Antisense oligonucleotides that modulate expression and / or function of RPS25 gene

Abstract

A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, which modulates the expression and / or function of the RPS25 gene, and which is characterized in that each nucleoside of the single-stranded antisense oligonucleotide is bonded via a phosphate group and / or a modified phosphate group, the single-stranded antisense oligonucleotide contains 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 being a nucleic acid comprising deoxyribose optionally containing a nucleic acid in which a sugar part is modified, and the gap region containing at least one 5 '-CP nucleic acid; the 3 '-wing region and the 5'-wing region are modified nucleic acids having a substituent at the 2 '-position, and the base length of the single-stranded antisense oligonucleotide is 12-30 mer. The base sequence of the single-stranded antisense oligonucleotide is a base sequence having 90%-100% sequence identity with respect to a base sequence complementary to at least one target region comprising the same base length as the single-stranded antisense oligonucleotide in the base sequence depicted in SEQ ID NO: 1. A nucleotide sequence complementary to a nucleotide sequence in which one or more nucleotides have been deleted, substituted, inserted or added in the target region; or a base sequence capable of hybridizing with the oligonucleotide having the target region under strict conditions.

Description

TECHNICAL FIELD

[0001] The present application relates to an antisense oligonucleotide that modulates the expression and / or function of the RPS25 gene, and an expression and / or function modulator of the RPS25 gene comprising the same. BACKGROUND

[0002] The ribosomal protein S25 (RPS25) gene is a gene that encodes one of the constituent proteins of the 40S subunit of the ribosome. The three-dimensional conformation of the constituent protein (RPS25 protein) encoded by the RPS25 gene in the 40S subunit of the ribosome has also been clarified. (Non-Patent Literature 1)

[0003] As part of the process of protein synthesis, the RPS25 protein binds to an RNA element that enables cap-independent translation, and regulates translation. The RNA element to which the RPS25 protein binds is referred to as an internal ribosome entry site (IRES). IRES is one of the cap-independent translation mechanisms that is widely present in viruses, in particular. (Non-Patent Literature 2)

[0004] In 2011, repeat-associated non-ATG translation (RAN translation) was first identified in a patient with spinocerebellar ataxia type 8 (SCA8). RAN translation refers to a mechanism in which a specific sequence is repeated and translated into a protein (DPR, etc.) without dependence on ATG. After that, it was reported that RAN translation is also involved in amyotrophic lateral sclerosis (ALS) with mutations in the C9orf72 gene (hereinafter, sometimes referred to as "C9orf72 ALS"), Huntington's disease, myotonic dystrophy, and various repeat diseases (diseases that develop due to the repetition of a specific gene sequence). The correlation between DPR produced by RAN translation and the disease condition was studied, and in fact, it was reported that the removal of DPR exerts an effect on the improvement of the disease condition. (Non-Patent Literature 4, Non-Patent Literature 5)

[0005] It was reported in 2019 that the RPS25 protein is a molecule that contributes greatly to the generation of dipeptide repeats caused by RAN translation. By knocking down the RPS25 gene, the generation of DPRs derived from GGGGCC repeats, which depend on RAN translation, or DPRs derived from CAG repeats was inhibited. It is known that GGGGCC repeats are abnormal elongation mutations of the C9orf72 gene, which is one of the familial mutations of amyotrophic lateral sclerosis. It is known that CAG repeats are abnormal elongation mutations of the Huntington gene and the ATXN2 gene. It has been clarified that in motor neurons derived from induced pluripotent stem cells (iPSCs) established from patients with mutations in the C9orf72 gene, the generation of DPRs derived from GGGGCC repeats was inhibited by knocking down the RPS25 gene, and motor neuron death was also inhibited. (Non-Patent Literature 6)

[0006] The antisense oligonucleotide against the RPS25 gene disclosed in Non-Patent Literature 6 is a gapmer in which the wing portions are modified with 2'-O-methylated RNA (2'-OMe nucleic acid), and the gap is phosphorothioated. Note that the base sequence of this antisense oligonucleotide includes a part of the base sequence of the sense strand of the RPS25 gene.

[0007] The present inventors and others have conducted intensive research, and as a result, reported a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that binds to the RPS25 gene and effectively regulates the expression of the RPS25 gene. (Patent Literature 7)

[0008] Antisense oligonucleotides commonly referred to as gapmers are compounds in which a gap region composed of deoxyribonucleic acids and modified nucleic acids developed to improve nuclease resistance, improve binding affinity to target RNA, specificity, and the like are arranged in the wing regions, and exhibit an effect by hybridizing to the RNA sequence that is the target to inhibit gene expression. In order to clinically apply gapmers, various modified nucleic acids have been developed, and many compounds (gapmers) to which they are applied have been developed and marketed. It is known that nucleic acid drugs containing gapmers, due to their properties, accumulate in specific organs such as the liver when systemically exposed, and in the research and development process, toxicity issues such as liver toxicity derived from nucleic acid drugs have become clear, and efforts to address the issue (mechanism elucidation, exploration of avoidance methods, etc.) have been made. On the other hand, in the case of local administration to the eye, brain, skin, and the like, the potential toxicity of gapmers to highly exposed sites has not been sufficiently clarified, and in addition, there are still issues such as elucidation of the expression mechanism, development of avoidance methods, and the like.

