points: no abnormalities; 1 point: abnormal hind limb function, tremors, decreased spontaneous movement; 2 points: dragging of hind limbs, tail or hind limb weakness; 3 points: complete hind limb dysfunction, hind limb paralysis, recumbency, prone position; 4 points: euthanasia. Pathological score: 0 points: no abnormalities; 1 point: abnormalities (single cell necrosis, vacuolation, etc.) For Example 51 in Table 9-1 and Reference Example 31 in Table 9-2, central nervous system toxicity was evaluated by the following method.

[0299] <<In Vivo Evaluation of Central Toxicity of Single-Stranded Antisense Oligonucleotides (2)>> The acute central toxicity (in vivo acute neuroTox) of the single-stranded antisense oligonucleotides of Example 51 in Table 9-1 and Reference Example 31 in Table 9-2 was evaluated by intracerebroventricular administration to ICR mice and evaluation one day after administration. The evaluation items were behavioral evaluation, and the behavioral evaluation of the mice was performed using functional observational battery (FOB). The evaluation items were 1) posture, 2) external abnormalities, 3) stereotypic behavior, 4) responsiveness to stimuli, 5) grip strength, 6) respiration, and 7) tremors / convulsions, each of which 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 under "in vivo acute neuroTox" in Tables 9-1 and 9-2.

[0300] <<In Vivo Evaluation of Central Toxicity of Single-Stranded Antisense Oligonucleotides (3)>> Delayed central toxicity (in vivo delayed neuroTox Clinical Sign Score) of the single-stranded antisense oligonucleotides of Example 51 in Table 9-1 and Reference Example 31 in Table 9-2 was evaluated by intracerebroventricular administration to ICR mice and evaluation was performed up to 28 days after administration. The evaluation item was behavioral evaluation, and the behavioral evaluation of the mice was performed using Functional Observation Battery (FOB). The evaluation items were 1) posture, 2) external abnormalities, 3) stereotypic behavior, 4) reactivity to stimuli, 5) grip strength, 6) respiration, and 7) tremors / convulsions, each of which was scored as normal (0), slightly abnormal (1), and extremely abnormal (2). The total score obtained for each item was entered in the "Clinical Sign Score" column under "in vivo delayed neuroTox" in Tables 9-1 and 9-2.

[0301]

[0302]

[0303] <<In silico evaluation of narrow off-target toxicity>> Narrow off-target toxicity evaluation for each single-stranded antisense oligonucleotide was performed using the high-speed base sequence search system (GGGenome) described above. 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 mismatches, deletions, or insertions were found during hybridization, the number of mismatches was calculated by summing the corresponding base numbers. For example, if the base sequence of the RNA targeted by the single-stranded antisense oligonucleotide is "AGCTGTAC," the mismatch (underlined base) for RNA with the base sequence "ATCTGTAC" is calculated as "1," and the number of mismatches is calculated as "1." Furthermore, if the base sequence of the target RNA is "AGCTGTAC," then for an RNA having the base sequence "ATCTG<G>TAC," the mismatch (underlined base) will be "1" and the deletion (base surrounded by angle brackets) will be "1," resulting in a calculated number of mismatches of "2." Furthermore, if the base sequence of the target RNA is "AGCTGTAC," then for an RNA having the base sequence "ATCTG*AC," the mismatch (underlined base) will be "1" and the insertion (position marked with *) will be "1," resulting in a calculated number of mismatches of "2." In this way, the number of mismatches upon hybridization to RNA other than the target (off-target RNA) is counted, and the number of RNAs with perfect matches, the number of RNAs with mismatches of 1 or less, and the number of RNAs with mismatches of 2 or less are calculated for each single-stranded antisense oligonucleotide. The fewer the number of RNAs with mismatches of 0, 1 or less, or 2 or less, the lower the risk of off-target toxicity in the narrow sense; conversely, the greater the number of RNAs with mismatches of 0, 1 or less, or 2 or less, the higher the risk of off-target toxicity in the narrow sense. The results are shown in Tables 10-1 to 10-4. When the base length is 18 or more, the number of RNAs that hybridize with the mismatch number of 1 or less is 10 or less, which is considered to have a low off-target risk in the narrow sense, and can be said to be a more preferable single-stranded antisense oligonucleotide.

