RPS25 gene expression and / or functional modifier

Abstract

The present invention provides a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof for regulating the expression and / or function of the RPS25 gene. In the single-stranded antisense oligonucleotide, each nucleotide is bonded by a phosphate group and / or a modified phosphate group. The single-stranded antisense oligonucleotide includes a gap region, a 3' wing region bonded to the 3' terminal of the gap region, and a 5' wing region bonded to the 5' terminal of the gap region. The gap region is a deoxyribose-constituted nucleic acid in which a sugar-moiety-modified nucleic acid may be included. The 3' wing region and the 5' wing region are modified nucleotides. The single-stranded antisense oligonucleotide has a base length of 12-30 mer. The base sequence of the antisense oligonucleotide is: a base sequence having a sequence identity of 90 to 100% inclusive with a base sequence that is complementary to at least one target region constituted at the same base length as the antisense oligonucleotide in the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2; a base sequence that is complementary to a base sequence obtained by deleting, substituting, inserting, or adding one or more bases in the target region; or a base sequence that hybridizes at a stringent condition with respect to an oligonucleotide having the above target region.

Description

[Technical Field]

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

[0002] The Ribosomal Protein S25 (RPS25) gene encodes one of the constituent proteins of the ribosome 40S subunit. The three-dimensional structure of the constituent protein encoded by the RPS25 gene (RPS25 protein) within the ribosome 40S subunit has also been elucidated. (Non-Patent Literature 1)

[0003] The RPS25 protein, as part of the protein synthesis process, binds to an RNA element that enables cap-independent translation initiation, thereby regulating translation. The RNA element to which the RPS25 protein binds is called the Internal Ribosome Entry Site (IRES). IRESs are one of the cap-independent translation mechanisms frequently observed, particularly in viruses. (Non-Patent Literature 2)

[0004] Repeat-associated non-ATG translation (RAN translation) was first identified in 2011 in patients with spinocerebellar ataxia type 8 (Non-Patent Literature 3). RAN translation refers to a mechanism in which the repetition of specific sequences is translated into proteins (such as dipeptide repeats (DPRs)) 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), including amyotrophic lateral sclerosis (ALS) with mutations in the C9orf72 gene (hereinafter sometimes referred to as "C9orf72 ALS"), Huntington's disease, and myotonic dystrophy. Research has progressed on the relationship between DPRs produced by RAN translation and disease states, and it has been reported that the removal of DPRs is effective in improving disease states (Non-Patent Literature 4, Non-Patent Literature 5).

[0005] In 2019, the RPS25 protein was reported as a molecule that contributes significantly to the production of dipeptide repeats via RAN translation. Knockdown of the RPS25 gene suppressed the production of DPR derived from GGGGCC repeat sequences or CAG repeat sequences, which are RAN translation-dependent. The GGGGCC repeat sequence is known as an abnormal elongation mutation in the C9orf72 gene, one of the familial mutations in amyotrophic lateral sclerosis (ALS). The CAG repeat sequence is known as an abnormal elongation mutation in the huntingtin and ATXN2 genes. In motor neurons derived from induced purulipotent stem cells (iPSCs) established from patients with the C9orf72 gene mutation, knockdown of the RPS25 gene suppressed the production of DPR derived from GGGGCC repeat sequences and also suppressed motor neuron cell death. (Non-patent Literature 6)

[0006] The antisense oligonucleotide for the RPS25 gene disclosed in Non-Patent Literature 6 is a gapmer in which the wing portion is modified with 2'-O-methylated RNA (2'-OMe nucleic acid) and the internucleosides are phosphorothioate-modified. The base sequence of this antisense oligonucleotide contains a portion of the base sequence of the sense strand of the RPS25 gene. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2019 / 084068 [Patent Document 2] International Publication No. 2011 / 052436 [Patent Document 3] International Publication No. 2014 / 046212 [Patent Document 4] International Publication No. 2015 / 125783 [Patent Document 5] International Publication No. 2020 / 158910 [Patent Document 6] International Publication No. 99 / 14226 [Non-patent literature]

[0008] [Non-Patent Document 1] Science(2011)331(6018):730-736. [Non-Patent Document 2] Genes Dev.(2009)23(23):2753-2764. [Non-Patent Document 3] Proc. Natl. Acad. Sci. USA(2011)108(1):260-265 [Non-Patent Document 4] Neuron (2015)88:667-677 [Non-Patent Document 5] Neuron (2020)105:645-662 [Non-Patent Document 6] Nat. Neurosci.(2019)22(9):1383-1388

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Summary of the Invention

Problems to be Solved by the Invention

[0009] Although Patent Document 1 reports that the production of DPR is suppressed by inhibiting the RPS25 gene, it is unclear whether a similar effect can be observed by an approach using nucleic acids such as antisense. In addition, the antisense oligonucleotide described in Non-Patent Document 5 suppresses the expression of the RPS25 gene, but the inhibitory effect is partial, and there is a need to create a more effective antisense oligonucleotide against the RPS25 gene for use as a pharmaceutical.

[0010] The present invention has been made in view of the above circumstances, and the problem to be solved by the present invention is to provide a single-stranded antisense oligonucleotide that regulates the expression and / or function of the RPS25 gene, and an agent for regulating the expression and / or function of the RPS25 gene containing the same.

Means for Solving the Problems

[0011] The present inventors, through diligent research to solve the above problems, have discovered 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 the expression of the RPS25 gene, and have completed the present invention. That is, the present invention is as follows.

[0012] [1] The antisense oligonucleotides of the present invention are single-stranded antisense oligonucleotides or pharmaceutically acceptable salts thereof that modulate the expression and / or function of the RPS25 gene, The above single-stranded antisense oligonucleotide has each nucleotide linked by a phosphate group and / or a modified phosphate group. The above single-stranded antisense oligonucleotide comprises 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. The above gap region is a nucleic acid composed of deoxyribose, which may contain nucleic acids with modified sugar moieties. The 3' wing region and 5' wing region described above are modified nucleic acids. The base lengths of the above single-stranded antisense oligonucleotides are 12-30 mers. The base sequence of the above single-stranded antisense oligonucleotide is: A nucleotide sequence having 90% to 100% sequence identity with respect to a nucleotide sequence complementary to at least one target region consisting of the same nucleotide length as the single-stranded antisense oligonucleotide in the nucleotide sequence described in Sequence ID No. 1 or Sequence ID No. 2, For a nucleotide sequence in which one or several bases are deleted, substituted, inserted, or added in the above target region, a complementary nucleotide sequence, or A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, which is a base sequence that hybridizes under stringent conditions to an oligonucleotide having the above-mentioned target region.

[0013] The antisense oligonucleotide of the present invention has modified nucleic acids arranged in the gap region, the 5' wing region, and the 3' wing region. When a deoxyribose containing a nucleic acid modified with a sugar moiety is arranged in the gap region, it is preferable that the modified nucleic acid is a 5'-CP nucleic acid (5'-cyclopropyl Nucleic Acid). The modified nucleic acids located in the 5' wing region and the 3' wing region are, as 2'-position 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 cross-linked modified nucleic acids, 2',4'-BNA (Locked Nucleic Acid, sometimes referred to as "LNA"), AmNA (Amido-bridged nucleic acid), GuNA (Guanidino-bridged nucleic acid), and / or scpBNA (2'-O,4'-C-Spirocyclopropylene bridged nucleic acid), and it is preferable that at least one of these modified nucleic acids is included. With this configuration, the antisense oligonucleotide of the present invention can be expected to have a high binding affinity to RPS25 mRNA or mRNA precursor. Furthermore, since the antisense oligonucleotide of the present invention is a so-called gapmer-type single-stranded antisense oligonucleotide, it functions as a catalyst in the degradation reaction of the RPS25 gene by RNA-degrading enzymes described later. Therefore, it is believed that the predetermined effect can be sustained even with a small dose.

[0014] [2] The base sequence of the above single-stranded antisense oligonucleotide is: It is preferable that the base sequence 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 described in Sequence ID No. 1 or Sequence ID No. 2, which consists of the same base length as the single-stranded antisense oligonucleotide.

[0015] [3] The base sequence of the above single-stranded antisense oligonucleotide is: It is preferable that the base sequence is complementary to at least one target region in the base sequence described in Sequence ID No. 1 or Sequence ID No. 2, which has the same base length as the single-stranded antisense oligonucleotide.

[0016] [4] The number of bases in the above gap region is 5 to 20 mers. The 3' wing region described above is a modified nucleic acid of 1-5 mers. The 5' wing region described above is preferably a modified nucleic acid of 1 to 5 mers.

[0017] [5] The base length of the single-stranded antisense oligonucleotide is preferably 14 to 22 mers.

[0018] [6] The modified nucleic acid in the 3' wing region includes at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA. The modified nucleic acid in the 5' wing region preferably includes at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

[0019] [7] Preferably, at least one internucleotide bond of the single-stranded antisense oligonucleotide is a phosphorothioate bond.

[0020] [8] Preferably, at least one internucleotide bond of the single-stranded antisense oligonucleotide is a phosphodiester bond.

[0021] [9] The base sequence of the above single-stranded antisense oligonucleotide is: A nucleotide sequence having 90% to 100% sequence identity with respect to a nucleotide sequence complementary to a target region consisting of 14 to 22 mers starting from nucleotides located at positions 8-10, 27-29, 34-40, 79, 98, 101-106, 123-129, 140, 160-161, 180-191, 208-221, 242-243, 255-268, 285-286, 292-304, 321-328, 340-344, 365, or 429-454 in the nucleotide sequence described in Sequence ID No. 1, counting from the 5' end. In the target region described above, a nucleotide sequence complementary to a nucleotide sequence in which one or several nucleotides are deleted, substituted, inserted, or added, or It is preferable that the base sequence hybridizes to the oligonucleotide having the above-mentioned target region under stringent conditions.

[0022]

[10] The nucleotide sequence of the single-stranded antisense oligonucleotide described above is a nucleotide sequence having 90% to 100% sequence identity with respect to a nucleotide sequence complementary to a target region consisting of 14 to 22 mers consecutively from the nucleotide position 8, 10, 28 to 29, 35 to 37, 101 to 104, 123 to 126, 129, 160, 180 to 187, 209 to 220, 258 to 267, 285, 295 to 297, 300 to 304, 321 to 327, 341, 344, 365, or 429 to 454 in the nucleotide sequence described in Sequence ID No. 1, counting from the 5' end. The above 3' wing region is 2-5mer. The above 5' wing region is preferably 2 to 5mer.

[0023]

[11] The nucleotide sequence of the single-stranded antisense oligonucleotide described above is preferably a nucleotide sequence complementary to a target region consisting of 14 to 22 mers consecutively from the nucleotide position 36, 102 to 103, 123 to 126, 185 to 187, 213 to 214, 220, 259 to 260, 263 to 265, 295 to 296, 300, 302 to 303, 322 to 327, 429 to 431, 435, or 438 to 454, counting from the 5' end in the nucleotide sequence described in Sequence ID No. 1.

[0024]

[12] The base sequences of the above single-stranded antisense oligonucleotides are: SEQ ID NOs: 18, 24-25, 28-29, 38, 48-49, 53, 58-59, 63-64, 66-68, 79-80, 84, 86-91, 93-95, 97, 99-105, 113-119, 121-123, 125, 127-130, 140, 162, 169, 171-173, 183, 188, 190, 304-306, 309, 310, 312, 31 It is preferable that the base sequence is one selected from the group consisting of the base sequences 3, 317, 321-323, 326-327, 331-332, 334, 337, 340-344, 346, 348, 349, 351, 353, 355-364, 366-367, 371-382, 385, 386, 388, 389, 391, 394, 396, 397, 407, 408, 410, 418-424, 426-427, and 431-432.

[0025]

[13] The nucleotide sequences of the above single-stranded antisense oligonucleotides are as follows, counting from the 5' end: 1, 75, 233, 261, 278-280, 390-392, 417-423, 445-447, 460-461, 510, 561-562, 589, 605, 626-628, 632-634, 696-697, 1034-1035, 1103-1107, 1128 A nucleotide sequence having 90% to 100% sequence identity with respect to a nucleotide sequence complementary to a target region consisting of 14 to 22 mers consecutively from a nucleotide located at nucleotides 1-1129, 1196-1197, 1398, 1408-1412, 1478-1480, 1715, 1749-1751, 2047-2049, 2121-2123, 2260-2268, 2342, 2406, or 2585-2587. In the target region described above, a nucleotide sequence complementary to a nucleotide sequence in which one or several nucleotides are deleted, substituted, inserted, or added, or It is preferable that the base sequence hybridizes to the oligonucleotide having the above-mentioned target region under stringent conditions.

[0026]

[14] The nucleotide sequence of the single-stranded antisense oligonucleotide described above is a nucleotide sequence having 90% to 100% sequence identity with respect to a nucleotide sequence complementary to a target region consisting of 14 to 22 mers consecutively from the nucleotide position 1, 278 to 279, 417 to 420, 561, 605, 627, 632 to 634, 697, 1035, 1128, 1196 to 1197, 1409 to 1410, 1478, 1715, 1750, 2047 to 2049, 2342, 2406, or 2585 to 2587 in the nucleotide sequence described in Sequence ID No. 2, counting from the 5' end. The above 3' wing region is 2-5mer. The above 5' wing region is preferably 2 to 5mer.

[0027]

[15] The double-stranded antisense oligonucleotide according to the present invention is a single-stranded antisense oligonucleotide, A double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, comprising a second-stranded oligonucleotide hybridized to the above single-stranded antisense oligonucleotide, The base sequence of the above-mentioned second-chain oligonucleotide is a base sequence that has a sequence identity of 90% to 100% relative to the base sequence of the above-mentioned single-chain antisense oligonucleotide, based on a base sequence complementary to it.

[0028]

[16] The antisense oligonucleotide complex according to the present invention comprises the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, An antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof, comprising the above-mentioned single-stranded antisense oligonucleotide or an adduct bound to the above-mentioned second-stranded oligonucleotide, The above-mentioned adducts are selected from the group consisting of polyethylene glycol, peptides, alkyl chains, ligand compounds, antibodies, proteins, and sugar chains.

[0029]

[17] The pharmaceutical product according to the present invention contains as an active ingredient a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, a double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or an antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof.

[0030]

[18] The expression and / or function modifier of the RPS25 gene according to the present invention comprises the above-mentioned single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the above-mentioned double-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.

[0031]

[19] The RAN translation-mediated dipeptide repeat production inhibitor according to the present invention comprises the above-mentioned single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the above-mentioned double-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.

[0032]

[20] The therapeutic agent for repeat disease according to the present invention comprises the above-mentioned single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the above-mentioned double-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.

[0033]

[21] The preventive agent for repeat disease according to the present invention comprises the above-mentioned single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the above-mentioned double-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.

[0034]

[22] In the therapeutic agent and the prophylactic agent described above, the repeat disease is preferably at least one selected from the group consisting of C9orf72 ALS, frontotemporal lobar degeneration (FTLD) with a mutation in the C9orf72 gene (hereinafter sometimes referred to as "C9orf72 FTLD"), Huntington's disease, spinocerebellar ataxia, dentatorubral-pallidoluysian atrophy, spinal-bulbar muscular atrophy, Friedreich ataxia, fragile X-associated ataxia tremor syndrome, and myotonic dystrophy.

[0035]

[23] A method for regulating the expression of the RPS25 gene according to the present invention comprises the step of administering the above-mentioned single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the above-mentioned double-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 to cells, tissues or individuals expressing the RPS25 gene.