[0009] Toxicity of antisense oligonucleotides can be classified into so-called "toxicity due to hybridization with non-target RNA (narrow sense off-target toxicity)" and "toxicity not due to hybridization with RNA (broad sense off-target toxicity)", and various methods have been employed in order to avoid these toxicities. In the broad sense off-target toxicity, as described above, a large number of studies have been conducted with toxicity in the liver, which is an accumulation organ, as an index. On the other hand, in recent years, development of antisense oligonucleotides for central nervous system diseases as target diseases based on intrathecal administration has also been actively conducted, and there has been a problem of toxicity to the central nervous system (hereinafter, sometimes referred to as "central nervous toxicity").

[0010] In the relationship between antisense oligonucleotides and central nervous toxicity, it has been known that intracerebral exposure within 1 hour shows central nervous system symptoms such as convulsions, and the correlation between intracellular free calcium concentration in nerve cells and central nervous toxicity has been disclosed. (Patent Literature 8, Non-Patent Literature 7) In addition, as a document evaluating central nervous toxicity caused by phosphorothioate modification in antisense oligonucleotides, it has been disclosed that central nervous toxicity can be reduced by replacing phosphorothioate bonds disposed in the wing portion with phosphodiester bonds in gapmers having phosphorothioate modification. (Non-Patent Literature 8)

[0011] For central nervous toxicity observed immediately after exposure of antisense oligonucleotides to the central nervous system (hereinafter, sometimes referred to as "acute central nervous toxicity", and also described as "in vivo acute neurotoxicity" in the present specification), it has been shown in Patent Literature 9 that, as a modified nucleic acid, having a substituent at the 5' position carbon atom of the sugar of the nucleoside has an effect of reducing central nervous toxicity. Note that, with respect to central nervous toxicity, in addition to acute central nervous toxicity, central nervous toxicity that occurs after a certain period of time after administration (hereinafter, sometimes referred to as "delayed neurotoxicity", and also described as "in vivo delayed neurotoxicity" in the present specification) is also known, and a decrease in spontaneous movement, abnormal gait and dysfunction of the hind limbs, tremor, weakness of the hind limbs or tail, loss of reflex of the hind limbs, decrease in body weight, and the like are observed on the day after exposure to the central nervous system and thereafter. For this delayed neurotoxicity, within the range known to the inventors, no specific solution has been reported.

[0012] Prior Art Documents

[0013] Patent Literature

[0014] Patent Literature 1: International Publication No. 2019 / 084068

[0015] Patent Literature 2: International Publication No. 2011 / 052436

[0016] Patent Literature 3: International Publication No. 2014 / 046212

[0017] Patent Literature 4: International Publication No. 2015 / 125783

[0018] Patent Literature 5: International Publication No. 2020 / 158910

[0019] Patent Literature 6: International Publication No. 99 / 14226

[0020] Patent Literature 7: International Publication No. 2022 / 097727

[0021] Patent Literature 8: International Publication No. 2016 / 127000

[0022] Patent Literature 9: International Publication No. 2022 / 234855

[0023] Non-Patent Literature

[0024] Non-Patent Literature 1: Science (2011) 331(6018): 730-736

[0025] Non-Patent Literature 2: Genes Dev. (2009) 23(23): 2753-2764

[0026] Non-Patent Literature 3: Proc. Natl. Acad. Sci. USA (2011) 108(1): 260-265

[0027] Non-Patent Literature 4: Neuron (2015) 88: 667-677

[0028] Non-Patent Literature 5: Neuron (2020) 105: 645-662

[0029] Non-Patent Literature 6: Nat. Neurosci. (2019) 22(9): 1383-1388

[0030] Non-Patent Literature 7: Mol. Ther. Nucleic Acids (2023) 31: 182-196

[0031] Non-Patent Literature 8: BioRxiv. doi: https: / / doi.org / 10.1101 / 2021.02.14.431096 (2021)

[0032] Non-Patent Literature 9: Mol. Gen. Genet. (1979) 169: 1-6

[0033] Non-Patent Literature 10: Curr. Opin. Struct. Biol. (2014) 24: 165-169

[0034] Non-patent literature 11: J. Med. Chem. (2016) 59: 9645-9667

[0035] Non-patent literature 12: Nature (2015) 518(7539): 409-412 SUMMARY

[0036] PROBLEMS TO BE SOLVED BY THE INVENTION

[0037] Although it is reported in patent literature 1 that the production of DPR is inhibited by inhibiting the RPS25 gene, it is not clear whether the same effect can be confirmed by a method using a nucleic acid such as an antisense nucleic acid. In addition, the antisense oligonucleotide described in non-patent literature 5 inhibits the expression of the RPS25 gene, but the inhibitory effect thereof is local. We reported in patent literature 7 a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that effectively regulates the expression of the RPS25 gene, but in order to be used as a drug, an antisense oligonucleotide against the RPS25 gene that has reduced toxicity (for example, delayed neurotoxicity) needs to be developed.