[0304]

[0305]

[0306]

[0307]

[0308] Although the embodiments and examples of the present invention have been described above, it is also planned from the beginning that the configurations of the above-described embodiments and examples may be appropriately combined.

[0309] The embodiments and examples disclosed herein are illustrative in all respects and should not be considered limiting. The scope of the present invention is defined by the claims, not by the embodiments and examples described above, and is intended to include any modifications within the scope of the claims and meanings equivalent to the claims.

Claims

1. A single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof for regulating the expression and / or function of RPS25 gene, wherein each nucleoside in the single-stranded antisense oligonucleotide is linked via a phosphate group and / or a modified phosphate group, the single-stranded antisense oligonucleotide comprises a gap region, a 3' wing region linked to the 3' end of the gap region, and a 5' wing region linked to the 5' end of the gap region, the gap region being 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 being modified nucleic acids having a substituent at the 2' position, the base length of the single-stranded antisense oligonucleotide is 12 to 30 mer, and the base sequence of the single-stranded antisense oligonucleotide is A single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof, which is a base sequence having 90% or more and 100% sequence identity based on a base sequence complementary to at least one target region having the same base length as the single-stranded antisense oligonucleotide 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 a base sequence which hybridizes under stringent conditions to an oligonucleotide having the target region.

2. A single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof for regulating the expression and / or function of RPS25 gene, wherein each nucleoside in the single-stranded antisense oligonucleotide is linked via a phosphate group and / or a modified phosphate group, the single-stranded antisense oligonucleotide comprises a gap region, a 3' wing region linked to the 3' end of the gap region, and a 5' wing region linked to the 5' end of the gap region, the gap region being 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 being modified nucleic acids having a substituent at the 2' position, the base length of the single-stranded antisense oligonucleotide is 12 to 30 mer, and the base sequence of the single-stranded antisense oligonucleotide is A single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof (excluding the single-stranded antisense oligonucleotides of Reference Examples 1 to 31), which is a base sequence having 90% to 100% sequence identity based on a base sequence complementary to at least one target region having the same base length as the single-stranded antisense oligonucleotide 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 have been deleted, substituted, inserted or added in the target region; or a base sequence which hybridizes under stringent conditions to an oligonucleotide having the target region.

3. A single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof for regulating the expression and / or function of RPS25 gene, wherein each nucleoside in the single-stranded antisense oligonucleotide is linked via a phosphate group and / or a modified phosphate group, the single-stranded antisense oligonucleotide comprises a gap region, a 3' wing region linked to the 3' end of the gap region, and a 5' wing region linked to the 5' end of the gap region, the gap region being 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 being modified nucleic acids having a substituent at the 2' position, the base length of the single-stranded antisense oligonucleotide is 12 to 30 mer, and the base sequence of the single-stranded antisense oligonucleotide is A single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof (excluding the single-stranded antisense oligonucleotides according to Reference Examples 1 to 31 and Design Examples 1 to 18), which is a base sequence having 90% to 100% sequence identity based on a base sequence complementary to at least one target region having the same base length as the single-stranded antisense oligonucleotide 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 have been deleted, substituted, inserted or added in the target region; or a base sequence which hybridizes under stringent conditions to an oligonucleotide having the target region.

4. The single-stranded antisense oligonucleotide according to any one of claims 1 to 3, or a pharma- ceutically acceptable salt thereof, wherein the base sequence of the single-stranded antisense oligonucleotide has a sequence identity of 95% or more and 100% or less based on a base sequence complementary to at least one target region having the same base length as the single-stranded antisense oligonucleotide in the base sequence set forth in SEQ ID NO:

1.

5. The single-stranded antisense oligonucleotide according to any one of claims 1 to 4, wherein 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, or a pharma- ceutical acceptable salt thereof.