[0036]

[24] The present invention provides a method for treating or preventing a repeat disease, comprising the step of administering to an individual suffering from the repeat disease the above-mentioned single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the above-mentioned double-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. 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, bulbar spinal muscular atrophy, Friedreich ataxia, fragile X-associated ataxia tremor syndrome, and myotonic dystrophy.

[0037]

[25] The present invention provides a method for treating or preventing C9orf72 ALS, comprising administering to an individual 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 complex or a pharmaceutically acceptable salt thereof.

[0038]

[26] The present invention provides single-stranded antisense oligonucleotides or pharmaceutically acceptable salts thereof, double-stranded antisense oligonucleotides or pharmaceutically acceptable salts thereof, or antisense oligonucleotide complexes or pharmaceutically acceptable salts thereof for use in the treatment or prevention of C9orf72 ALS.

[0039]

[27] The present invention provides single-stranded antisense oligonucleotides or pharmaceutically acceptable salts thereof, double-stranded antisense oligonucleotides or pharmaceutically acceptable salts thereof, or antisense oligonucleotide complexes or pharmaceutically acceptable salts thereof, for use in producing therapeutic or prophylactic agents for C9orf72 ALS. [Effects of the Invention]

[0040] According to the present invention, it is possible to provide single-stranded antisense oligonucleotides that regulate the expression and / or function of the RPS25 gene, and RPS25 gene expression and / or function modifiers containing the same. [Brief explanation of the drawing]

[0041] [Figure 1] Figure 1 is a schematic diagram showing an example of the structure of a single-stranded antisense oligonucleotide according to this embodiment. [Figure 2] Figure 2 is a schematic diagram illustrating the mechanism by which the expression of the RPS25 gene is suppressed when using a single-stranded antisense oligonucleotide according to this embodiment. [Figure 3] Figure 3 is a schematic diagram showing the chemical structure of the single-stranded antisense oligonucleotide used in Example 412. [Figure 4] Figure 4 is a schematic diagram showing the chemical structure of the single-stranded antisense oligonucleotide used in Example 413. [Figure 5] Figure 5 is a schematic diagram showing the chemical structure of the single-stranded antisense oligonucleotide used in Example 414. [Figure 6] Figure 6 is a schematic diagram showing the chemical structure of the single-stranded antisense oligonucleotide used in Example 415. [Figure 7] Figure 7 is a schematic diagram showing the chemical structure of the single-stranded antisense oligonucleotide used in Example 416. [Modes for carrying out the invention]

[0042] The following describes one embodiment of the present invention (which may be referred to as "this embodiment"). However, this embodiment is not limited thereto. In this specification, the notation "I~J" means the upper and lower limits of a range (i.e., I or more and J or less). If no unit is specified for I, but a unit is specified only for J, the unit for I and the unit for J are the same.

[0043] Single-stranded antisense oligonucleotides that regulate the expression and / or function of the RPS25 gene. The single-stranded antisense oligonucleotide of this embodiment is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that modulates the expression and / or function of the RPS25 gene. The above single-stranded antisense oligonucleotide has each nucleotide linked by a phosphate group and / or a modified phosphate group. The above single-stranded antisense oligonucleotide comprises 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. The above gap region is a nucleic acid composed of deoxyribose, which may contain nucleic acids with modified sugar moieties. The 3' wing region and 5' wing region described above are modified nucleic acids. The base lengths of the above antisense oligonucleotides are 12-30 mer. The base sequence of the above antisense oligonucleotide is: A nucleotide sequence having 90% to 100% sequence identity with respect to a nucleotide sequence complementary to at least one target region in the nucleotide sequence described in Sequence ID No. 1 or Sequence ID No. 2, which has the same nucleotide length as the antisense oligonucleotide described above. For a nucleotide sequence in which one or several bases are deleted, substituted, inserted, or added in the above target region, a complementary nucleotide sequence, or A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, which is a base sequence that hybridizes under stringent conditions to an oligonucleotide having the above-mentioned target region. Further details are provided below.

[0044] <Definitions of Terms, etc.> First, the definitions of terms used in this specification are explained below.

[0045] (RPS25 gene) In this embodiment, the "RPS25 gene" can be defined by Mol. Gen. Genet. (1979) 169:1-6 (Non-Patent Literature 7) and Curr. Opin. Struct. Biol. (2014) 24:165-169 (Non-Patent Literature 8). Synonyms for "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.

[0046] (Single-stranded antisense oligonucleotide) In this embodiment, "single-stranded antisense oligonucleotide" or "antisense oligonucleotide" (hereinafter sometimes referred to as "ASO") means an oligonucleotide complementary to the mRNA, mRNA precursor, or ncRNA (non-coding RNA) of a target gene (hereinafter these three may be collectively referred to as "target RNA"), or a pharmacologically acceptable salt thereof. Antisense oligonucleotides are composed of DNA, RNA, and / or analogs thereof. Antisense oligonucleotides inhibit the function of the target mRNA, mRNA precursor, or ncRNA by forming a double helix with the target mRNA, mRNA precursor, or ncRNA. Antisense oligonucleotides include those having a base sequence that is completely complementary to the base sequence of the target mRNA, mRNA precursor, or ncRNA, those having a base sequence in which one or more bases are deleted, substituted, inserted, or added in the complementary base sequence, and those containing bases that form fluctuating 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 portion is a modified sugar" (sugar-modified modified nucleotides) as described later. Examples of modified nucleotides known in the art include, in addition to sugar-modified modified nucleotides, phosphate-modified modified nucleotides and nucleic acid base-modified modified nucleotides, as described later. In this embodiment, the structure of both ends of the antisense oligonucleotide is not particularly limited; for example, it may be -OH or -OR (where R represents an alkyl chain, a phosphate ester, or an adduct described later). Furthermore, the single-stranded antisense oligonucleotide in this embodiment may be in a single-stranded form, or it may hybridize with a second-stranded oligonucleotide, as described later, to take a double-stranded form. A double-stranded oligonucleotide consisting of the above-mentioned single-stranded antisense oligonucleotide and a second-stranded oligonucleotide hybridized to the above-mentioned single-stranded antisense oligonucleotide may be referred to as a "double-stranded antisense oligonucleotide."

[0047] (oligonucleotide) In this embodiment, "oligonucleotide" means a polymer of nucleotides in which 2 to 30 identical or different nucleotides are linked together by phosphate diester bonds or other bonds. The above oligonucleotide can also be understood as being composed of a nucleic acid base portion, a phosphate portion, and a sugar portion, as shown in the following structural formula.

[0048] [ka]

[0049] The above oligonucleotides are broadly classified into natural oligonucleotides and unnatural oligonucleotides. "Natural oligonucleotides" refer to oligonucleotides consisting of naturally occurring nucleotides. "Unnatural oligonucleotides" refer to oligonucleotides containing at least one modified nucleotide as a constituent unit, as described below. Examples of "unnatural oligonucleotides" include: modified sugar derivatives in which the sugar portion is modified; phosphorothioate derivatives in which one non-crosslinked oxygen atom of the phosphate diester bond is replaced with a sulfur atom; phosphorodithioate derivatives in which two non-crosslinked oxygen atoms of the phosphate diester bond are replaced with sulfur atoms; ester derivatives in which the phosphate diester bond is triesterinated; phosphoamide derivatives in which the phosphate diester bond is amidated; boranophosphate derivatives in which the phosphate diester bond is boronic acid esterified; alkylphosphonate (e.g., methylphosphonate, methoxypropylphosphonate, etc.) derivatives in which the non-crosslinked oxygen atom of the phosphate diester bond is replaced with an alkyl group; amide derivatives in which the phosphate diester bond is replaced with an amide bond; and modified base derivatives in which the nucleic acid base is modified. More preferably, the non-natural oligonucleotides include: cross-linked modified sugar derivatives in which the sugar moiety is modified; phosphorothioate derivatives in which one non-crosslinked oxygen atom of the phosphate diester bond is replaced with a sulfur atom; ester derivatives in which the phosphate diester bond is esterified; and alkylphosphonate derivatives in which the sugar moiety is modified with a modified sugar (e.g., a cross-linked sugar) described later, and one non-crosslinked oxygen atom of the phosphate diester bond is replaced with a sulfur atom, or the non-crosslinked oxygen atom of the phosphate diester bond is replaced with an alkyl group.

[0050] (Nucleoside) In this embodiment, "nucleoside" refers to a compound in which a purine base or pyrimidine base is bonded to a sugar. Nucleosides that exist naturally are sometimes called "natural nucleosides." Modified nucleosides that do not exist naturally are sometimes called "modified nucleosides." Modified nucleosides in which the sugar portion is modified are sometimes called "modified sugar nucleosides."

[0051] (nucleotide) In this embodiment, "nucleotide" refers to a compound in which a phosphate group is bonded to the sugar portion of the above-mentioned nucleoside. Naturally occurring nucleotides are sometimes called "natural nucleotides." Modified nucleotides that do not exist naturally are sometimes called "modified nucleotides" or "modified nucleic acids." Examples of "modified nucleotides" or "modified nucleic acids" include compounds in which a phosphate group is bonded to the sugar portion of the above-mentioned modified nucleoside, compounds in which a modified phosphate group described later is bonded to the sugar portion of the above-mentioned modified nucleoside, and compounds in which a modified phosphate group described later is bonded to the sugar portion of a natural nucleoside.

[0052] (sugar modification, modified sugar) In this embodiment, "sugar modification" means that the sugar portion of the nucleotide is modified. The modified sugar portion is sometimes specifically referred to as "modified sugar." Modified nucleotides that have undergone sugar modification can be used as modified nucleic acids, and examples include AmNA, GuNA, scpBNA, 2'-O-alkyl (e.g., 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, etc.), 2'-F, 5'-methyl-DNA, LNA, 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 LNA include structures represented by the symbols "A(L)", "5(L)", "G(L)", and "T(L)" described later. Examples of AmNA include structures represented by the symbols "A(Y)", "5(Y)", "G(Y)", and "T(Y)" described later. Examples of GuNA include structures represented by the symbols "A(Gx)", "5(Gx)", "G(Gx)", and "T(Gx)" described later. Examples of scpBNA include structures represented by the symbols "A(S)", "5(S)", "G(S)", and "T(S)" described later. Examples of 2'-MOE nucleic acids include structures represented by the symbols "A(m)", "5(m)", "G(m)", and "T(m)" described later. Examples of 5'-CP nucleic acids include structures represented by the symbols "A(5'-CP)", "5(5'-CP)", "G(5'-CP)", and "T(5'-CP)" described later. Examples of 2'-OMe nucleic acids include those containing structures represented by the symbols "A(M)", "C(M)", "G(M)", and "U(M)" as described later. Examples of MCE nucleic acids include those containing structures represented by the symbols "A(Mx)", "C(Mx)", "G(Mx)", and "U(Mx)" as described later.

[0053] (Modifications of nucleotides known in this field other than sugar modification) Nucleotide modifications known in the art other than the sugar modifications described above can be used as modified nucleic acids for producing single-stranded antisense oligonucleotides of the present invention. Known nucleotide modifications include phosphate group modifications and nucleic acid base modifications, which will be described later. Examples of such nucleotide modifications include those described in W. Brad Wan et. Al. J. Med. Chem. (2016) 59:9645-9667. (Non-Patent Literature 9), etc. These nucleotide modifications can be carried out based on methods known in the art as described in the literature cited in the above-mentioned document.

[0054] (Phosphate group) In this embodiment, "phosphate group" means that the phosphate portion of the nucleotide is bonded in a naturally occurring manner, such as a phosphodiester bond (a bond indicated by the symbol "-" later described below).

[0055] (Phosphate group modification, modified phosphate group) In this embodiment, "phosphate group modification" means that the phosphate portion of the nucleotide is modified. The modified phosphate portion is sometimes specifically referred to as a "modified phosphate group." Examples of bonding modes including the modified phosphate group include phosphorothioate bonds (bonds indicated by the symbol "∧" later described), phosphorodithioate bonds, phosphoamidate bonds (bonds indicated by the symbol "=" later described), or boranophosphate bonds (bonds indicated by the symbol "×" later described), alkylphosphonates, and the like.

[0056] (Nucleic acid base modification, modified nucleic acid bases) In this embodiment, "nucleic acid base modification" means that the nucleic acid base portion of the nucleotide is modified. The modified nucleic acid base portion is sometimes specifically referred to as a "modified nucleic acid base." Examples of modified nucleic acid bases include 5-methylcytosine, 5-hydroxymethylcytosine, and 5-propynylcytosine.

[0057] (An analogue of DNA or RNA) The above-mentioned DNA or RNA analogs refer to molecules that have a structure similar to DNA or RNA. Examples include peptide nucleic acids (pNA) and morpholino nucleic acids.

[0058] (ncRNA) In this embodiment, "ncRNA" refers to a general term for RNA that is not involved in protein translation. Examples of ncRNA include ribosomal RNA, transfer RNA, miRNA, and natural antisense transcript (NAT).

[0059] (The nucleic acid base portion of an oligonucleotide) Examples of nucleic acid bases in the above oligonucleotides include thyminyl group, cytosinyl group, adeninyl group, guanyl group, 5-methylcytosinyl group, urasilyl group, 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidine-1-yl group, 2-oxo-4-amino-1,2-dihydropyrimidine-1-yl group, 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidine-1-yl group, and 2-oxo-4-hydroxy-1,2-dihydropyrimidine-1-yl group. Preferably, examples of nucleic acid bases include thyminyl group, cytosinyl group, adeninyl group, guanyl group, 5-methylcytosinyl group, and urasilyl group. Among these nucleic acid bases, uracil (U) and thymine (T) are interchangeable. Both uracil (U) and thymine (T) can form base pairs with adenine (A) in the complementary strand. The same is true for the nucleic acid base portion of antisense oligonucleotides.

[0060] (Target RNA) In this embodiment, "target RNA" means RNA whose function is suppressed by the binding of the single-stranded antisense oligonucleotide described above. In other words, in this embodiment, target RNA means RPS25 mRNA and mRNA precursor. Examples of the target RNA include human RPS25 mRNA having the nucleotide sequence described in SEQ ID NO: 1 (hereinafter sometimes referred to as "hRPS25"), human RPS25 mRNA precursor having the nucleotide sequence described in SEQ ID NO: 2 (hereinafter sometimes referred to as "hpRPS25"), monkey RPS25 mRNA having the nucleotide sequence described in SEQ ID NO: 3 (hereinafter sometimes referred to as "cRPS25"), monkey RPS25 mRNA precursor having the nucleotide sequence described in SEQ ID NO: 4, mouse RPS25 mRNA having the nucleotide sequence described in SEQ ID NO: 5 (hereinafter sometimes referred to as "mRPS25"), and mouse RPS25 mRNA precursor having the nucleotide sequence described in SEQ ID NO: 6.

[0061] (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 through complementarity with the target RNA. The double-stranded nucleic acid only needs to be formed in at least a portion of the target RNA. The strength of the binding to the target RNA can be measured, for example, by an indicator of thermal stability. An example of such an indicator of thermal stability is the melting temperature (Tm value) of the double-stranded nucleic acid. The Tm value is preferably 40 to 90°C, and more preferably 50 to 70°C.

[0062] (target area) The above-mentioned target region refers to the region in the RPS25 mRNA and mRNA precursor that binds to the single-stranded antisense oligonucleotide. The above-mentioned target region includes the target region consisting of the indicated nucleotide sequence and the region on the RPS25 mRNA precursor.