[0038] The present invention was completed 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 in which delayed neurotoxicity is reduced, and an expression and / or function regulator of the RPS25 gene containing the same.

[0039] SOLUTION TO THE PROBLEM

[0040] The present inventors and others made intensive studies in order to solve the above problem, and as a result, found that, in a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that binds to the RPS25 gene and effectively regulates the expression of the RPS25 gene (hereinafter, sometimes referred to as "antisense oligonucleotide of the present invention"), by disposing a modified nucleic acid having a cyclopropane ring at the 5' position of the nucleoside sugar to the gap region, delayed neurotoxicity is reduced, and the present invention was completed. That is, the present invention is as described below.

[0041] [1] The antisense oligonucleotide of the first mode of the present invention is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that regulates the expression and / or function of the RPS25 gene,

[0042] each nucleoside of the above single-stranded antisense oligonucleotide is bonded by a phosphate group and / or a modified phosphate group,

[0043] the above single-stranded antisense oligonucleotide contains a gap region, a 3' wing region that binds to the 3' terminal end of the above gap region, and a 5' wing region that binds to the 5' terminal end of the above gap region,

[0044] The gap region is a deoxyribose-constituted nucleic acid optionally containing a sugar moiety-modified nucleic acid,

[0045] The gap region contains at least one 5'-CP nucleic acid,

[0046] The 3' wing region and the 5' wing region are modified nucleic acids having a substituent at the 2' position,

[0047] The single-stranded antisense oligonucleotide has a base length of 12 to 30 mers,

[0048] The base sequence of the single-stranded antisense oligonucleotide is a base sequence as follows:

[0049] A base sequence having 90% or more and 100% or less sequence identity, based on a base sequence complementary to at least one target region constituted by the same base length as the single-stranded antisense oligonucleotide in the base sequence recited in SEQ ID NO: 1;

[0050] A base sequence complementary to a base sequence in which one or several bases are deleted, substituted, inserted, or added in the target region; or

[0051] A base sequence capable of hybridizing to an oligonucleotide having the target region under stringent conditions.

[0052] [2] The antisense oligonucleotide of the second aspect of the present application is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, which is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that modulates the expression and / or function of an RPS25 gene, wherein the single-stranded antisense oligonucleotide of Reference Examples 1 to 31 is excluded,

[0053] The nucleotides of the single-stranded antisense oligonucleotide are bonded by a phosphate group and / or a modified phosphate group,

[0054] The single-stranded antisense oligonucleotide contains 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,

[0055] The gap region is a deoxyribose-constituted nucleic acid optionally containing a sugar moiety-modified nucleic acid,

[0056] The gap region contains at least one 5'-CP nucleic acid,

[0057] The 3' wing region and the 5' wing region are modified nucleic acids having a substituent at the 2' position,

[0058] The single-stranded antisense oligonucleotide has a base length of 12 to 30 mers,

[0059] The base sequence of the above single-stranded antisense oligonucleotide is as follows:

[0060] Based on a base sequence that is complementary to at least one target region consisting of the same base length as the single-stranded antisense oligonucleotide described in sequence number 1, a base sequence having more than 90% and less than 100% sequence identity.

[0061] A base sequence complementary to a base sequence that has one or more bases missing, substituted, inserted, or added in the target region described above; or

[0062] A base sequence that can hybridize with oligonucleotides having the above-mentioned target regions under strict conditions.

[0063] [3] The third-party antisense oligonucleotide of the present invention is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that regulates the expression and / or function of the RPS25 gene, except for the single-stranded antisense oligonucleotides of Reference Examples 1-31 and Design Examples 1-18.

[0064] The nucleosides of the aforementioned single-stranded antisense oligonucleotides are bonded together via phosphate groups and / or modified phosphate groups.

[0065] The aforementioned single-stranded antisense oligonucleotide includes a gap region, a 3' wing region that binds to the 3' end of the gap region, and a 5' wing region that binds to the 5' end of the gap region.

[0066] The aforementioned interstitial regions are nucleic acids composed of deoxyribose, which may contain glycosidic modified nucleic acids.

[0067] The aforementioned interstitial region contains at least one 5'-CP nucleic acid.

[0068] The aforementioned 3' wing region and 5' wing region are modified nucleic acids with substituents at the 2' position.

[0069] The base length of the aforementioned single-stranded antisense oligonucleotides is 12–30 meters.

[0070] The base sequence of the above single-stranded antisense oligonucleotide is as follows:

[0071] Based on a base sequence that is complementary to at least one target region consisting of the same base length as the single-stranded antisense oligonucleotide described in sequence number 1, a base sequence having more than 90% and less than 100% sequence identity.