6. The single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 5, wherein the gap region has a length of 5 to 20 nucleotides, the 3' wing region is a 3 to 5 nucleotide modified nucleic acid having a substituent at the 2' position, and the 5' wing region is a 3 to 5 nucleotide modified nucleic acid having a substituent at the 2' position.

7. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 6, wherein the 5'-CP nucleic acid is positioned at least at the second position counting from the 5' side of the gap region.

8. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 7, wherein two or more of the 5'-CP nucleic acids are arranged in the gap region.

9. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 8, wherein 2 to 5 of the 5'-CP nucleic acids are arranged in the gap region.

10. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 7, wherein the 5'-CP nucleic acid is positioned at least one-ninth of the gap region.

11. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 10, wherein the 5'-CP nucleic acid is positioned at least one-fifth of the gap region.

12. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 11, wherein the 5'-CP nucleic acid is arranged in a continuous 2-4 mer sequence in at least one position in the gap region.

13. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 12, wherein the 5'-CP nucleic acid is positioned on the 5'-terminal side of the gap region.

14. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 13, wherein the 5'-CP nucleic acid is positioned on the 5'-terminal and 3'-terminal sides of the gap region.

15. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 14, wherein the base length of the single-stranded antisense oligonucleotide is 15 to 20 mer.

16. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 15, wherein the base length of the single-stranded antisense oligonucleotide is 18 to 20 mer.

17. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 16, wherein 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, and 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.

18. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 17, wherein 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, and the modified nucleic acid having a substituent at the 2' position in the 5' wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

19. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 18, wherein 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, and the modified nucleic acid having a substituent at the 2' position in the 5' wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA.

20. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 19, wherein at least one internucleoside linkage of the single-stranded antisense oligonucleotide is a phosphorothioate linkage.

21. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 20, wherein at least one internucleoside bond of the single-stranded antisense oligonucleotide is a phosphodiester bond.

22. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 21, wherein the ratio of phosphorothioate bonds in the internucleoside linkages constituting the single-stranded antisense oligonucleotide is 50% to 80%.

23. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 22, wherein the ratio of phosphorothioate bonds in the internucleoside linkages constituting said single-stranded antisense oligonucleotide is 50% to 70%.

24. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 23, wherein in 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 a phosphorothioate bond (except when the 5'-CP nucleic acid is positioned at the 3' end of the gap region).

25. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 24, wherein in 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 a phosphorothioate bond (excluding the case where the 5'-CP nucleic acid is positioned at the 3' end of the gap region and the case where the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is a 5'-CP nucleic acid).

26. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 25, wherein in the gap region, 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.

27. The single-stranded antisense oligonucleotide according to any one of claims 1 to 26, or a pharma- ceutically acceptable salt thereof, wherein the base sequence of the single-stranded antisense oligonucleotide is a base sequence having 90% to 100% sequence identity based on a base sequence complementary to a target region consisting of a continuous 14-22 mer starting 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 have been deleted, substituted, inserted or added in the target region, or a base sequence which hybridizes under stringent conditions to an oligonucleotide having the target region.

28. The single-stranded antisense oligonucleotide according to any one of claims 1 to 27, or a pharma- ceutically acceptable salt thereof, wherein the base sequence of the single-stranded antisense oligonucleotide is: a base sequence having 90% to 100% sequence identity based on a base sequence complementary to a target region consisting of a continuous 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; a base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted or added in the target region; or a base sequence which hybridizes under stringent conditions to an oligonucleotide having the target region.

29. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 28, wherein the base sequence of the single-stranded antisense oligonucleotide is a base sequence having a sequence identity of 90% or more and 100% or less based on a base sequence complementary to a target region consisting of 18 to 22 mers consecutively consisting of 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 to 5 mer, and the 5' wing region is a 3 to 5 mer.

30. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 29, wherein the base sequence of the single-stranded antisense oligonucleotide is a base sequence having a sequence identity of 90% or more and 100% or less based on a base sequence complementary to a target region consisting of a consecutive 18-20 mer starting from the base located at position 450 to 451 or 453 counting from the 5' end in the base sequence set forth in SEQ ID NO:

1.