[0063] (mRNA precursor) The above mRNA precursor refers to the primary transcript of RNA transcribed from DNA. That is, the mRNA precursor is RNA containing exon regions, intron regions, and untranslated regions (UTRs). The mRNA precursor can also be understood as RNA before post-transcriptional splicing occurs. When the mRNA precursor is spliced, it becomes mRNA.

[0064] (Binding to the target region) The binding to the target region described above means that the single-stranded antisense oligonucleotide of the present invention forms a double helix with the target region. However, the single-stranded antisense oligonucleotide of the present invention does not necessarily need to form a double helix with the entire target region; it is sufficient if it forms a double helix with a portion of the target region. In other words, it is preferable that the single-stranded antisense oligonucleotide of the present invention has complete complementarity with the target region, but it is sufficient if it is complementary with at least a portion of the target region insofar as it binds to the target RNA of RPS25.

[0065] (Part of the target region) The above-mentioned portion of the target region refers to the region within the target region that is 10 to 15 nucleotides long.

[0066] (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. This also includes being complementary to the bases of the region on the mRNA or mRNA precursor corresponding to at least a portion of the target region.

[0067] <Base sequence of single-stranded antisense oligonucleotide> The base sequence of the single-stranded antisense oligonucleotide according to this embodiment is: (A) A nucleotide sequence having 90% to 100% sequence identity with respect to a nucleotide sequence complementary to a target region consisting of 12 to 30 mers (preferably 14 to 22 mers) consecutively from nucleotides located at positions 8 to 10, 27 to 29, 34 to 40, 79, 98, 101 to 106, 123 to 129, 140, 160 to 161, 180 to 191, 208 to 221, 242 to 243, 255 to 268, 285 to 286, 292 to 304, 321 to 328, 340 to 344, 365, or 429 to 454 in the nucleotide sequence described in Sequence ID No. 1, counting from the 5' end, or In the base sequence described in Sequence ID No. 2, counting from the 5' end, the nucleotides are 1, 75, 233, 261, 278-280, 390-392, 417-423, 445-447, 460-461, 510, 561-562, 589, 605, 626-628, 632-634, 696-697, 1034-1035, 1103-1107, 1128-1129, 1196-1197, and 139 A base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to a target region consisting of 12 to 30 mers (preferably 14 to 22 mers) consecutively from a base located at position 8, 1408 to 1412, 1478 to 1480, 1715, 1749 to 1751, 2047 to 2049, 2121 to 2123, 2260 to 2268, 2342, 2406, or 2585 to 2587, (B) For a nucleotide sequence in which one or more nucleotides are deleted, substituted, inserted, or added in the target region described above, a complementary nucleotide sequence, or (C) A base sequence that hybridizes under stringent conditions to an oligonucleotide having the above target region. Furthermore, in this embodiment, each base sequence shown in the sequence listing is used only to indicate the sequence information of the nucleic acid base portion. Structural information of oligonucleotides, including the sugar portion and phosphate portion in addition to the nucleic acid base portion, is shown in the format shown in Tables 3-1 to 3-17 and Tables 4-1 to 4-5 described later.

[0068] In this embodiment, "sequence identity" refers to the percentage (%) of identical bases in the total overlapping base sequences when two base sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm may consider introducing gaps into one or both sequences for optimal alignment). The "sequence identity" of base sequences can be easily confirmed by those skilled in the art. For example, NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) can be used.

[0069] The base sequence of the single-stranded antisense oligonucleotide according to this embodiment preferably has 95% to 100% sequence identity with a base sequence complementary to the predetermined target region in the base sequence described in Sequence ID No. 1 or Sequence ID No. 2, more preferably 98% to 100%, and even more preferably 100% sequence identity.

[0070] In this embodiment, "a nucleotide sequence in which one or more bases are deleted, substituted, inserted, or added" can refer to, for example, nucleotide sequences that, as a result of the deletion, substitution, insertion, or addition, have 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity with respect to the nucleotide sequence before the deletion, substitution, insertion, or addition. The specific number of "one or more bases" may be such that the above-mentioned deletions, substitutions, insertions, or additions occur independently in one, two, three, four, or five locations, or they may occur in combination.

[0071] In this embodiment, "stringent conditions" refers to conditions in which the sample is incubated at room temperature for 12 hours in a solution containing 6×SSC (composition of 1×SSC: 0.15M NaCl, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5×Denhardt, 100 μg / mL denatured salmon sperm DNA, and 50% (v / v) formamide, and then washed with 0.5×SSC at a temperature of 50°C or higher. Furthermore, even more stringent conditions are included, such as incubation at 45°C or 60°C for 12 hours, washing with 0.2×SSC or 0.1×SSC, and washing at a temperature of 60°C or 65°C or higher.

[0072] In one aspect of this embodiment, the target region is preferably a nucleotide sequence consisting of 12-30 mers or 14-22 mers consecutively, starting from the 8th, 10th, 28th-29th, 35th-37th, 101st-104th, 123rd-126th, 129th, 160th, 180th-187th, 209th-220th, 258th-267th, 285th, 295th-297th, 300th-304th, 321st-327th, 341st, 344th, 365th, or 429th-454th nucleotides in the nucleotide sequence described in Sequence ID No. 1, counting from the 5' end. In one aspect of this embodiment, the target region is a nucleotide sequence consisting of 12 to 30 mers or 14 to 22 mers consecutively, starting from the nucleotides located at positions 1, 278 to 279, 417 to 420, 561, 605, 627, 632 to 634, 697, 1035, 1128, 1196 to 1197, 1409 to 1410, 1478, 1715, 1750, 2047 to 2049, 2342, 2406, or 2585 to 2587 in the nucleotide sequence described in Sequence ID No. 2, counting from the 5' end, and preferably the 3' wing region is 2 to 5 mers and the 5' wing region is 2 to 5 mers.

[0073] In another aspect of this embodiment, the target region is preferably a nucleotide sequence consisting of 12-30mer, 14-22mer, or 14-20mer sequences starting from the nucleotide positions 36, 102-103, 123-126, 185-187, 213-214, 220, 259-260, 263-265, 295-296, 300, 302-303, 322-327, 429-431, 435, or 438-454 in the nucleotide sequence described in Sequence ID No. 1, counting from the 5' end.

[0074] The single-stranded antisense oligonucleotide described above can bind to the target region of RPS25. In this specification, "binding to the target region of RPS25" of the single-stranded antisense oligonucleotide of the present invention includes direct binding of the single-stranded antisense oligonucleotide of the present invention to the mRNA of RPS25 and direct binding of RPS25 to the mRNA precursor.

[0075] One embodiment of the single-stranded antisense oligonucleotide of the present invention is a single-stranded oligonucleotide that regulates the expression of the RPS25 gene, having one of the nucleotide sequences listed in Tables 1-1 to 1-9, and said single-stranded oligonucleotide is complementary to the target region in human RPS25 mRNA listed in Tables 1-1 to 1-9. The single-stranded oligonucleotide may extend by 1 to 5 nucleotides each to the 3' and / or 5' sides, provided it contains the nucleotide sequences listed in Tables 1-1 to 1-9. The target region is, in particular, a region in human RPS25 mRNA that is related to the regulation of human RPS25 gene expression (for example, a region of mRNA secondary structure to which antisense nucleotides readily bind). For example, if the 5' end position is "8" and the 3' end position is "22" in Table 1-1, then the nucleotide sequence from the 8th to the 22nd nucleotide from the 5' end in the nucleotide sequence described in Sequence ID No. 1 is the target region in human RPS25 mRNA targeted by the corresponding single-stranded antisense oligonucleotide (sequence name "h8-22").

[0076] Table 1-1

[0077] Table 1-2

[0078] Table 1-3

[0079] Table 1-4

[0080] Table 1-5

[0081] Table 1-6

[0082] Table 1-7

[0083] Table 1-8

[0084] Table 1-9

[0085] In Tables 1-1 to 1-9 above and Tables 2-1 to 2-5 described later, the symbols "A'", "C'", "G'", and "T'" are selected from natural nucleosides (a, A, c, C, g, G, t, and U, described later) or modified nucleosides (including modified sugar nucleosides). Furthermore, as modified sugar nucleosides, the symbol "A'" is selected from A(M), A(m), A(L), A(Y), A(Gx), A(5'-CP), A(Mx), or A(S) as described later; 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 later; 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 later; 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 later.

[0086] One embodiment of the single-stranded antisense oligonucleotide of the present invention is a single-stranded oligonucleotide that regulates the expression of the RPS25 gene, having one of the nucleotide sequences listed in Tables 2-1 to 2-5, and said single-stranded oligonucleotide is complementary to the target region in the human RPS25 mRNA precursor listed in Tables 2-1 to 2-5. The single-stranded oligonucleotide may extend by 1 to 5 nucleotides each to the 3' and / or 5' sides, provided it contains the nucleotide sequences listed in Tables 2-1 to 2-5. The above-mentioned target region is, in particular, a region in the human RPS25 mRNA precursor that is related to the regulation of human RPS25 gene expression (for example, a region exhibiting a secondary structure of the mRNA precursor to which antisense nucleotides readily bind). For example, if the 5' end position is "1" and the 3' end position is "15" in Table 2-1, then the nucleotide sequences from the 1st to the 15th nucleotides from the 5' end in the nucleotide sequence described in Sequence ID No. 2 will be the target region in the human RPS25 mRNA precursor targeted by the corresponding single-stranded antisense oligonucleotide (sequence name "hp1-15").

[0087] [Table 2-1]

[0088] [Table 2-2]

[0089] [Table 2-3]

[0090] [Table 2-4]

[0091] [Table 2-5]

[0092] In one aspect of this embodiment, the base sequences of the single-stranded antisense oligonucleotides are: SEQ ID NOs: 7, 9, 11-12, 17-19, 23-25, 27-29, 31, 33, 36-38, 46-50, 52-53, 58-61, 63-68, 70, 73-74, 79-81, 84-107, 111, 113-130, 136, 140, 143, 148, 150, 159, 161-162, 169-173, 18 It is preferable that the base sequence is one selected from the group consisting of base sequences 3-186, 188-190, 203, 208-212, 229, 232-234, 238, 298-300, 303-313, 317, 319, 321-323, 326-338, 340-349, 351-367, 370-382, 385-398, 400-411, 413, 415, 416, 418-427, and 430-432.

[0093] In one aspect of this embodiment, the base sequences of the single-stranded antisense oligonucleotides are as follows: SEQ ID NOs: 18, 24-25, 28-29, 38, 48-49, 53, 58-59, 63-64, 66-68, 79-80, 84, 86-88, 91, 93-95, 97, 99, 101-105, 113-119, 121-123, 125, 127-130, 140, 162, 169, 171-173, 183, 188, 190, 304-306, 309, 310, It is more preferable that the base sequence is one selected from the group consisting of the base sequences 312, 313, 317, 321-323, 326, 327, 331, 332, 334, 337, 340-342, 344, 346, 348, 349, 351, 353, 355-364, 366-367, 371-382, 385, 386, 388, 389, 391, 394, 396, 397, 407, 408, 410, 418-424, 426, 427, 431, and 432.

[0094] 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: 24-25, 28, 64, 80, 91, 94, 97, 113-114, 116, 119, 127, 129-130, 162, 172, 183, 305, 306, 309, 312, 322, 323, 326, 331, 340, 341, 346, 348, 349, 351, 355, 358-361, 363, 366, 367, 372-379, 381, 382, ​​386, 394, 397, 419, 421-424, 426, 431, and 432.

[0095] <Pharmacologically acceptable salts> The single-stranded antisense oligonucleotide according to this embodiment may be in the form of a pharmacologically acceptable salt. Here, "pharmacologically acceptable salt" means a salt of the single-stranded antisense oligonucleotide of the present invention that is physiologically acceptable, that is, a salt that retains the desired biological activity of the single-stranded antisense oligonucleotide and does not retain any undesirable toxicological effects. The same applies to the double-stranded antisense oligonucleotide and antisense oligonucleotide complex described later.

[0096] <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 salt" means a pharmaceutically acceptable salt as described above that is also an acid-added salt or a base-added salt. Examples of acid-added salts include inorganic salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate, as well as organic 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 salt, potassium salt, calcium salt, magnesium salt, barium salt, and aluminum salt, 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. Furthermore, examples of base addition salts include salts (amino acid salts) with basic amino acids or acidic amino acids such as arginine, lysine, ornithine, aspartic acid, or glutamic acid. The same applies to double-stranded antisense oligonucleotides and antisense oligonucleotide complexes, which will be discussed later.

[0097] <Structure of a single-stranded antisense oligonucleotide> The single-stranded antisense oligonucleotide according to this embodiment includes a gap region, a 3' wing region bound to the 3' end of the gap region, and a 5' wing region bound to the 5' end of the gap region (see, for example, Figure 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-stranded oligonucleotide described later to take a double-stranded form (double-stranded antisense oligonucleotide). The base sequence of the second-stranded oligonucleotide is preferably a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to the base sequence of the single-stranded antisense oligonucleotide.

[0098] The single-stranded antisense oligonucleotide described above is a so-called gapmer-type single-stranded antisense oligonucleotide. This gapmer-type single-stranded antisense oligonucleotide inhibits 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, the RNA-degrading enzyme RNaseH recognizes and binds to the complex of the single-stranded antisense oligonucleotide and the target RNA (center of Figure 2). Subsequently, the target RNA is cleaved and degraded by the enzymatic degradation reaction by RNaseH. At this time, the single-stranded antisense oligonucleotide is unaffected by the enzymatic degradation by RNaseH (bottom of Figure 2). Therefore, the single-stranded antisense oligonucleotide can bind to another target RNA and cleave and degrade that RNA. Thus, because the gapmer-type single-stranded antisense oligonucleotide functions as a catalyst in the enzymatic degradation reaction by RNaseH described above, it is thought that it can exert a sustained effect even with a small dose.

[0099] Furthermore, in this embodiment, the single-stranded antisense oligonucleotide can be suitably used to regulate the expression of the RPS25 gene through the mechanism described above (including acting by regulating the maturation of the RPS25 mRNA precursor). Moreover, according to this embodiment, the effect of regulating the expression of the RPS25 gene by the single-stranded antisense oligonucleotide can be exerted even in intrathecal administration, which is the usual route of administration used in clinical applications. Here, "regulating the expression of the RPS25 gene" means at least suppressing the expression of the RPS25 gene, and as a result, at least suppressing the function of the RPS25 protein (such as RAN translation).

[0100] (Gap area) The gap region described above is preferably a nucleic acid composed of 5-20 mer deoxyribose, which may contain a modified sugar moiety. In other words, the gap region can also be understood as a nucleic acid containing 5-20 mer deoxyribose, which may contain a modified sugar moiety. Alternatively, the gap region can be understood as being composed of 5-20 mer natural nucleotides, non-natural nucleotides, or both, in which the sugar moiety is deoxyribose. The gap region, by having a deoxyribose or modified deoxyribose sugar moiety, can form a complex recognizable by RNaseH together with the target RNA, such as the mRNA of RPS25. Here, an example of a nucleic acid containing a modified deoxyribose is 5'-CP nucleic acid.

[0101] The number of bases in the gap region is preferably 5 to 20 mers, more preferably 6 to 17 mers, even more preferably 7 to 13 mers, and even more preferably 7 to 11 mers.