[0072] A base sequence complementary to a base sequence that has one or more bases missing, substituted, inserted, or added in the target region described above; or

[0073] A base sequence that can hybridize with oligonucleotides having the above-mentioned target regions under strict conditions.

[0074] In the antisense oligonucleotides of the present invention, the modified nucleic acid is disposed in the interstitial region, the 5'-wing region, and the 3'-wing region. The interstitial region contains at least one 5'-CP nucleic acid (5'-cyclopropyl nucleic acid). Regarding the modified nucleic acids with substituents at the 2' position configured in the 5' and 3' wing regions, the non-bridging modified nucleic acids are 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 the bridging modified nucleic acids are 2',4'-BNA / LNA (2',4'-bridged nucleic acid / locked nucleic acid, hereinafter sometimes referred to as "LNA"), AmNA (amide-bridged modified nucleic acid), GuNA (guanidine-bridged modified nucleic acid), and / or scpBNA (2'-O,4'-C-spirocyclopropylene bridged nucleic acid), preferably containing at least one of these modified nucleic acids. By employing this configuration, it is expected that the antisense oligonucleotides of the present invention will exhibit high binding affinity for RPS25 mRNA or mRNA precursors while reducing delayed neurotoxicity.

[0075] Furthermore, the antisense oligonucleotide of the present invention is a so-called interstitial type single-stranded antisense oligonucleotide, and therefore functions as a catalyst in the degradation reaction of the RPS25 gene using RNA-degrading enzymes, as described later. Therefore, it is believed that even small doses can sustainably exert the desired effect.

[0076] [4] In any of the above [1] to [3], the base sequence of the single-stranded antisense oligonucleotide is a base sequence with more than 95% and less than 100% sequence identity based on 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 recorded in Serial No. 1.

[0077] [5] In any of [1] to [4] above, the base sequence of the single-stranded antisense oligonucleotide is a base sequence complementary to at least one target region in the base sequence recorded in Serial No. 1, which consists of the same base length as the single-stranded antisense oligonucleotide.

[0078] [6] In any of the above [1] to [5], the number of bases in the above interstitial region is 5 to 20.

[0079] The aforementioned 3' wing region refers to modified nucleic acids with substituents at the 2' position, which are 3-5 mers.

[0080] The aforementioned 5' wing region is a modified nucleic acid with a substituent at the 2' position in the 3-5mer.

[0081] [7] In any of the above [1] to [6], the above 5'-CP nucleic acid is configured at least in the second position from the 5' side of the above gap region.

[0082] [8] In any of the above [1] to [7], the above 5'-CP nucleic acid is configured in two or more in the above interstitial region.

[0083] [9] In any of the above [1] to [8], the above 5'-CP nucleic acid is configured in 2 to 5 units in the above interstitial region.

[0084]

[10] In any of the above [1] to [7], more than 1 / 9 of the above 5'-CP nucleic acid is configured in the above-mentioned interstitial region.

[0085]

[11] In

[10] above, more than 1 / 5 of the above 5'-CP nucleic acid was configured in the above-mentioned interstitial region.

[0086]

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

[11] , the above 5'-CP nucleic acid is continuously configured with 2 to 4 meters at at least one location in the above interstitial region.

[0087]

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

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

[0088]

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

[13] , the above 5'-CP nucleic acid is disposed on the 5' end side and the 3' end side of the above gap region.

[0089]

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

[14] , the base length of the single-stranded antisense oligonucleotide is 15 to 20 mere.

[0090]

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

[15] , the base length of the above single-stranded antisense oligonucleotide is 18 to 20 mere.

[0091]

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

[16] , the modified nucleic acid with a substituent at the 2' position in the 3' wing region comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA and scpBNA.

[0092] The modified nucleic acid with a substituent at the 2' position in the 5' wing region mentioned above includes at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

[0093]

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

[17] , the modified nucleic acid with a substituent at the 2' position in the above 3' wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA and scpBNA.

[0094] The modified nucleic acid with a substituent at the 2' position in the aforementioned 5' wing region consists of modified nucleic acids selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

[0095]

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

[18] , the modified nucleic acid with a substituent at the 2' position in the above 3' wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA and scpBNA.

[0096] The modified nucleic acid with a substituent at the 2' position in the aforementioned 5' wing region consists of modified nucleic acids selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA.

[0097]

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

[19] , at least one nucleoside bond of the above single-stranded antisense oligonucleotide is a thiophosphate bond.

[0098]

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

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

[0099]

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

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

[0100]

[23] In the above

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

[0101]

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

[23] , in the above interstitial region, the bond between the above 5'-CP nucleic acid and the adjacent nucleoside on the 3' side of the above 5'-CP nucleic acid is a thiophosphate bond, except when the above 5'-CP nucleic acid is located at the 3' end of the interstitial region.