31. The single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 30, wherein the base sequence of the single-stranded antisense oligonucleotide is one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 5 to 47, and 49 to 56.

32. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 31, wherein the base sequence of the single-stranded antisense oligonucleotide is one 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.

33. The single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 32, wherein the base sequence of the single-stranded antisense oligonucleotide is one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 41, 47, 50, and 53 to 55.

34. An antisense oligonucleotide conjugate or a pharma- ceutically acceptable salt thereof, comprising: a single-stranded antisense oligonucleotide according to any one of claims 1 to 33, or a pharma-ceutically acceptable salt thereof; and an additional substance bound to the single-stranded antisense oligonucleotide, wherein the additional substance is selected from the group consisting of polyethylene glycol, a peptide, an alkyl chain, a nucleic acid, a ligand compound, an antibody, a protein, and a sugar chain.

35. A pharmaceutical comprising as an active ingredient the single-stranded antisense oligonucleotide according to any one of claims 1 to 33 or a pharma- ceutically acceptable salt thereof, or the antisense oligonucleotide complex according to claim 34 or a pharma- ceutically acceptable salt thereof.

36. The pharmaceutical product of claim 35, wherein the single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof, or the antisense oligonucleotide conjugate or a pharma- ceutically acceptable salt thereof, is administered so as to be exposed to the central nervous system.

37. The pharmaceutical product of claim 35 or claim 36, wherein the single-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof, or the antisense oligonucleotide conjugate or a pharma- ceutically acceptable salt thereof, is administered to a subject susceptible to delayed central nervous system toxicity.

38. An agent for regulating the expression and / or function of the RPS25 gene, comprising as an active ingredient the single-stranded antisense oligonucleotide according to any one of claims 1 to 33 or a pharma- ceutically acceptable salt thereof, or the antisense oligonucleotide complex according to claim 34 or a pharma- ceutically acceptable salt thereof.

39. An inhibitor of the production of dipeptide repeats by RNA translation, comprising as an active ingredient a single-stranded antisense oligonucleotide according to any one of claims 1 to 33 or a pharma- ceutically acceptable salt thereof, or an antisense oligonucleotide complex according to claim 34 or a pharma- ceutically acceptable salt thereof.

40. A therapeutic agent for repeat disease comprising as an active ingredient the single-stranded antisense oligonucleotide according to any one of claims 1 to 33 or a pharma- ceutically acceptable salt thereof, or the antisense oligonucleotide complex according to claim 34 or a pharma- ceutically acceptable salt thereof.

41. A preventive agent for repeat disease, comprising as an active ingredient the single-stranded antisense oligonucleotide according to any one of claims 1 to 33 or a pharma- ceutically acceptable salt thereof, or the antisense oligonucleotide complex according to claim 34 or a pharma- ceutically acceptable salt thereof.

42. The therapeutic agent for a repeat disease according to claim 40, or the preventive agent for a repeat disease according to claim 41, wherein 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, tremor ataxia syndrome associated with fragile X, and myotonic dystrophy.

43. A method for treating or preventing a repeat disease, comprising administering to an individual the single-stranded antisense oligonucleotide according to any one of claims 1 to 33 or a pharma- ceutically acceptable salt thereof, or the antisense oligonucleotide complex according to claim 34 or a pharma- ceutically acceptable salt thereof.

44. The method for treating or preventing a repeat disease according to claim 43, wherein 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.

45. A single-stranded antisense oligonucleotide according to any one of claims 1 to 33 or a pharma- ceutically acceptable salt thereof, or an antisense oligonucleotide conjugate according to claim 34 or a pharma- ceutically acceptable salt thereof, for use in the treatment or prevention of C9orf72 ALS.

46. ​​A single-stranded antisense oligonucleotide according to any one of claims 1 to 33 or a pharma- ceutically acceptable salt thereof, or an antisense oligonucleotide complex according to claim 34 or a pharma- ceutically acceptable salt thereof, for use in producing an agent for treating or preventing C9orf72 ALS.