[0102] Examples of natural nucleotides whose sugar moiety is deoxyribose include deoxyadenosine monophosphate, deoxyguanosine monophosphate, thymidine monophosphate, deoxycytidine monophosphate, and deoxy-5-methylcytidine monophosphate (also called 5-methyldeoxycytidine). In other words, natural nucleotides that constitute the above gap region include those containing structural formulas corresponding to the symbols a, g, t, and c described later.

[0103] Examples of non-natural nucleotides whose sugar moiety is deoxyribose or modified deoxyribose include 5'-CP nucleic acids and 2-thio-thymidine. Monophosphate , 2-aminoadenosine Monophosphate , 7-deazaguanosine Monophosphate These are some examples.

[0104] Furthermore, the gap region described above may be a modified sugar in part of a natural nucleotide whose sugar portion is deoxyribose, 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 part of the sugar portion is deoxyribose and the other part of the sugar portion is a modified sugar (for example, modified deoxyribose).

[0105] (3' Wing Area) The 3' wing region described above is a modified nucleic acid. In other words, the 3' wing region can be understood as being composed of modified nucleotides. Preferably, the modified nucleic acid in the 3' wing region includes at least one selected from the group consisting of 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, and MCE nucleic acid as 2'-position modified nucleic acids, and LNA, AmNA, GuNA, and scpBNA as cross-linked modified nucleic acids. It is believed that by having the 3' wing region and the 5' wing region described later composed of the predetermined modified nucleotides, a high binding affinity to the target RNA can be expected, and consequently, the function of the target RNA can be effectively suppressed. In one aspect of this embodiment, the 3' wing region may be a modified nucleic acid in which the sugar portion is a modified sugar. Examples of modified nucleic acids in which the sugar portion is a modified sugar include those listed above under (sugar modification, modified sugar). In one aspect of this embodiment, the modified nucleic acid in the 3' wing region may consist only of 2'-MOE nucleic acid. Furthermore, the modified nucleic acids in the 3' wing region described above may include multiple types within a single-stranded antisense oligonucleotide.

[0106] The number of bases in the 3' wing region described above is preferably 1 to 5 mers, more preferably 2 to 5 mers, even more preferably 2 to 4 mers, and even more preferably 3 to 4 mers.

[0107] (5' Wing area) The 5' wing region described above is a modified nucleic acid. In other words, the 5' wing region can be understood as being composed of modified nucleotides. Preferably, the modified nucleic acid in the 5' wing region includes at least one selected from the group consisting of 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, and MCE nucleic acid as 2'-position modified nucleic acids, and LNA, AmNA, GuNA, and scpBNA as cross-linked modified nucleic acids. In one aspect of this embodiment, the 5' wing region may be a modified nucleic acid in which the sugar portion is a modified sugar. Examples of modified nucleic acids in which the sugar portion is a modified sugar include those listed above under (sugar modification, modified sugar). In one aspect of this embodiment, the modified nucleic acid in the 5' wing region may consist only of 2'-MOE nucleic acid. Note that multiple types of modified nucleic acids may be included in a single single-stranded antisense oligonucleotide in the 5' wing region. In another aspect of this embodiment, the modified nucleic acids in the 3' wing region and the 5' wing region may consist only of 2'-MOE nucleic acid.

[0108] The number of bases in the 5' wing region is preferably 1 to 5mers, more preferably 2 to 5mers, and even more preferably 2 to 4mers.

[0109] In one aspect of this embodiment, it is preferable that the number of bases in the gap region is 6 to 17mers, the number of bases in the 3' wing region is 2 to 4mers, and the number of bases in the 5' wing region is 2 to 4mers.

[0110] In one aspect of this embodiment, it is more preferable that the number of bases in the gap region is 7 to 13mers, the number of bases in the 3' wing region is 2 to 4mers, and the number of bases in the 5' wing region is 2 to 4mers.

[0111] In one aspect of this embodiment, it is even more preferable that the number of bases in the gap region is 7 to 11mer, the number of bases in the 3' wing region is 2 to 4mer, and the number of bases in the 5' wing region is 2 to 4mer.

[0112] (Other configurations) In one aspect of this embodiment, the single-stranded antisense oligonucleotide may further contain a native nucleotide bound to the 3' end of the 3' wing region. The number of bases of the native nucleotide bound to the 3' end of the 3' wing region may be one or several, or it may be just one.

[0113] (Notation for gapmer-type structures) The single-stranded antisense oligonucleotide of the present invention is of the gapmer type. The structure of the gapmer type may be represented using the notation "XYZ" or "XYZW". In the above notation, "X" indicates the number of bases in the 5' wing region, "Y" indicates the number of bases in the gap region, "Z" indicates the number of bases in the 3' wing region, and "W" indicates the number of bases in the natural nucleoside bound to the 3' end of the 3' wing region.

[0114] As for "XYZ", 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 Examples include -12-5, 3-13-3, 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, 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, when written as "2-8-4", it 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.

[0115] As for "XYZW", the combinations are 2-8-4-1, 2-8-3-1, 2-8-5-1, 2-9-2-1, 2-9-3-1, 2-9-4-1, 2-9-5-1, 2-10-3-1, 2-10-4-1, 2-10-5-1, 2-11-3-1, 2-11-4-1, 2-11-5-1, 2-12-3-1, 2-12-4-1 , 2-12-5-1, 3-8-2-1, 3-8-3-1, 3-8-4-1, 3-8-5-1, 3-9-2-1, 3-9-3-1, 3-9-4-1, 3-9-5-1, 3-10-3-1, 3-10-4-1, 3-10-5-1, 3-11-3-1, 3-11-4-1, 3-11-5-1, 3-12-3-1, 3- 12-4-1, 3-12-5-1, 4-8-2-1, 4-8-3-1, 4-8-4-1, 4-8-5-1, 4-9-3-1, 4-9-4-1, 4-9-5-1, 4-10-3-1, 4-10-4-1, 4-10-5-1, 4-11-2-1, 4-11-3-1, 4-11-4-1, 4-11-5-1, 5-8- Examples include 2-1, 5-8-3-1, 5-8-4-1, 5-8-5-1, 5-9-2-1, 5-9-3-1, 5-9-4-1, 5-9-5-1, 5-10-2-1, 5-10-3-1, 5-10-4-1, 5-10-5-1, 5-11-2-1, 5-11-3-1, 5-11-4-1, 5-11-5-1, etc. For example, when written as 2-8-4-1, it means that the 5' wing region is a 2-mer oligonucleotide, the gap region is an 8-mer oligonucleotide, the 3' wing region is a 4-mer oligonucleotide, and the natural nucleoside bound to the 3' end of the 3' wing region is a 1-nucleotide.

[0116] The base length of the single-stranded antisense oligonucleotide of the present invention is 12 to 30 mers, preferably 12 to 22 mers, more preferably 14 to 20 mers, even more preferably 14 to 18 mers, and particularly preferably 15 to 17 mers. When the base length of the single-stranded antisense oligonucleotide of the present invention is 12 to 22 mers, 14 to 20 mers, 14 to 18 mers, or 15 to 17 mers, the binding of RPS25 to mRNA or to the mRNA precursor of RPS25 is particularly strong, and the regulation of RPS25 gene expression can be performed more effectively. If a natural nucleoside is further bound to the 3' end of the 3' wing region, the base length of the antisense oligonucleotide shall be counted including the number of bases of the natural nucleoside.

[0117] In this embodiment, it is preferable that each nucleoside in the single-chain antisense oligonucleotide is linked by a phosphate group and / or a modified phosphate group, and that these links are phosphodiester bonds or phosphorothioate bonds.

[0118] 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 2 to 5 mers, and a 3' wing region consisting of 2 to 5 mers. Here, the gap region is located between the 5' wing region and the 3' wing region. 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 also contain 2'-O-alkylated or 2'-F-alkylated nucleotides. As the 2'-O-alkylated nucleotide, a 2'-O-alkylated (e.g., 2'-O-methylated, etc.) nucleotide of D-ribofuranose may be used. Furthermore, the above gapmer-type single-stranded antisense oligonucleotide may form a double helix by hybridization with a second-strand oligonucleotide.

[0119] <Double-stranded antisense oligonucleotide> The double-stranded antisense oligonucleotide according to this embodiment is a single-stranded antisense oligonucleotide, A double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, comprising a second-stranded oligonucleotide hybridized to the single-stranded antisense oligonucleotide described above. Preferably, the base sequence of the second-stranded oligonucleotide has a sequence identity of 90% to 100% relative to a base sequence complementary to the base sequence of the single-stranded antisense oligonucleotide.

[0120] The above double-stranded antisense oligonucleotide can be dissociated in solution to separate into the above single-stranded antisense oligonucleotide and the above second-stranded oligonucleotide. The separated single-stranded antisense oligonucleotide can bind to the target RNA described above. The above single-stranded antisense oligonucleotide can also be understood as the "first-stranded oligonucleotide" in relation to the above second-stranded oligonucleotide. Although the first-stranded oligonucleotide that constitutes the above double-stranded antisense oligonucleotide has an antisense strand against the above-mentioned target RNA, the double-stranded oligonucleotide consisting of the first-stranded oligonucleotide and the above second-stranded oligonucleotide will be referred to as the "double-stranded antisense oligonucleotide" for convenience.

[0121] ≪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-phase support using a commercially available automated nucleic acid synthesizer. Next, the single-stranded oligonucleotide synthesized from the solid-phase support is cleaved using a basic substance, etc., and deprotected to obtain a crude single-stranded oligonucleotide. Subsequently, the obtained crude single-stranded oligonucleotide is purified using HPLC or the like. Not limited to the above production method, the single-stranded antisense oligonucleotides of the present invention can be produced by appropriately changing the base sequence, modification sites, etc. of the nucleic acid according to methods known to those skilled in the art. Furthermore, AmNA, GuNA, and scpBNA can be produced by the methods described in International Publication No. 2011 / 052436 (Patent Document 2), International Publication No. 2014 / 046212 (Patent Document 3), and International Publication No. 2015 / 125783 (Patent Document 4), respectively. 2'-MOE nucleic acids can be produced using amidites that are available for purchase as reagents. 5'-CP nucleic acid can be produced by the method described in International Publication No. 2020 / 158910 (Patent Document 5). LNA can be produced by the method described in International Publication No. 99 / 14226 (Patent Document 6).

[0122] ≪Method for producing double-stranded antisense oligonucleotides≫ The double-stranded antisense oligonucleotide of the present invention is first produced using the same manufacturing method as for the single-stranded antisense oligonucleotide described above, to produce an oligonucleotide (second-stranded oligonucleotide) having a predetermined sequence identity based on a base sequence complementary to the single-stranded antisense oligonucleotide. Subsequently, it can be produced by hybridizing the single-stranded antisense oligonucleotide and the second-stranded oligonucleotide.

[0123] Antisense oligonucleotide complex The antisense nucleotide complex according to this embodiment is The above single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the above double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, The adduct bonded to the above single-stranded antisense oligonucleotide or the above second-stranded oligonucleotide, The above-mentioned adduct is selected from the group consisting of polyethylene glycol, peptides, alkyl chains (e.g., saturated aliphatic hydrocarbons, etc.), ligand compounds, antibodies, proteins, and sugar chains (e.g., carbohydrates, polysaccharides, etc.).

[0124] In one aspect of this embodiment, the antisense oligonucleotide complex is The above single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, The device comprises an adduct bonded to the single-stranded antisense oligonucleotide, the adduct being selected from the group consisting of polyethylene glycol, peptides, alkyl chains (e.g., saturated aliphatic hydrocarbons), ligand compounds, antibodies, proteins, and sugar chains (e.g., carbohydrates, polysaccharides).

[0125] In another aspect of this embodiment, the antisense oligonucleotide complex is The above double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, The product comprises an adduct bonded to the above-mentioned single-strand antisense oligonucleotide or the above-mentioned second-strand oligonucleotide, wherein the adduct is selected from the group consisting of polyethylene glycol, peptides, alkyl chains (e.g., saturated aliphatic hydrocarbons, etc.), ligand compounds, antibodies, proteins, and sugar chains (e.g., carbohydrates, polysaccharides, etc.).

[0126] In this embodiment, "adductor" means a substance bound to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide, which is used to impart a predetermined effect. The adductor may be bound to the 5' end, the 3' end, or both the 5' and 3' ends of the single-stranded antisense oligonucleotide. The adductor may also be bound to the 5' end, the 3' end, or both the 5' and 3' ends of the second-stranded oligonucleotide. In one aspect of this embodiment, it is preferable that the adductor is bound to either the 5' end or the 3' end of the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide. The adductor may also be directly covalently bonded to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide. The adductor may also be bonded to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide via a linker substance. Examples of the linker substance include linkers composed of alkyl groups, polyethylene glycol, peptides, disulfides, nucleic acids, and / or combinations thereof. A method for attaching the above-mentioned adduct to the above-mentioned single-chain antisense oligonucleotide or the above-mentioned second-chain oligonucleotide is, for example, the method described in the examples below.

[0127] Examples of peptides used as adducts include, but are not limited to, the following: CPPs (Cell Penetrating Peptides), nuclear-transport peptides, TAT (Trans-Activator of Transcription Protein), polyarginine, glucagon-like peptide-1 analogues, synthetic cyclic RGD peptides, and brain-transport peptides.

[0128] Examples of ligand compounds used as the above-mentioned adducts include, but are not limited to, the following: N-acetylgalactosamine (GalNAc), sugars (glucose, mannose, etc.), lipids (cholesterol, palmitic acid, docosahexaenoic acid, etc.), vitamins (folic acid, vitamin A, vitamin E (tocopherol), etc.), amino acids, and monoamine receptor ligands (indatraline, etc.).

[0129] Examples of antibodies that can be used as the above-mentioned adducts include, but are not limited to, the following: anti-insulin receptor antibodies, anti-transferrin receptor antibodies, anti-LDL receptor-related protein antibodies, anti-CD22 antibodies, anti-CD30 antibodies, and anti-HER2 antibodies.

[0130] Examples of proteins that can be used as the above-mentioned adduct include, but are not limited to, the following: albumin

[0131] ≪RPS25 gene expression regulator≫ The RPS25 gene expression regulator according to this embodiment comprises the single-stranded antisense oligonucleotide of the present invention, the double-stranded antisense oligonucleotide described above, or the antisense oligonucleotide complex described above 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 for RAN translation. In yet another aspect of this embodiment, the expression regulator can also be understood as an expression inhibitor for dipeptide repeats via the inhibition of RAN translation. The single-stranded antisense oligonucleotide of the present invention inhibits the expression of the RPS25 gene by binding to RPS25 mRNA or mRNA precursor, thereby inhibiting RAN translation by its translation product. Any method of administration and formulation of the RPS25 gene expression regulator of the present invention known in the art can be used.

[0132] ≪Pharmaceutical compositions containing single-stranded antisense oligonucleotides, etc., as active ingredients≫ The pharmaceutical composition according to this embodiment contains, as an active ingredient, a 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 complex or a pharmaceutically acceptable salt thereof. Any method of administration and formulation known in the art can be used for the pharmaceutical composition of this embodiment. Hereinafter, the pharmaceutical composition may be referred to as "pharmaceutical composition such as antisense oligonucleotide."