[0102]

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

[24] , in the above interstitial region, the bond between the above 5'-CP nucleic acid and the adjacent nucleoside on the 3' side of the above 5'-CP nucleic acid is a thiophosphate bond, except for the case where the above 5'-CP nucleic acid is located at the 3' end of the interstitial region and the case where the adjacent nucleoside on the 3' side of the above 5'-CP nucleic acid is a 5'-CP nucleic acid.

[0103]

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

[25] , in the above interstitial region, the bond between the above 5'-CP nucleic acid and the adjacent nucleoside on the 5' side of the above 5'-CP nucleic acid is a phosphodiester bond.

[0104]

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

[26] , the base sequence of the above single-stranded antisense oligonucleotide is as follows:

[0105] Based on the base sequence complementary to the target region consisting of 14 to 22 mers consecutively starting from the 5' end at positions 123, 124, 185, 186, 263, 264, 324, 325, 442, 443, or 447 to 453, the base sequence having a sequence identity of more than 90% and less than 100%;

[0106] A base sequence complementary to a base sequence that has one or more bases missing, substituted, inserted, or added in the target region described above; or

[0107] A base sequence that can hybridize with oligonucleotides having the above-mentioned target regions under strict conditions.

[0108]

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

[27] , the base sequence of the above single-stranded antisense oligonucleotide is as follows:

[0109] Based on the base sequence complementary to the target region consisting of 14 to 22 consecutive bases starting from the 324th, 325th, or 448th to 453rd positions from the 5' end, the base sequence having more than 90% and less than 100% sequence identity.

[0110] A base sequence complementary to a base sequence that has one or more bases missing, substituted, inserted, or added in the target region described above; or

[0111] A base sequence that can hybridize with oligonucleotides having the above-mentioned target regions under strict conditions.

[0112]

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

[28] , the base sequence of the single-stranded antisense oligonucleotide is a base sequence that has more than 90% and less than 100% sequence identity with the target region consisting of 18 to 22 consecutive bases starting from the 448th to 453rd position from the 5' end in the base sequence recorded in Serial No. 1.

[0113] The aforementioned 3' wing region is 3–5mer.

[0114] The aforementioned 5' wing region is 3 to 5mer.

[0115]

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

[29] , the base sequence of the single-stranded antisense oligonucleotide is a base sequence that has more than 90% and less than 100% sequence identity with the target region consisting of 18 to 20 meristems of the base sequence recorded in sequence number 1, which is complementary to the base sequence starting from the 450th to 451st or 453rd position from the 5' end.

[0116]

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

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

[0117]

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

[31] , the base sequence of the single-stranded antisense oligonucleotide is one base sequence selected from the group consisting of the base sequences of sequence numbers 6, 9, 12, 15 to 18, 22, 24, 27 to 29, 31 to 36, 38, 40 to 42, 47 and 49 to 54.

[0118]

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

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

[0119]

[34] The antisense oligonucleotide complex of the present invention is an antisense oligonucleotide complex having any one of the above [1] to

[33] , or a pharmaceutically acceptable salt thereof, and an additional substance bound to the above single-stranded antisense oligonucleotide, wherein,

[0120] The above-mentioned additional substances are selected from the group consisting of polyethylene glycol, peptides, alkyl chains, nucleic acids, ligand compounds, antibodies, proteins, and sugar chains.

[0121]

[35] The medicament of the present invention comprises a single-stranded antisense oligonucleotide of any one of [1] to

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

[34] above, or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0122]

[36] In the above

[35] , the above single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the above antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof, is administered in a manner that exposes the central nervous system.

[0123]

[37] In

[35] or

[36] above, the above single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the above antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof, is administered to a subject sensitive to delayed neurotoxicity.

[0124]

[38] The expression and / or function regulator of the RPS25 gene of the present invention comprises a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof of any one of [1] to

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

[34] above as an active ingredient.

[0125]

[39] The inhibitor of dipeptide repeat generation caused by RAN translation of the present invention comprises a single-stranded antisense oligonucleotide of any one of [1] to

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

[34] above or a pharmaceutically acceptable salt thereof as an active ingredient.

[0126]

[40] The therapeutic agent for recurrent diseases of the present invention comprises a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof of any one of [1] to

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

[34] above as an active ingredient.

[0127]

[41] The preventive agent for recurrent diseases of the present invention comprises a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof of any one of [1] to

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

[34] above as an active ingredient.

[0128]

[42] In the therapeutic agent of

[40] and the preventive agent of

[41] above, the aforementioned recurrent disease is selected from at least one of the following groups: C9orf72 ALS, C9orf72 FTLD, Huntington's disease, spinocerebellar ataxia, dentate nucleus-rubra-pallidus hypothalamic atrophy, spinobulbar muscular atrophy, Friedreich ataxia, fragile X-related tremor / ataxia syndrome and myotonic dystrophy.

[0129]

[43] The treatment or prevention method for recurrent diseases of the present invention includes the following steps: administering to an individual a single-stranded antisense oligonucleotide of any one of [1] to

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

[34] above or a pharmaceutically acceptable salt thereof.