[0133] The above pharmaceutical composition is used for the treatment or prevention of diseases related to the RPS25 gene, that is, diseases that may be caused by dipeptide repeats produced by RAN translation. In other words, the above pharmaceutical composition can be used for the treatment or prevention of diseases in which improvement of symptoms can be expected by suppressing the expression of the RPS25 gene. Such diseases are sometimes referred to as "repeat diseases." Specific examples of repeat diseases include, for example, C9orf72 ALS, C9orf72 FTLD, Huntington's disease, spinocerebellar ataxia (types 1, 2, 3, 6, 7, 8, 12, and 17), dentatorubral-pallidoluysian atrophy, bulbar spinal muscular atrophy, Friedreich ataxia, fragile X-associated ataxia tremor syndrome, myotonic dystrophy, and various other neuropsychiatric and muscular diseases.

[0134] ≪Therapeutic and preventive agents for recurrent disease≫ The therapeutic agent for repeat diseases according to this embodiment comprises the above-mentioned single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the above-mentioned double-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. The prophylactic agent for repeat diseases according to this embodiment comprises the above-mentioned single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the above-mentioned double-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. The above-mentioned 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, bulbar spinal muscular atrophy, Friedreich ataxia, fragile X-associated ataxia tremor syndrome, and myotonic dystrophy.

[0135] <C9orf72 ALS> The above-mentioned C9orf72 ALS refers to ALS characterized by an abnormal repetition and extension of the GGGGCC sequence in the intron region between exon 1a and exon 1b of the C9orf72 gene. The C9orf72 gene is the most frequently causative gene for ALS, accounting for approximately 6% of sporadic ALS and approximately 40% of familial ALS. 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 nerve dysfunction. Specifically, brainstem, cervical spinal cord, thoracic spinal cord, lumbosacral spinal cord If findings indicating upper or lower motor nerve dysfunction are present in one of the four regions and electromyographic findings are observed in the other regions, the diagnosis is laboratory-supported probable; if findings are present in two regions, it is probable; and if findings are present in three regions, it is definite.

[0136] <C9orf72 FTLD> The above-mentioned C9orf72 FTLD refers to FTLD characterized by an abnormal repetition and extension of the GGGGCC sequence in the intron region between exon 1a and exon 1b of the C9orf72 gene. The above-mentioned FTLD is characterized by progressive abnormal behavior and cognitive impairment, which interfere with daily life, as well as the presence of three or more symptoms, including disinhibited behavior or apathy / apathy or persistence / stereotyped behavior or lip-proneness and changes in eating habits. The above-mentioned FTLD is diagnosed as behavioral FTLD when imaging findings show atrophy of the frontal or anterior temporal lobe or decreased metabolism and blood flow, and it can be differentiated from a specific disease. The above-mentioned FTLD is diagnosed as semantic dementia FTLD when it shows impairment in object naming and word comprehension, impairment in knowledge of objects or symptoms such as superficial alexia / agraphia, atrophy predominantly in the anterior temporal lobe, and it can be differentiated from a specific disease.

[0137] Huntington's disease Huntington's disease, as described above, refers to a hereditary neurodegenerative disorder that exhibits an autosomal dominant inheritance pattern and is caused by abnormal repetition and elongation of the CAG sequence in the exon 1 region of the huntingtin gene. Huntington's disease presents with motor impairment characterized by involuntary movements, psychiatric symptoms, and cognitive symptoms. A diagnosis of Huntington's disease is made when specific neurological findings are observed and an abnormal elongation mutation of the CAG sequence is confirmed by genetic testing, or when a progressive course is observed, there is a family history of autosomal dominant inheritance, specific neurological findings, and clinical laboratory findings, and similar diseases are ruled out in differential diagnosis.

[0138] <Spinocerebellar ataxia (types 1, 2, 3, 6, 7, 8, 12, and 17), and dentateburubral-pallidoluysian atrophy> The above-mentioned spinocerebellar ataxias (types 1, 2, 3, 6, 7, 8, 12, 17), and dentatorubral-pallidoluysian atrophy refer to hereditary neurodegenerative diseases that show an autosomal dominant inheritance pattern and are caused by abnormal repeated expansions of specific three-base sequences (CAG or CTG) existing on the responsible genes for each disease. In spinocerebellar ataxias types 1, 2, 3, 6, 7, 12, and 17, as well as dentatorubral-pallidoluysian atrophy, CAG repeat sequences are observed. In spinocerebellar ataxia type 8, CTG repeat sequences are observed. The above-mentioned spinocerebellar ataxias and dentatorubral-pallidoluysian atrophy mainly present with cerebellar or posterior columnar motor ataxia or spastic paraplegia, and are basically slowly progressive, and are diagnosed by combining genetic diagnosis or neuropathological diagnosis, etc.

[0139] <Bulbospinal muscular atrophy> The above-mentioned bulbospinal muscular atrophy refers to a hereditary disease caused by abnormal repeated expansion of the CAG sequence existing in the exon region of the androgen receptor gene. The above-mentioned bulbospinal muscular atrophy is diagnosed by combining neurological findings (bulbar symptoms, lower motor neuron signs, finger tremors, reduced limb tendon reflexes), clinical findings and examination findings, genetic diagnosis, etc.

[0140] <Friedreich ataxia> The above-mentioned Friedreich ataxia refers to a hereditary neurodegenerative disease that shows an autosomal recessive inheritance pattern caused by mutations in the frataxin gene. Most cases of the above-mentioned Friedreich ataxia are due to abnormal repeated expansion of the GAA sequence existing in the first intron.

[0141] <Fragile X-associated tremor / ataxia syndrome> The above-mentioned fragile X-associated tremor / ataxia syndrome refers to a hereditary neurodegenerative disease caused by abnormal repeated expansion of the CGG sequence existing in the 5’UTR of the FMP1 gene. The above-mentioned fragile X-associated tremor / ataxia syndrome is diagnosed by combining clinical symptoms (cerebellar ataxia, tremors during movement, parkinsonism, dementia, intellectual disability), middle cerebellar peduncle signs by MRI examination, genetic diagnosis, etc.

[0142] <Myotonic dystrophy> The above-mentioned myotonic dystrophy refers to a hereditary muscle disorder that follows an autosomal dominant inheritance pattern and is caused by abnormal repetition and elongation of the CUG sequence located in the 3'UTR of the DMPK gene.

[0143] <Individual> The term "individual" refers to a mammal. Preferably, the individual is a human, monkey, marmoset, dog, pig, rabbit, guinea pig, rat, or mouse. More preferably, the individual is a human.

[0144] The method of administration and dosage form of the single-stranded antisense oligonucleotide or its pharmaceutical composition (including therapeutic and prophylactic agents for C9orf72 ALS) of the present invention are not particularly limited. That is, any known method of administration and formulation in the art can be used as the method of administration 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, vaginal administration, rectal administration, intranasal administration, transdermal administration, intravenous injection, intradrip infusion, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration by aspiration or inhalation, intrathecal administration, and intraventricular administration.

[0145] The formulations of the present invention, such as antisense oligonucleotides, include excipients, binders, wetting agents, disintegrants, lubricants, diluents, flavoring agents, fragrances, solubilizers, suspending agents, emulsifiers, stabilizers, and preservatives. Isotonic agent Various pharmaceutical additives can be mixed in as needed.

[0146] When administering the pharmaceutical composition of the present invention, such as the antisense oligonucleotide, topically, formulations such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders can be used.

[0147] When administering the pharmaceutical composition of the present invention, such as the antisense oligonucleotide, orally, formulations such as powders, granules, suspensions or solutions dissolved in water or a non-aqueous medium, capsules, powders, tablets, etc., can be used.

[0148] When administering the pharmaceutical composition of the present invention, such as the antisense oligonucleotide, parenterally, intrathecally, or intraventricularly, a formulation such as a sterile aqueous solution can be used.

[0149] The effective dose of the single-stranded antisense oligonucleotide of the present invention can be arbitrarily determined depending on the sex, age, weight, symptoms, etc. of the individual being administered the drug. Furthermore, it can also be arbitrarily determined depending on the method, route, frequency, etc. of administration. For example, the dose can be 0.01 to 100 mg / kg. Preferably, it is 0.1 to 50 mg / kg, and more preferably 0.1 to 10 mg / kg.

[0150] Methods for regulating the expression of the RPS25 gene The method for regulating the expression of the RPS25 gene in this embodiment includes the step of administering the above-mentioned single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the above-mentioned double-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 to cells, tissues, or individuals expressing the RPS25 gene.

[0151] In this embodiment, the method for administering the single-stranded antisense oligonucleotide, etc., to cells, tissues, or organisms may be performed in vitro or in vivo. When administered in vivo, the administration route described above is used.

[0152] In this embodiment, "cells expressing the RPS25 gene" include, for example, nerve cells constituting the central nervous system, nerve cells constituting the peripheral nervous system, and other cells constituting skin tissue.

[0153] The method for treating or preventing repeat disease in this embodiment includes the step of administering to an individual suffering from repeat disease the above-mentioned single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the above-mentioned double-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.

[0154] Examples of recurring illnesses include the neuropsychiatric disorders and muscular disorders mentioned above. The dosage form, route of administration, and dosage when administering to an individual can be appropriately adopted from those described above.

[0155] The antisense oligonucleotide according to this embodiment has been described above. The single-stranded antisense oligonucleotide having the above-described configuration can regulate the expression of the RPS25 gene. The suppressive activity (knockdown activity) on the expression of the RPS25 gene can be measured by known methods. For example, a method for measuring knockdown activity can be found in Nature (2015) 518(7539):409-12 (Non-Patent Literature 10). It can also be measured by transfection of HEK293T cells with the antisense oligonucleotide, as described later.

[0156] ≪Method for evaluating the repressive activity of the RPS25 gene on expression≫ <Introduction of antisense oligonucleotides into cells> Cells expressing the RPS25 gene are treated with antisense oligonucleotides for 6 hours to 3 days using methods such as lipofection, electroporation, or direct addition. Any cells expressing the RPS25 gene can be used, such as HEK293T cells, more preferably nerve cells, and even more preferably human-derived nerve cells. The cells treated with the antisense oligonucleotide may be harvested immediately after treatment, or the antisense oligonucleotide may be removed and the cells continued to be cultured.

[0157] <Evaluation of RPS25 mRNA level> Reverse transcription reaction is performed on the total RNA extracted from the collected cells, and real-time PCR or the like is performed using a probe specific to the RPS25 gene for the obtained complementary DNA to measure the amount of RPS25 mRNA. Examples of the probe used for real-time PCR include Taqman probes. Examples of the reaction method include a method of repeating three steps of "(denaturation of cDNA)-(annealing)-(extension reaction)" or two steps of "(denaturation of cDNA)-(annealing and extension reaction)" any number of times. The number of repetitions of the two- or three-step cycle is, for example, 25 to 45 times, preferably 35 to 40 times. The (cDNA denaturation) temperature is, for example, 90°C to 98°C, preferably 92°C to 95°C. The (annealing) temperature is, for example, 40°C to 70°C, preferably 50°C to 60°C. The (extension reaction) temperature is, for example, 65°C to 75°C, preferably the optimal temperature of the polymerase used in the reaction. The (annealing and extension reaction) temperature is, for example, 55°C to 70°C.

[0158] <Evaluation of RPS25 protein level> The collected cells are lysed to obtain an extract. Immunochemical methods such as Western blotting and ELISA (Enzyme-Linked Immuno Sorbent Assay) are used to evaluate the amount of RPS25 protein contained in the above extract. In Western blotting, any device can be used for each step of electrophoresis, transfer, and detection. The reaction time and reaction temperature of the membrane with the primary antibody or secondary antibody can be arbitrarily set, for example, overnight at 4°C or 1 to 3 hours at room temperature.

[0159] Note that the present invention is not limited to the above-described embodiments. For example, the single-stranded antisense oligonucleotide includes the following embodiments.

[0160] One embodiment of the present invention is a single-stranded antisense oligonucleotide that modulates the expression of the RPS25 gene or a pharmaceutically acceptable salt thereof. The above single-stranded antisense oligonucleotide has each nucleotide linked by a phosphate group and / or a modified phosphate group. The above single-stranded antisense oligonucleotide comprises 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. The above gap region is a nucleic acid composed of deoxyribose, which may contain nucleic acids with modified sugar moieties. The 3' wing region and 5' wing region described above are modified nucleic acids. The base lengths of the above single-stranded antisense oligonucleotides are 12-30 mers. The base sequence of the above single-stranded antisense oligonucleotide is: A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, having a sequence identity of 90% or more and 100% or less based on a sequence complementary to at least one target region having the same base length as the single-stranded antisense oligonucleotide in the base sequence described in Sequence ID No. 1 or Sequence ID No. 2.

[0161] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, the base sequence of the single-stranded antisense oligonucleotide is: The base sequence is one that has 95% to 100% sequence identity with respect to a base sequence complementary to at least one target region consisting of the same base length as the single-stranded antisense oligonucleotide in the base sequence described in Sequence ID No. 1 or Sequence ID No. 2.

[0162] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, the nucleotide sequence of the single-stranded antisense oligonucleotide is a nucleotide sequence complementary to at least one target region in the nucleotide sequence described in Sequence ID No. 1 or Sequence ID No. 2, which has the same nucleotide length as the single-stranded antisense oligonucleotide.

[0163] Another embodiment of the single-stranded antisense oligonucleotide of the present invention is: The number of bases in the above gap region is a nucleic acid composed of deoxyribose, which may contain 5-20 mer nucleic acids with modified sugar moieties. The 3' wing region described above is a modified nucleic acid consisting of 1 to 5 members. The modified nucleic acid in the 3' wing region is a 2'-modified nucleic acid and / or a cross-linked modified nucleic acid. The above 5' wing region is a modified nucleic acid consisting of 1 to 5 mers. The modified nucleic acid in the 5' wing region described above is a 2'-modified nucleic acid and / or a cross-linked modified nucleic acid.

[0164] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, the base length of the single-stranded antisense oligonucleotide is 14 to 22 mers. The number of bases in the above gap region is a nucleic acid composed of deoxyribose, which may contain 6-17mer nucleic acids with modified sugar moieties. The 3' wing region described above is a modified nucleic acid of 2-5 mers. The modified nucleic acid in the 3' wing region described above includes at least one selected from the group consisting of LNA, AmNA, GuNA, and scpBNA. The above 5' wing region is a modified nucleic acid consisting of 2-5 mers. The modified nucleic acid in the 5' wing region described above includes at least one selected from the group consisting of LNA, AmNA, GuNA, and scpBNA. At least one internucleotide bond in the above single-stranded antisense oligonucleotide is a phosphorothioate bond.

[0165] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, the chain length of the single-stranded antisense oligonucleotide is 14 to 20 mers. The number of bases in the above gap region is a nucleic acid composed of deoxyribose, which may contain 7-13 mer nucleic acids with modified sugar moieties. The nucleic acid in the above gap region includes at least one selected from the group consisting of 5-methyldeoxycytidine and 5'-CP nucleic acids. The 3' wing region described above is a modified nucleic acid consisting of 2-4 mers. The modified nucleic acid in the 3' wing region described above includes at least one selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA. The above 5' wing region is a modified nucleic acid consisting of 2-4 mers. The modified nucleic acid in the 5' wing region described above includes at least one selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA.

[0166] In another embodiment of the single-stranded antisense oligonucleotide of the present invention, the base length of the single-stranded antisense oligonucleotide is 14 to 18 mers. The above gap region is 7-11mer. The 3' wing region described above is a modified nucleic acid consisting of 2-4 mers. The modified nucleic acid in the 3' wing region described above includes at least one selected from the group consisting of AmNA, GuNA, and scpBNA. The above 5' wing region is a modified nucleic acid consisting of 2-4 mers. The modified nucleic acid in the 5' wing region described above includes at least one selected from the group consisting of AmNA, GuNA, and scpBNA. [Examples]

[0167] The following describes examples of the present invention, but the present invention is not limited to these examples.

[0168] <<Production of single-stranded antisense oligonucleotides for the RPS25 gene>> First, single-stranded antisense oligonucleotides were designed as shown in Tables 3-1 to 3-17 and Tables 4-1 to 4-5. For some of the designed oligonucleotides, single-stranded antisense oligonucleotides targeting the RPS25 gene were produced using the following procedure.

[0169] Single-stranded antisense oligonucleotides containing modified nucleic acids such as 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, AmNA, scpBNA, 5'-CP nucleic acid, and / or GuNA, and / or nucleic acids whose nucleic acid base is 5-methylcytosine, were synthesized on a 0.2 μmol scale using an automated nucleic acid synthesizer (nS-8 type, manufactured by Gene Design Co., Ltd.). Chain lengthening was performed using a standard phosphoramidite protocol. CPG resin was used as the solid support. For phosphorothioate (PS) skeleton formation, sulfurization was performed using DDTT (((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazaoline-3-thione), etc. Single-stranded antisense oligonucleotides containing 2'-MOE nucleic acid, AmNA, and / or scpBNA were obtained with the terminal 5' hydroxyl group not protected by a DMTr(4,4'-dimethoxytrityl) group and the 3' position supported on a solid phase. Subsequently, the single-stranded antisense oligonucleotides were cleaved from the solid phase support by alkali treatment and recovered in solution. The crude product was then obtained by distillation off the solvent from the recovered solution. The obtained crude product was purified by reverse-phase HPLC to obtain purified single-stranded antisense oligonucleotides. The purity and structure of each obtained single-stranded antisense oligonucleotide were confirmed by LC-MS (Waters).

[0170] Examples 412 to 416 in Table 3-14 were synthesized according to the above protocol using phosphoramidites having the corresponding adducts (for example, the compound synthesis in Figures 3 to 7). As the phosphoramidites having adducts, α-Tocopherol-TEG phosphoramidite (Glen Research, product code: 10-1977-02) was used in Example 412, and 5'-Palmitate-C6 CE-Phosphoramidite (Link Technologies Ltd, item number: 2199) was used in Examples 413 and 414. In Examples 415 and 416, oligonucleotides having an amino group at the 5'-position (amino ASOs) were first synthesized according to the protocol described above using the corresponding phosphoramidite (5'-amino-modifier C6 (Glen Research, catalog no. 10-1906-02)). In Example 415, the compound was synthesized using 2,5-dioxopyrrolidine-1-yl-8-(((1R,3S)-3-(3,4-dichlorophenyl)-2,3-dihydro-1H-inden-1-yl)(methyl)amino)-8-oxooctanoate, which was synthesized according to the method described below, on the obtained amino ASO. In Example 416, the compound was synthesized by condensing the above amino ASO with docosahexaenoic acid (DHA, Fujifilm, catalog no. 90310).

[0171] Synthesis of 2,5-dioxopyrrolidin-1-yl 8-(((1R,3S)-3-(3,4-dichlorophenyl)-2,3-dihydro-1H-inden-1-yl)(methyl)amino)-8-oxooctanoate [ka] A suspension of N,N'-disuccinimidyl suberic acid (101 mg, 0.274 mmol) and N-methylmorpholine (0.040 mL, 0.365 mmol) in N,N-dimethylacetamide (1 mL) was mixed with a solution of indatraline hydrochloride (10 mg, 0.030 mmol) in N,N-dimethylacetamide at 0°C. The reaction mixture was then heated to 40°C and stirred for 2 hours. The resulting reaction mixture was dried under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain 7.0 mg of a mixture of the title compound and N,N'-disuccinimidyl suberic acid. LC / MS:545[M+H]

[0172] Tables 3-1 to 3-17 and 4-1 to 4-5 below list the single-stranded antisense oligonucleotides produced by the method described above. The single-stranded antisense oligonucleotides shown in Tables 3-1 to 3-17 are single-stranded antisense oligonucleotides against human RPS25 mRNA (SEQ ID NO: 1). Examples 463 and 464 serve as negative controls.

[0173] [Table 3-1]

[0174] [Table 3-2]

[0175] [Table 3-3]

[0176] [Table 3-4]

[0177] [Table 3-5]

[0178] Table 3-6

[0179] Table 3-7

[0180] Table 3-8

[0181] Table 3-9

[0182] Table 3-10

[0183] Table 3-11

[0184] Table 3-12

[0185] Table 3-13

[0186] Table 3-14

[0187] Table 3-15

[0188]

Table 3-16

[0189]

Table 3-17

[0190] The single-stranded antisense oligonucleotides shown in Tables 4-1 to 4-5 are single-stranded antisense oligonucleotides against the mRNA precursor of human RPS25 (SEQ ID NO: 2).

[0191]

Table 4-1

[0192]

Table 4-2

[0193]

Table 4-3

[0194]

Table 4-4

[0195]

Table 4-5

[0196] In this specification, the corresponding structures may be represented using the following symbols or notations.

[0197]

Chemical Structure

[0198]

Chemical Structure

[0199] In the structural formula shown above, R

[0201] , , and R 2 each independently represents a hydrogen atom, or a linear or branched alkyl group having 1 to 3 carbon atoms. Here, in the bond represented by the above-mentioned "=", R 1 and R 2 both represent methyl groups. R 3 , R 4 , and R 5 each independently represents 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. Here, in the GuNA represented by the above-mentioned "Gx", R 3 and R 5 are both hydrogen atoms, and when R 4 is a methyl group, it is denoted as "Gm", and when R 3 is a hydrogen atom, and R 4 and R 5 are both methyl groups, it is denoted as "Gdm", and when R 3 and R 5 are hydrogen atoms, and R 4 is a tert-butyl group, it is denoted as "GtB".

[0200] ≪Evaluation of RPS25 gene expression≫ The evaluation of RPS25 gene expression was carried out separately into the expression evaluation using human fetal kidney cells and the expression evaluation using primary cultured mouse neurons according to the manufactured single-stranded antisense oligonucleotides. Also, the evaluation of the above RPS25 gene expression can be carried out using human iPS cell-derived neurons. The gene expression evaluation in this example means evaluating the mRNA amount by measuring the amount of complementary DNA (cDNA) obtained by reverse transcription reaction. Hereinafter, the specific procedures for each expression evaluation will be described.

[0201] <Expression evaluation using human fetal kidney cells> Human embryonic kidney cells HEK293T (ATCC® CRL-3216®) were cultured in culture medium at 37°C and 5% CO2. The culture medium used for HEK293T cells had the following composition.

[0202] Culture medium composition of HEK293T cells Dulbecco's Modified Eagle Medium (DMEM): SIGMA Corporation, Cat#D6429 10% Fetal Bovine Serum (FBS): BioWest, Cat#S1820 100-fold diluted penicillin-streptomycin mixed solution: Manufactured by Nacalai Tesque, Cat#09367-34 (Penicillin 10,000 units / ml, Streptomycin 10,000 μg / ml, containing stabilizer)

[0203] First, 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 cultured overnight at 37°C and 5% CO2. Subsequently, each single-stranded antisense oligonucleotide (final concentrations of 0.5 nM, 5 nM, 15 nM, or 50 nM) diluted in phosphate-buffered saline (PBS) was transfected into the aforementioned cells by lipofection. As a negative control group, cells transfected with PBS in which the single-stranded antisense oligonucleotide was not dissolved were used. The transfected cells were cultured in growth medium at 37°C and 5% CO2 for 48 hours. After that, the growth medium was removed, and the extracted total RNA was reverse transcribed using the Taqman Fast Cells-to-CT Kit (Thermo Fisher Scientific, Cat#4399003). Using the complementary DNA (cDNA) obtained from this reverse transcription reaction, real-time PCR was performed using Taqman gene expression assays (Applied Biosystems) with pre-designed gene-specific probes (see below) (40 cycles of 3 seconds at 95°C and 30 seconds at 60°C).

[0204] List of gene-specific probes used in the evaluation of human RPS25 gene expression human RPS25: Hs01568661_g1 human GAPDH: 4326317E (internal control)

[0205] Tables 5-1 to 5-7, 6-1, and 6-2 show the expression ratios of human RPS25 mRNA for each single-stranded antisense oligonucleotide against human RPS25 mRNA, as determined by the method described above. The expression ratio of human RPS25 mRNA in the negative control group was set to 1.00. Single-stranded antisense oligonucleotides with an expression ratio of 0.80 or less were considered capable of suppressing human RPS25 mRNA expression. In the tables, "-" indicates that measurement was not performed. Generally, suppression of mRNA expression is thought to suppress subsequent protein translation, etc. Therefore, single-stranded antisense oligonucleotides with an expression ratio of 0.80 or less can be considered capable of regulating the function of the human RPS25 gene.

[0206] [Table 5-1]

[0207] [Table 5-2]

[0208] [Table 5-3]

[0209] [Table 5-4]

[0210] [Table 5-5]

[0211] [Table 5-6]

[0212] [Table 5-7]

[0213] [Table 6-1]

[0214] [Table 6-2]

[0215] Tables 7-1 to 7-5 show the expression ratios of human RPS25 mRNA in each single-stranded antisense oligonucleotide relative to human RPS25 mRNA precursor using a similar method. Each single-stranded antisense oligonucleotide used in the lipofection method was adjusted to a final concentration of 50 nM or 100 nM.

[0216] [Table 7-1]

[0217] [Table 7-2]

[0218] [Table 7-3]

[0219] [Table 7-4]

[0220] [Table 7-5]

[0221] <Evaluation of expression using primary mouse neuronal cells> Primary mouse nerve cells were cultured in culture medium at 37°C and 5% CO2. The culture medium used for primary mouse nerve cells had the following composition.

[0222] Culture medium composition of primary mouse neuronal cells B-27 Electrophysiology Kit: Manufactured by gibco, Cat#A1413701 100-fold diluted 200 mM-L-glutamine solution: Manufactured by Nacalai Tesque: Cat#16948-04 100-fold diluted penicillin-streptomycin mixed solution: Manufactured by Nacalai Tesque, Cat#09367-34 (Penicillin 10,000 units / ml, Streptomycin 10,000 μg / ml, containing stabilizer)

[0223] First, primary mouse neuronal cells (derived from mouse fetal brain) were seeded in 96-well plates at a rate of 40,000 cells / well and cultured for 5 days at 37°C and 5% CO2. Then, single-stranded antisense oligonucleotides (final concentrations of 0.01 μM, 0.1 μM, or 1 μM) diluted in phosphate-buffered saline (PBS) were added to the culture medium. For the negative control group, PBS without dissolved single-stranded antisense oligonucleotides was added to the culture medium. The cells were cultured in the culture medium at 37°C and 5% CO2 for 48 hours. Afterward, the culture medium was removed, and the extracted total RNA was reverse transcribed using the Taqman Fast Cells-to-CT Kit (Thermo Fisher Scientific, Cat#4399003). Using the complementary DNA (cDNA) obtained from this reverse transcription reaction, real-time PCR was performed using Taqman gene expression assays (Applied Biosystems) with pre-designed gene-specific probes (see below) (40 cycles of 3 seconds at 95°C and 30 seconds at 60°C).

[0224] List of gene-specific probes used to evaluate mouse RPS25 gene expression mouse Rps25: Mm02342783_g1 mouse GAPDH: 4352339E (Internal Control)

[0225] Table 8 shows the expression ratios of mouse RPS25 mRNA for each single-stranded antisense oligonucleotide determined by the method described above. The expression ratio of mouse RPS25 mRNA determined in the negative control group was set to 1.00. Generally, suppression of mRNA expression is thought to suppress subsequent protein translation, etc. Therefore, if the expression ratio is 0.80 or less, it can be determined that the single-stranded antisense oligonucleotide is capable of regulating the function of the mouse RPS25 gene. The target region to which the single-stranded antisense oligonucleotide h451-465-A binds is a region whose sequence is conserved between the human RPS25 gene and the mouse RPS25 gene.

[0226] [Table 8]

[0227] ≪Evaluation using human iPS cell-derived motor neurons≫ Human iPS cells were differentiated into motor neurons and used for evaluation. Cell maintenance and differentiation induction were carried out in the culture medium described below, under conditions of 37°C and 5% CO2.

[0228] List of culture medium compositions (Cell culture medium for SNL cells) DMEM (manufactured by Sigma-Aldrich, Cat#D6429), 100-fold diluted penicillin-streptomycin mixture (ThermoFisher Scientific, Cat#15140-122) 10-fold diluted fetal bovine serum (ThermoFisher Scientific, Cat#10437-028) (Culture medium for iPS cells) Primate ES / iPS cell culture medium (REPROCELL, Cat#RCHEMD001B) 100-fold diluted penicillin-streptomycin mixture (ThermoFisher Scientific, cat#15140-122) (Mixed medium A) DMEM / Ham's F12 GlutaMAX (manufactured by ThermoFisher Scientific, Cat#10565-018) 2 mM L-glutamine (ThermoFisher Scientific, Cat#25030-081) Non-Essential Amino Acid (NEAA) (manufactured by ThermoFisher Scientific, Cat#11140-050) 100-fold diluted penicillin-streptomycin mixture (ThermoFisher Scientific, cat#15140-122) 2μg / mL Heparin (Sigma-Aldrich, H-4784) N2 supplement (manufactured by ThermoFisher Scientific, Cat#17502-048) (Mixed medium B) Neurobasal medium (ThermoFisher Scientific, cat#21103-049) 2 mM L-glutamine (ThermoFisher Scientific, Cat#25030-081) Non-Essential Amino Acid (NEAA) (manufactured by ThermoFisher Scientific, Cat#11140-050) Antibiotic-Antimycotic (manufactured by ThermoFisher Scientific, cat#15240-062) 2μg / mL Heparin (Sigma-Aldrich, H-4784) N2 supplement (manufactured by 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 (manufactured by R&D Systems, cat#212-GD-050) B27 supplement (manufactured by ThermoFisher Scientific, cat#12587010) 200 μM Ascorbic acid (Sigma-Aldrich, Cat#A5960) 10 ng / mL Human BDNF (Peprotech, Cat#450-02) (Culture medium for nerve cells) Neurobasal medium Electro (manufactured by ThermoFisher Scientific, cat#A14098-01) 2 mM L-glutamine (ThermoFisher Scientific, Cat#25030-081) Non-Essential Amino Acid (NEAA) (manufactured by ThermoFisher Scientific, Cat#11140-050) Antibiotic-Antimycotic (manufactured by ThermoFisher Scientific, cat#15240-062) 2μg / mL Heparin (Sigma-Aldrich, H-4784) N2 supplement (manufactured by 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 (manufactured by R&D Systems, cat#212-GD-050) B27 supplement, Electro (manufactured by ThermoFisher Scientific, cat#A14097-01) 200 μM Ascorbic acid (Sigma-Aldrich, Cat#A5960) 10 ng / mL Human BDNF (Peprotech, Cat#450-02) 25μM 2-mercaptoethanol (ThermoFisher Scientific, cat#21985-0123) 0.1% Bovine serum albumin (Sigma-Aldrich, cat#A9576)

[0229] <Mitomycin treatment of feeder cells> Mitomycin-treated SNL cells were used as feeder cells for seeding human iPS cells. Cell Bio Lab A medium (manufactured by Iwaki Corporation, Cat#CBA-316) was prepared. Mitomycin treatment of SNL cells was performed as follows: First, 0.1% gelatin (manufactured by Fujifilm Wako Pure Chemical Industries, Cat#190-15805) was added to a 10 cm petri dish (manufactured by Iwaki Corporation, Cat#3020-100), and the petri dish was left standing in an incubator at 37°C and 5% CO2 for more than 1 hour (hereinafter, this operation may be referred to as "gelatin treatment"). After that, excess gelatin was aspirated and removed from the petri dish, and thawed SNL cells were placed in a medium for SNL cells, 1 to 2 × 10⁶ cells per petri dish. 6 The seeds were sown to form individual cells. The cells were subcultured every 3-4 days, diluted 8-16 times, until the required number of cells was reached. Next, 2-4 × 10 cells were placed in 15cm petri dishes (Iwaki Co., Ltd., Cat#3030-150) treated with 0.1% gelatin. 6Nine SNL cells were seeded. After culturing the cells until 80-90% confluence, mitomycin C (Kyowa Kirin Co., Ltd., YJ code 4231400D1031), diluted to 0.4 mg / mL in SNL cell medium, was added to the petri dish to a final concentration of 6.2 μg / mL. The petri dish was incubated for 2 hours and 15 minutes at 37°C under 5% CO2 conditions. After that, the medium was removed from the petri 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%) and added to the SNL cells, and left to stand at room temperature for 1 minute. After that, the SNL cells were collected in a tube, centrifuged, suspended in Cellbanker(R) (ZENOAQ Resource, Cat#CB011), and cryopreserved.