[0130]

[44] In the treatment or prevention methods for the recurrent disease described in

[43] above, the recurrent disease is selected from at least one of the following groups: C9orf72 ALS, C9orf72 FTLD, Huntington's disease, spinocerebellar ataxia, dentate nucleus-rubra-pallidus-thalamic atrophy, spinobulbar muscular atrophy, Friedreich ataxia, fragile X-related tremor / ataxia syndrome, and myotonic dystrophy.

[0131]

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

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

[34] above, or a pharmaceutically acceptable salt thereof, for the treatment or prevention of C9orf72 ALS.

[0132]

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

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

[34] above, or a pharmaceutically acceptable salt thereof, for preparing a therapeutic or preventative agent of C9orf72 ALS.

[0133] The effects of the invention

[0134] According to the present invention, a single-stranded antisense oligonucleotide that reduces delayed neurotoxicity and regulates the expression and / or function of the RPS25 gene, and an RPS25 gene expression and / or function regulator comprising the oligonucleotide, may be provided. Attached Figure Description

[0135] Figure 1 This is a schematic diagram illustrating an example of the structure of a single-stranded antisense oligonucleotide of this embodiment.

[0136] Figure 2 This is a schematic diagram illustrating the mechanism by which the expression of the RPS25 gene is suppressed when using the single-stranded antisense oligonucleotide of this embodiment. Detailed Implementation

[0137] Hereinafter, one embodiment of the present invention (hereinafter sometimes referred to as "this embodiment") will be described. However, this embodiment is not limited thereto. In this specification, the expression "I to J" refers to the upper and lower limits of the range (i.e., above I and below J). When no unit is stated in I but only in J, the unit of I is the same as the unit of J.

[0138] Single-stranded antisense oligonucleotides regulating the expression and / or function of the RPS25 gene

[0139] The single-stranded antisense oligonucleotide in this embodiment is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that regulates the expression and / or function of the RPS25 gene.

[0140] The nucleosides of the aforementioned single-stranded antisense oligonucleotides are bonded together via phosphate groups and / or modified phosphate groups.

[0141] The aforementioned single-stranded antisense oligonucleotide includes a gap region, a 3' wing region that binds to the 3' end of the gap region, and a 5' wing region that binds to the 5' end of the gap region.

[0142] The aforementioned interstitial regions are nucleic acids composed of deoxyribose, which may contain glycosidic modified nucleic acids.

[0143] The aforementioned interstitial region contains at least one 5'-CP nucleic acid.

[0144] The aforementioned 3' wing region and 5' wing region are modified nucleic acids with substituents at the 2' position.

[0145] The base length of the aforementioned single-stranded antisense oligonucleotides is 12–30 meters.

[0146] The base sequence of the above single-stranded antisense oligonucleotide is as follows:

[0147] Based on a base sequence that is complementary to at least one target region consisting of the same base length as the single-stranded antisense oligonucleotide described in sequence number 1, a base sequence having more than 90% and less than 100% sequence identity.

[0148] A base sequence complementary to a base sequence that has one or more bases missing, substituted, inserted, or added in the target region described above; or

[0149] A base sequence that can hybridize with oligonucleotides containing the above-mentioned target regions under stringent conditions. This will be explained in detail below.

[0150] <Definitions of terms, etc.>

[0151] First, the following explains the definitions of the terms used in this specification.

[0152] (RPS25 gene)

[0153] In this embodiment, the "RPS25 gene" can be defined by Mol. Gen. Genet. (1979) 169: 1-6 (Non-Patent Literature 9) and Curr. Opin. Struct. Biol. (2014) 24: 165-169 (Non-Patent Literature 10). Synonyms for "RPS25" include 40S ribosomal protein S25, ribosomal protein S25, ribosomal small subunit protein eS25, Rps25, 2810009D21Rik, ribosomal protein s25, ribosomal protein S25, S25, eS25, and ribosomal protein, etc.

[0154] (Single-stranded antisense oligonucleotide)

[0155] In this embodiment, a "single-stranded antisense oligonucleotide" or "antisense oligonucleotide" (hereinafter, sometimes referred to as "ASO") refers to an oligonucleotide or a pharmacologically acceptable salt thereof that is complementary to the mRNA, mRNA precursor, or ncRNA (non-coding RNA) of a target gene (hereinafter, these three are sometimes collectively referred to as "target RNA"). Antisense oligonucleotides are composed of DNA, RNA, and / or analogs thereof. Antisense oligonucleotides inhibit the action of the target mRNA, mRNA precursor, or ncRNA by forming a double strand with the target mRNA, mRNA precursor, or ncRNA. Antisense oligonucleotides include: antisense oligonucleotides having a base sequence that is completely complementary to the base sequence of the target mRNA, mRNA precursor, or ncRNA; antisense oligonucleotides having a base sequence in which one or more bases are deleted, substituted, inserted, or added in the complementary base sequence; and antisense oligonucleotides containing bases that form a wobble base pair in the base sequence.

[0156] Furthermore, the antisense oligonucleotides of the present invention may further include modified nucleotides known in the art, other than the "modified nucleic acids with a modified sugar portion" (sugar-modified modified nucleotides) described later. Examples of modified nucleotides known in the art, besides sugar-modified modified nucleotides, include, for example, phosphate-modified modified nucleotides and nucleic acid base-modified modified nucleotides described later.

[0157] It should be noted that the structure of the two ends of the antisense oligonucleotide in this embodiment is not particularly limited. For example, it can be -OH or -OR (where R represents an alkyl chain, phosphate ester, or additional substances described later).

[0158] Furthermore, the single-stranded antisense oligonucleotide in this embodiment can be in a single-stranded form, or it can hybridize with the second-stranded oligonucleotide described later to form a double-stranded form. Sometimes, a double-stranded oligonucleotide composed of the above-mentioned single-stranded antisense oligonucleotide and a second-stranded oligonucleotide that can hybridize with the above-mentioned single-stranded antisense oligonucleotide is called a "double-stranded antisense oligonucleotide".

[0159] (Oligonucleotides)

[0160] In this embodiment, "oligonucleotide" refers to a polymer of nucleosides consisting of 2 to 30 identical or different nucleosides linked by phosphodiester bonds or other bonds. As shown in the following structural formula, the oligonucleotide can also be understood as being composed of a nucleic acid base portion, a phosphate portion, and a sugar portion.

[0161]

[0162] The aforementioned oligonucleotides are broadly classified into natural oligonucleotides and non-natural oligonucleotides. "Natural oligonucleotides" refer to oligonucleotides composed of naturally occurring nucleotides. "Non-natural oligonucleotides" refer to oligonucleotides containing at least one modified nucleotide (described later) as a structural unit. Preferred examples of "non-natural oligonucleotides" include: modified sugar derivatives with modified sugar moieties; thiophosphate derivatives formed by replacing one sulfur atom with a non-bridging oxygen atom in the phosphodiester bond; dithiophosphate derivatives formed by replacing two sulfur atoms with a non-bridging oxygen atom in the phosphodiester bond; ester derivatives with triesterified phosphodiester bonds; phosphoramide derivatives with amidated phosphodiester bonds; boron phosphate derivatives with borate-enhanced phosphodiester bonds; alkylphosphonates (e.g., methylphosphonate, methoxypropylphosphonate, etc.) derivatives formed by replacing an alkyl atom with a non-bridging oxygen atom in the phosphodiester bond; amide derivatives with amide bonds replacing phosphodiester bonds; and modified base derivatives with modified nucleic acid bases. More preferably, the aforementioned non-natural oligonucleotides may include: bridging modified sugar derivatives with modified sugar portions; thiophosphate derivatives formed by replacing one sulfur atom with a non-bridging oxygen atom of the phosphodiester bond; ester derivatives with esterified phosphodiester bonds; and alkylphosphonate derivatives formed by modifying the sugar portion with the modified sugar (e.g., bridging sugar) described later, and replacing one sulfur atom with a non-bridging oxygen atom of the phosphodiester bond, or replacing one alkyl atom with a non-bridging oxygen atom of the phosphodiester bond.

[0163] (nucleoside)

[0164] In this embodiment, "nucleoside" refers to a compound formed by the combination of a purine or pyrimidine base and a sugar. Sometimes, naturally occurring nucleosides are called "natural nucleosides." Sometimes, non-naturally occurring modified nucleosides are called "modified nucleosides." In particular, modified nucleosides with modified sugar moieties are sometimes called "modified glyconucleosides."

[0165] (nucleotides)

[0166] In this embodiment, "nucleotide" refers to a compound in which a phosphate group is bonded to the sugar of the aforementioned nucleoside. Sometimes, naturally occurring nucleotides are referred to as "natural nucleotides." Sometimes, modified nucleotides that are not naturally occurring are referred to as "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 aforementioned modified nucleoside, compounds in which a modified phosphate group (described later) is bonded to the sugar portion of the aforementioned modified nucleoside, and compounds in which a modified phosphate group (described later) is bonded to the sugar portion of a natural nucleoside.

[0167] (Sugar modification, modified sugar)

[0168] In this embodiment, "sugar modification" refers to the modification of the sugar portion of the aforementioned nucleotide. Sometimes, the modified sugar portion is specifically referred to as "modified sugar." The modified nucleotide that has undergone sugar modification can be used as a modified nucleic acid, and examples include LNA, AmNA, GuNA, scpBNA, 2'-O-alkyl (e.g., 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, etc.), 2'-F, 5'-methyl-DNA, ENA (2'-O,4'-C-ethylene-bridged nucleic acid), S-cEt (2',4'-conformation-restricted ethyl nucleic acid), 5'-CP nucleic acid (5'-cyclopropyl nucleic acid), etc.