[0230] <Maintenance of human iPS cells> 0.1% gelatin was added to a 10 cm petri dish and left to stand in an incubator at 37°C and 5% CO2 for at least 1 hour. Mitomycin-treated SNL cells were suspended in SNL cell medium. Then, 1.5 × 10⁶ 6 The above SNL cells were seeded in a 10 cm petri dish and cultured for 2-3 days. Subsequently, the SNL cell medium was removed from the petri dish and the SNL cells were washed with PBS. Then, human iPS cells (201B7 strain, obtained from iPS Academia Japan, AJ-H1-01) suspended in iPS cell medium containing 1 / 1000 of Y-27632 (Tocris, Cat#1254) were seeded in the petri dish. The culture medium was changed daily from two days after seeding until differentiation induction began.

[0231] <Induction of differentiation from human iPS cells to motor neurons> Human iPS cells were exposed to Y-27632 (final concentration 10 μM) for more than 1 hour by adding Y-27632 to the cell culture medium. After removing the culture supernatant and washing the cells with PBS, cell dissociation was performed. solution(CTK solution) (REPROCELL, Cat#RCHETP002) was added and the mixture was reacted at room temperature for 1 minute. The CTK solution was removed, the cells were washed twice with PBS, and then 1 mL of iPS cell medium was added. The cells were detached with a cell scraper, and the cell aggregates were dispersed through a cell strainer (Becton Dickinson, Cat#352350) to obtain a cell suspension. The obtained suspension was transferred to a 6-well plate (Corning, Cat#3471). The 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 cells were cultured in an incubator at 37°C and 5% CO2 (Day 0 of culture).

[0232] On days 2 and 4 of incubation, the culture medium was removed by pipette and replaced with fresh medium containing 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 by pipette and replaced with fresh medium containing mixed medium A supplemented with LDN193189 (final concentration 0.3 μM), SB431542 (final concentration 2 μM), CHIR-99021 (final concentration 3 μM), Purmorphamine (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Cat#166-23991) (final concentration 0.5 μM), and Retinoic Acid (manufactured by Sigma-Aldrich, Inc., Cat#R2625) (final concentration 0.1 μM). On days 14 and 16 of culture, the culture medium was removed by pipette and replaced with 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).

[0233] On day 18 of culture, the mixed medium B was replaced with fresh medium containing 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 aggregate was washed with PBS, then centrifuged and the supernatant was removed. Accutase (Innovative Cell Technologies, Cat#AT104) and Y27632 (final concentration 10 μM) were added to the cell aggregate and incubated at 37°C for 10 minutes. After cooling the cell aggregate on ice, it was dispersed by pipetting. After centrifugation (300 × g, 5 minutes, 4°C), the precipitated cells were collected and suspended in mixed medium B, and this procedure was repeated twice. By performing the above procedure, motor neurons derived from iPS cells were obtained. The obtained motor neurons were suspended in Cell Banker(R), aliquoted, and stored cryopreserved.

[0234] <Culture of human iPS cell-derived motor neurons and evaluation of single-stranded antisense nucleotides> A 96-well Optical Btm Plt Polybase Black w / Lid Cell Culture Sterile PS (ThermoFisher Scientific, Cat#165305) was mixed with a 6.66-fold dilution of Poly-L-Ornitine Solution (PLO) (Sigma-Aldrich, Cat#P4957) in PBS and allowed to stand at room temperature for at least 2 hours. After washing three times with PBS, iMatrix diluted in PBS was added at a concentration of 0.5 μg / cm³. 2The cells were added to the plate and left to stand overnight at 4°C. Next, the human iPS cell-derived motor neurons that had been cryopreserved in the previous step were thawed and suspended in neuronal cell culture medium. The supernatant was then removed by centrifugation, and the cells were resuspended in neuronal cell culture medium containing 1 / 100 volume of Culture One Supplement (ThermoFisher Scientific, A3320201) and Compound E (final concentration 0.1 μM). These cells were seeded at a density of 30,000 cells / well on a coated 96-well plate and cultured for 28 days in an incubator at 37°C and 5% CO2. Half of the neuronal cell culture medium was replaced every 2-3 days. For the first 7 days of culture, the neuronal cell culture medium contained Culture One Supplement and Compound E.

[0235] After seeding, single-stranded antisense oligonucleotides (final concentrations 0.01 μM, 0.1 μM, and 1 μM), diluted in PBS, were added to the culture medium on days 1, 11, 18, and 26 of culture (denoted as D1, D11, D18, and D26, respectively). As a negative control group, cells were cultured in PBS without dissolved single-stranded antisense oligonucleotides. The cells were cultured in culture medium at 37°C and 5% CO2 for 48 or 72 hours, after which the medium containing the single-stranded antisense nucleotides was removed and the cells were continued in neuronal cell culture medium (half-volume medium change every 2-3 days). Subsequently, the above culture medium was removed, and the extracted total RNA was reverse transcribed using the Taqman Fast Cells-to-CT Kit (Thermo Fisher Scientific, Cat#4399003). Table 9 shows the results of performing real-time PCR using Taqman gene expression assays (Applied Biosystems) with pre-designed gene-specific probes (see below) (40 cycles of 3 seconds at 95°C and 30 seconds at 60°C) with the complementary DNA (cDNA) obtained from this reverse transcription reaction. The "expression repression rate" in Table 9 is calculated using the following formula (1). (Repression rate) = 1 - (Expression ratio) (Equation (1)) The higher the expression suppression rate, the more effective the single-stranded antisense oligonucleotide was in suppressing human RPS25 mRNA expression.

[0236] List of gene-specific probes used in the evaluation of human RPS25 gene expression human RPS25: Hs01568661_g1 human GAPDH: 4326317E (internal control)

[0237] [Table 9]

[0238] <<Evaluation of RPS25 protein expression>> RPS25 protein expression was evaluated using human embryonic kidney cells, depending on the single-stranded antisense oligonucleotides produced. In this example, protein expression level evaluation refers to the evaluation of the amount of protein translated from mRNA. The specific procedure for expression evaluation is described below.

[0239] <Evaluation of expression using human fetal kidney cells> Human embryonic kidney cells HEK293T (ATCC® CRL-3216®) were cultured in culture medium at 37°C and 5% CO2. The culture medium used for HEK293T cells had the following composition.

[0240] Culture medium composition of HEK293T cells Dulbecco's Modified Eagle Medium (DMEM): SIGMA Corporation, Cat#D6429 10% Fetal Bovine Serum (FBS): BioWest, Cat#S1820 100-fold diluted penicillin-streptomycin mixed solution: Manufactured by Nacalai Tesque, Cat#09367-34 (Penicillin 10,000 units / ml, Streptomycin 10,000 μg / ml, containing stabilizer)

[0241] <Quantitative analysis of proteins by Western blotting> First, HEK293T cells (500,000 cells / well) were seeded in a 6-well plate and cultured overnight at 37°C and 5% CO2. Subsequently, each single-chain antisense oligonucleotide (final concentration 50 nM) diluted in phosphate-buffered saline (PBS) was transfected into the aforementioned cells using lipofection. As a negative control group, cells transfected with PBS in which the single-chain antisense oligonucleotide was not dissolved were used. The transfected cells were cultured in growth medium at 37°C and 5% CO2 for 48 hours. After that, the growth medium was removed, the cells were washed with PBS, and the cells were collected using a cell scraper. The collected solution was centrifuged at 2700 × 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 volume of Protease Inhibitor (ThermoFisher Scientific, Cat#1860932) was added, and the cells were disrupted using an ultrasonic lysator. The cells were then centrifuged at 15000 × g for 10 minutes at 4°C, and the supernatant was used as the sample. The collected samples were subjected to protein quantification using the Pierce® BCA Protein Assay Kit (ThermoScientific, Cat#23225). After adjusting the concentration of each sample to be constant, Pierce® Lane Marker Reducing Sample Buffer (ThermoFisher Scientific, Cat#39000) was added, and the samples were heat-treated at 95°C for 5 minutes. The prepared samples were stratified so that the protein content was 10 μg to 20 μg / lane, and electrophoresis was performed. Electrophoresis was performed using Criterion® TDX® precast gels 4-15% (BIO-RAD, Cat#5671085J10) under constant voltage conditions of 200V for 30 minutes. Running Buffer Solution (10×) for SDS-PAGE (Nacalai Tesque, Cat#30329-61), diluted to 1× concentration, was used as the electrophoresis buffer.

[0242] 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 BIO-RAD Trans-Blot Turbo Transfer System Standard protocol (30 minutes) was used for the transfer apparatus. After transfer, the membrane was washed with TBST. The TBST composition was 1× concentration Tris-buffered saline (pH 7.4) (Nacalai Tesque, Cat#35438-81) containing 0.06% polyoxyethylene sorbitan monolaurate (Tween-20) (Nacalai Tesque, Cat#28353-85). After washing, blocking was performed by shaking with Blocking One (Nacalai Tesque, Cat#03953-95) or PVDF Blocking Reagent (Toyobo Co., Ltd., Cat#NYPBR01) for 1 hour at room temperature. After blocking, the sample was washed with TBST, diluted primary antibody was added, and the sample was 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 (manufactured by Toyobo Co., Ltd., Cat#NKB-101) ·β-actin Antibody: β-Actin (13E5) Rabbit mAb (HRP conjugate) (Cell Signaling, Cat#5125) Solvent: Blocking One

[0243] After shaking with the primary antibody, the mixture was washed with TBST, and the diluted secondary antibody was added and shaken at room temperature for 1 hour. The secondary antibody and diluent are as follows: RPS25 Antibody: Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody (manufactured by Invitrogen, Cat#A24537) Solvent: Canget Signal Solution 2 (manufactured by Toyobo Co., Ltd., Cat#NKB-101) After secondary antibody shaking, the samples were washed with TBST and detected using ECL prime (Amersham, Cat#RPN2232). An Amersham Imager680 was used for detection and analysis.

[0244] Table 10 shows the expression ratios of human RPS25 protein in each single-stranded antisense oligonucleotide, as determined by the method described above. The expression ratio of human RPS25 protein in the negative control group was set to 1.00. Since the protein expression ratio is below 0.80, it can be concluded that these are single-stranded antisense oligonucleotides capable of regulating the function of the RPS25 gene.

[0245] [Table 10]

[0246] ≪Evaluation of Cytotoxicity of Single-Stranded Antisense Oligonucleotides≫ HeLa-S3 cells, representing human cervical cancer cells, were cultured in growth medium at 37°C and 5% CO2. The growth medium used had the following composition:

[0247] Composition of growth medium used for cytotoxicity evaluation 10% Fetal Bovine Serum (FBS): GIBCO, CAT#10437028 1% Non-essential amino acids (NEAA): Manufactured by GIBCO, Cat#11140050 Dulbecco's Modified Eagle Medium Low Glucose (Contains L-Glutamine and Phenol Red) (Manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Cat#041-29775)

[0248] The day before the experiment, the above cells (1.0 × 10⁶) were placed in a 96-well plate. 4Cells were seeded (cells / well). The seeded cells were cultured overnight at 37°C and 5% CO2. Then, single-chain antisense oligonucleotides (final concentration: 1-200 nM) that had formed a complex with Lipofectamine 3000 (Thermo Fisher Scientific, Cat#L3000-015) were added to Opti-Minimum Essential Medium (Thermo Fisher Scientific, Cat#31985070), and the cells were cultured for 24 hours at 37°C and 5% CO2. Subsequently, Caspase activity and cell viability were evaluated by adding Caspase-Glo 3 / 7 Assay System (Promega, Cat#G8093) or Celltiter-Glo 2.0 Assay (Promega, Cat#G9242) to the growth medium. The cytotoxicity evaluation results (cell viability) for each single-stranded antisense oligonucleotide obtained by the method described above are shown in Tables 11-1 to 11-3.

[0249] [Table 11-1]

[0250] [Table 11-2]

[0251] [Table 11-3]

[0252] <<Evaluation of Serum Stability of Single-Stranded Antisense Oligonucleotides>> To a 4 μL solution of Tris-EDTA buffer (pH=8.0) containing 400 pmol of single-stranded antisense oligonucleotides, mouse serum (20 μL) or human serum (20 μL) is mixed, and then mineral oil (15 μL) is added. After incubating this solution at 37°C, 8 mol / L urea solution (10 μL) is mixed in to inactivate the nucleases in the serum. After adding ultrapure water (10 μL), the solution is separated into an aqueous layer containing single-stranded antisense oligonucleotides and a mineral oil layer by centrifugation. The aqueous layer is analyzed by LC-MS (Waters), and the remaining single-stranded antisense oligonucleotides are calculated from the area intensity of the UV chromatogram obtained. "Remaining oligonucleotides (%)" refers to the percentage of undegraded single-stranded antisense oligonucleotides remaining after 72 hours compared to the undegraded single-stranded antisense oligonucleotides analyzed immediately after mixing with serum.

[0253] A stable single-stranded antisense oligonucleotide is determined to have a survival rate of 50% or more after 72 hours.

[0254] <<In vivo expression evaluation of the RPS25 gene>> The expression of the RPS25 gene was evaluated by administering it intracerebroventricularly to mice and measuring the amount of mRNA in each region of the prefrontal cortex. In this example, gene expression evaluation means evaluating 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.

[0255] FVB mice (Clea Nippon) were anesthetized using isoflurane (Pfizer, Cat#114133403). Next, 10 μL / individual antisense oligonucleotides dissolved in artificial cerebrospinal fluid (Tocris Bioscience, Cat#3525 / 25 mL) were administered to the anesthetized FVB mice using a two-stage needle (TOP, medical device approval number 15800BZZ01460000) attached to a 50 μL Hamilton syringe (Hamilton, Cat#705LT). Negative control mice were administered only artificial cerebrospinal fluid at a dose of 10 μL / individual.

[0256] After a certain rearing period following administration, the FVB mice were euthanized, and tissue samples were collected from three locations: the brain, cervical spinal cord, and lumbar spinal cord. The collected tissues were immersed in RNA later (Applied Biosystems, Cat#AM7024) and left to stand overnight, then stored at -80°C. RNA extraction from the stored tissue samples was performed using the RNeasy Mini Kit (QIAGEN, Cat#74106). Reverse transcription of the extracted mRNA was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cat#4368814). For the reverse transcription, 1 μg of mRNA was diluted in 20 μL and used. Using the complementary DNA (cDNA) obtained from this reverse transcription reaction, real-time PCR was performed using Taqman expression assays (Applied Biosystems) with pre-designed gene-specific probes (see below) (40 cycles of 3 seconds at 95°C and 30 seconds at 60°C).

[0257] List of gene-specific probes used to evaluate mouse RPS25 gene expression mouse Rps25: Mm02342783_g1 mouse GAPDH: 4352339E (Internal Control)

[0258] Table 12 shows the expression ratios of mouse RPS25 mRNA for each single-stranded antisense oligonucleotide, as determined by the method described above. The expression ratio of mouse RPS25 RNA in the negative control group was set to 1.00. Generally, suppression of mRNA expression is thought to suppress subsequent protein translation, etc. Therefore, if the expression ratio is 0.80 or less, it can be determined that the single-stranded antisense oligonucleotide is capable of regulating the function of the mouse RPS25 gene. The target regions to which the single-stranded antisense oligonucleotides listed in Table 12 bind are regions whose sequences are conserved between the human RPS25 gene and the mouse RPS25 gene.

[0259] [Table 12]

[0260] ≪Evaluation of RAN translation inhibitory effect in CAG repeat-expressing neurons≫ CAG repeat-expressing neurons were generated by introducing a lentiviral vector containing the CAG102 repeat into pan-neurons differentiated from healthy iPS cells. Conditions were established to detect RAN translation products using these generated CAG repeat-expressing neurons, and the amount of RAN translation products in the CAG repeat-expressing neurons after each antisense oligonucleotide treatment was measured to evaluate RAN translation inhibitory activity. The evaluation procedure is described below.

[0261] The composition of the culture medium used is described below. (StemFit AK03N): Manufactured by Ajinomoto Co., Ltd. (Neural Induction Medium) Neurobasal Medium (manufactured by ThermoFisher Scientific, Cat#21103-49) Neural Induction Supplement (ThermoFisher Scientific, Cat#A1647801): 1 / 50 of the total amount

[0262] (Neural Expansion Medium) Neurobasal Medium (manufactured by ThermoFisher Scientific, Cat#21103-49) Advanced DMEM (manufactured by ThermoFisher Scientific, Cat#12634) Neural Induction Supplement (ThermoFisher Scientific, Cat#A1647801) 1 / 50

[0263] (NB medium) Neurobasal Medium (manufactured by ThermoFisher Scientific, Cat#21103049) 1 / 50 SM1 Neural Supplement (StemCell, Cat#05711) 1 / 100 N2 Neural Supplement (StemCell, Cat#07152) 10 ng / mL Human BDNF (Peprotech, Cat#450-02) 10 ng / mL Human GDNF (manufactured by R&D Systems, cat#212-GD-050) 100μM Ascorbic Acid (manufactured by Tokyo Kasei Co., Ltd., Cat#A0537) 100 μM N6,2'-O-Dibutyryladenosine-3',5'-cyclic Monophosphate Sodium Salt (Nacalai, Cat#11540-61) 100-fold diluted penicillin-streptomycin mixture (ThermoFisher Scientific, cat#15140-122) 0.1 μM Compound E (Calbiochem, Cat#56790)

[0264] (BP medium) BrainPhys Neuronal Medium (manufactured by StemCell, Cat#05790) 1 / 50 SM1 Neural Supplement (StemCell, Cat#05711) 1 / 100 N2 Neural Supplement (StemCell, Cat#07152) 10 ng / mL Human BDNF (Peprotech, Cat#450-02) 10 ng / mL Human GDNF (manufactured by R&D Systems, cat#212-GD-050) 100μM Ascorbic Acid (manufactured by Tokyo Kasei Co., Ltd., Cat#A0537) 100 μM N6,2'-O-Dibutyryladenosine-3',5'-cyclic Monophosphate Sodium Salt (Nacalai, Cat#11540-61) 100-fold diluted penicillin-streptomycin mixture (ThermoFisher Scientific, cat#15140-122) 0.1 μM Compound E (Calbiochem, Cat#56790)

[0265] <Maintenance of human iPS cells> iMAtrix-511 (Nippi, Cat#892012), diluted 100-fold in PBS, was added to a 6-well plate and the plate was coated. 200,000 cells / plate of iPS cells (201B7 strain, obtained from iPS Academia Japan, AJ-H1-01) thawed in StemFitAK-03N medium (Ajinomoto) containing 10 μM Y-27632 (Tocris, Cat#1254) were seeded into the plate. The medium was replaced with StemFitAK-03N medium without Y-27632 the day after seeding, and thereafter, the medium was changed once every 2-3 days with AK-03 medium without Y-27632. Cell culture was performed by subculturing once a week. Cell subculturing was carried out according to the following procedure. First, cells were detached from plates from which the culture medium had been removed by adding 350 μL / well of Accutase (Funakoshi Co., Ltd., Cat#AT104) and treating at 37°C for 5 minutes. Next, the cells were dispersed using PBS, and after counting the number of cells, they were seeded in StemFitAK03N medium containing 10 μM Y-27632 and 1 / 150 volume iMAtrix to a concentration of 5000 to 15000 cells / well.

[0266] <Induction of pan-neuron differentiation from human iPS cells> The iPS cells were passaged by the above method and seeded in a 6-well plate at 300000 cells / well. The next day after seeding, it was confirmed that the cells were 15 - 25% confluent, and the medium was replaced with PSC Neural Induction Medium. The medium was changed once every two days with PSC Neural Induction Medium. The passage of these cells was performed on the 7th day after the first replacement with PSC Neural Induction Medium. Specifically, first, Geltrex™ hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix was added to a 10-cm petri dish and allowed to stand at 37°C for 1 hour or more to coat it. The medium was removed from the cultured cells, and after washing with PBS, Accutase was added to detach the cells. The detached cells were collected through a cell strainer (Falcon, Cat#352360) and centrifuged at 900 rpm for 4 minutes using himacCF7D2 manufactured by Hitachi, Ltd. After resuspending with PBS, the cell number was counted, and the required number of cells was aliquoted and centrifuged again at 900 rpm for 4 minutes. Neural Expansion Medium containing 5 μM of Y-27632 was added to the precipitated cells, and they were resuspended and seeded at 3 - 6×10 6 cells / dish. The next day after seeding, the medium was changed to Neural Expansion Medium without Y-27632, and cell culture was continued. Cell stocks were prepared at the second or subsequent passages. Specifically, after detaching the cells by the same procedure as passage, the cells were suspended in Bambanker (manufactured by Nippon Genetics Co., Ltd., Cat#CS-04-001) to a concentration of 1×10 7 cells / mL and frozen.

[0267] <Maintenance culture of pan-neuron> Frozen pan-neuron stocks (frozen stocks) were awakened. Specifically, 6-well plates were first coated with geltrex at 37°C for at least 1 hour. Next, Neural Expansion Medium (containing 5 μM Y-27632) warmed to 37°C was added to the frozen stocks to thaw the cells, and the entire volume was seeded into 1 well. The following day, the medium was replaced with Neural Expansion Medium without Y-27632. Three days after thawing the cells, the medium was removed, washed with PBS, and Accutase was added and allowed to stand at room temperature for 1 minute. Accutase was removed, the cells were detached with PBS, and the number of cells was counted. After separating the required number of cells, they were centrifuged at 300 × g for 4 minutes, the supernatant was removed, and 1 × 10⁶ cells were collected. 5 The cells were resuspended in NB medium containing 5 μM Y-27632 to a concentration of cells / mL. After suspension, 100 μL / well of cells were seeded into a 96-well U-type plate (ThermoFisher Scientific, Cat#174929), centrifuged at 1000 rpm for 4 minutes, and then cultured at 37°C under 5% CO2 conditions. Simultaneously with cell seeding, a lentivirus solution containing the CAG102 repeat (5 μL) and an antisense oligonucleotide solution (final concentration 1 μM) were added. Two days after cell seeding, 100 μL / well of BP medium without Y-27632 was added, and thereafter, half of the medium was changed with BP medium without Y-27632 every 2-3 days. On days 7 and 14 of culture, antisense oligonucleotides were added to a final concentration of 1 μM.

[0268] <Method for preparing lentiviruses> A partial HTT gene sequence containing 120 (CAG) repeats + 3x epitope tag (Myc, Flag, V5) (Table 13, SEQ ID NO: 788) was inserted between NheI and SwaI of the pCDH-EF1-MCS plasmid vector (System Biosciences, Cat#CD502A-1-SBI). HEK293T cells were transfected with this plasmid vector and a lentivirus packaging plasmid vector (Invitrogen, Cat#ViraPower Packaging mix K497500) (Takara Bio, Cat#TransIT-293 V2700). Plasmid vector introduction conditions were followed according to the manufacturer's instructions. After culturing the transfected cells for 3 days, the culture supernatant was collected. The lentivirus in the culture supernatant was concentrated using PEG-it (System Biosciences LV825A-1) and suspended in PBS. The prepared lentivirus was used to infect HEK293T, and immunohistochemical staining was used to confirm the production of the target molecule.

[0269] [Table 13]

[0270] <Immunostaining and Observation> On day 23 of cell culture, cells were fixed with 4% paraformaldehyde for immunostaining (incubated at room temperature for 15 minutes). After fixation, the cells were washed with PBS (incubated for 5 minutes three times), and blocking buffer (3% BSA [Sigma, Cat#A9576-50ML], 0.3% Triton X-100 [Nacalai Tesque, Cat#:35501-02] in PBS) was added, and the cells were incubated at room temperature for 1 hour to block them. After blocking, the following primary antibodies diluted with blocking buffer were added to the cells and incubated overnight at 4°C. • Anti-cMyc antibody (Abcom, Cat#ab9106) 1190-fold dilution • Anti-V5 antibody (ThermoFisher Scientific, Cat#R960-25) 1000-fold dilution

[0271] The following day, the cells were washed with PBS-T (3% BSA, 0.3% TritonX-100 in PBS) (after adding the solution and letting it stand for 5 minutes, three times), and then the following secondary antibodies, diluted with blocking buffer, were added and left to stand overnight at 4°C. ·Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus (Invitrogen, Cat#A32766) 1000 times diluted Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Invitrogen, Cat# A32766), 1000x dilution. Hoechst 33342 solution (1 mg / mL) (WAKO code:346-07951)

[0272] The following day, the cells were washed with PBS-T (3% BSA, 0.3% TritonX-100 in PBS) (after 5 minutes of standing, 3 times), and then observed using a confocal quantitative image cytometer CellVoyager CQ1 (Yokogawa Electric Corporation). Peptides produced by normal translation (detected by Myc tag) were compared between the cell group treated with antisense oligonucleotides and the untreated cell group, but there was no difference in the amount of peptides produced. On the other hand, peptides produced by RAN translation (detected by V5 tag) were specifically reduced in the cell group treated with antisense oligonucleotides compared to the untreated cell group.

[0273] As described above, embodiments and examples of the present invention have been explained, but it is also intended from the outset that the configurations of each of the above embodiments and examples may be combined as appropriate.

[0274] The embodiments and examples disclosed herein should be considered in all respects to be illustrative and not restrictive. The scope of the present invention is indicated by the claims rather than the embodiments and examples described above, and all modifications within the scope of the claims are intended to be included in the meaning of equivalents and within the scope.

Claims

1. A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that suppresses the expression and / or function of the RPS25 gene, The single-stranded antisense oligonucleotide is such that each nucleotide is linked by a phosphate group and / or a modified phosphate group. The single-stranded antisense oligonucleotide comprises 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. The gap region is a nucleic acid composed of deoxyribose, which may contain nucleic acids with modified sugar moieties. The 3' wing region and the 5' wing region are modified nucleic acids. The base length of the single-stranded antisense oligonucleotide is 12 to 30 mer. The base sequences of the aforementioned single-stranded antisense oligonucleotides are SEQ ID NOs: 18, 24-25, 28-29, 38, 48-49, 53, 58-59, 63-64, 66-68, 79-80, 84, 86-91, 93-95, 97, 99-105, 113-119, 121-123, 125, 127-130, 140, 162, 169, 171-173, 183, 188, 190, 304 A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, which is one base sequence selected from the group consisting of the base sequences 306, 310, 340, 343, 346, 351, 353, 355, 357-364, 372-374, 376-380, 382, ​​386, 388, 389, 407, 408, 410, 418-424, 426, 427, 431, and 432.

2. The number of bases in the gap region is 5 to 20 mer. The 3' wing region is a modified nucleic acid of 1 to 5 mer. The 5' wing region is a 1-5 mer modified nucleic acid, as described in claim 1, for a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof.

3. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1 or claim 2, wherein the base length of the single-stranded antisense oligonucleotide is 14 to 22 mer.

4. The modified nucleic acid in the 3' wing region comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA. The modified nucleic acid in the 5' wing region comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA, according to any one of claims 1 to 3, a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof.

5. A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein at least one internucleotide bond of the single-stranded antisense oligonucleotide is a phosphorothioate bond.

6. A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein at least one internucleotide bond of the single-stranded antisense oligonucleotide is a phosphodiester bond.

7. The 3' wing region is 2 to 5 meters, The 5' wing region is 2 to 5 mer, the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6.

8. A single-stranded antisense oligonucleotide according to any one of claims 1 to 7, A double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, comprising a second-stranded oligonucleotide hybridized to the single-stranded antisense oligonucleotide, A double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the base sequence of the second-stranded oligonucleotide has a base sequence that exhibits 90% to 100% sequence identity with respect to a base sequence complementary to the base sequence of the single-stranded antisense oligonucleotide.

9. A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, or a double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 8, An antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof, comprising the single-stranded antisense oligonucleotide or an adduct bound to the second-stranded oligonucleotide, The adduct is an antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof, selected from the group consisting of polyethylene glycol, peptides, alkyl chains, ligand compounds, antibodies, proteins, and sugar chains.

10. A pharmaceutical product comprising as an active ingredient a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, a double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 8, or an antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof according to claim 9.

11. An RPS25 gene expression and / or function inhibitor comprising, as an active ingredient, a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, a double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 8, or an antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof according to claim 9.

12. An inhibitor of dipeptide repeat production by RAN translation, comprising as an active ingredient a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, a double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 8, or an antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof according to claim 9.

13. A therapeutic agent for recurrent disease comprising as an active ingredient a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, a double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 8, or an antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof according to claim 9.

14. A preventive agent for recurrent disease, comprising as an active ingredient a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, a double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 8, or an antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof according to claim 9.

15. The therapeutic agent for a repeat disease according to claim 13, or the preventive agent for a repeat disease according to claim 14, 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, bulbar spinal muscular atrophy, Friedreich ataxia, fragile X-associated ataxia tremor syndrome, and myotonic dystrophy.

16. C9orf72 Use of a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, a double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 8, or an antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof according to claim 9, for the manufacture of a therapeutic or prophylactic agent for ALS.

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