[0169] Examples of LNAs that include structures represented by the symbols "A(L)", "5(L)", "G(L)", and "T(L)" (described later) can be listed. Examples of AmNAs that include structures represented by the symbols "A(Y)", "5(Y)", "G(Y)", and "T(Y)" (described later) can be listed. Examples of GuNAs that include structures represented by the symbols "A(Gx)", "5(Gx)", "G(Gx)", and "T(Gx)" (described later) can be listed. Examples of scpBNAs that include structures represented by the symbols "A(S)", "5(S)", "G(S)", and "T(S)" (described later) can be listed. Examples of 2'-MOE nucleic acids that include structures represented by the symbols "A(m)", "5(m)", "G(m)", and "T(m)" (described later) can be listed. Examples of 5'-CP nucleic acids include those with structures represented by the symbols "A(5'-CP)", "5(5'-CP)", "G(5'-CP)", and "T(5'-CP)" (described below). Examples of 2'-OMe nucleic acids include those with structures represented by the symbols "A(M)", "5(M)", "G(M)", "U(M)", and "T(M)" (described below). Examples of MCE nucleic acids include those with structures represented by "A(Mx)", "5(Mx)", "G(Mx)", and "U(Mx)" (described below).

[0170] (Modified nucleic acids with substituents at the 2' position)

[0171] In this embodiment, "a modified nucleic acid having a substituent at the 2' position" refers to a modified nucleic acid having a substituent at the 2' position of the sugar portion of the aforementioned nucleotide. For example, examples of non-bridging modified nucleic acids include 2'-O-alkyl (e.g., 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, MCE nucleic acid, etc.) and 2'-F, while examples of bridging modified nucleic acids include LNA, AmNA, GuNA, scpBNA, ENA, S-cEt, etc.

[0172] (Nucleic acid modifications known in this field, excluding sugar modifications)

[0173] The aforementioned modifications to nucleotides known in the art, excluding sugar modifications, can be used as modified nucleic acids for preparing the single-stranded antisense oligonucleotides of the present invention. Phosphate group modifications and nucleic acid base modifications, described later, are known as nucleotide modifications. Examples of such nucleotide modifications include those described in W. Brad Wan et al., A. J. Med. Chem. (2016) 59: 9645-9667. (Non-Patent Literature 11). These nucleotide modifications can be performed based on methods known in the art as described in the aforementioned documents.

[0174] (phosphate group)

[0175] In this embodiment, "phosphate group" refers to the phosphate group of the nucleotide being bonded by a naturally occurring phosphodiester bond (the bond indicated by the symbol "-" in the following description).

[0176] (Phosphate group modification, modified phosphate group)

[0177] In this embodiment, "phosphate modification" refers to the modification of the phosphate portion of the nucleotide. Sometimes, the modified phosphate portion is specifically referred to as a "modified phosphate group". Examples of bonding methods that include the modified phosphate group include thiophosphate bonds (bonds indicated by the symbol "∧" in the following description), dithiophosphate bonds, phosphoramidate bonds (bonds indicated by the symbol "=" in the following description), or boron phosphate bonds (bonds indicated by the symbol "×" in the following description), alkyl phosphonates, etc.

[0178] (Nucleic acid base modification, modification of nucleic acid bases)

[0179] In this embodiment, "nucleic acid base modification" refers to the modification of the nucleic acid base portion of the aforementioned nucleotide. Sometimes, the modified nucleic acid base portion is specifically referred to as a "modified nucleic acid base." Examples of modified nucleic acid bases include 5-methylcytosine, 5-hydroxymethylcytosine, and 5-propynylcytosine.

[0180] (DNA or RNA analogs)

[0181] The aforementioned 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.

[0182] (ncRNA)

[0183] In this embodiment, "ncRNA" refers to all RNAs that do not participate in protein translation. Examples of ncRNAs include ribosomal RNA, transfer RNA, miRNA, and natural antisense transcripts (NAT).

[0184] (The nucleic acid base portion of an oligonucleotide)

[0185] Examples of nucleic acid bases that can be used as the oligonucleotide bases include thymine, cytosine, adenine, guanine, 5-methylcytosine, uracil, 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl, 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl, and 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl. Preferably, examples of nucleic acid bases include thymine, cytosine, adenine, guanine, 5-methylcytosine, and uracil. Uracil (U) and thymine (T) are interchangeable in these nucleic acid bases. Both uracil (U) and thymine (T) can form base pairs with adenine (A) on the complementary strand. Additionally, cytosine (C) and 5-methylcytosine (5(x)) are interchangeable and can both form base pairs with guanine (G) on the complementary strand. The same applies to the nucleic acid base portion of antisense oligonucleotides.

[0186] (Target RNA)

[0187] In this embodiment, "target RNA" refers to RNA that inhibits the function of the aforementioned single-stranded antisense oligonucleotide. In other words, in this embodiment, target RNA refers to RPS25 mRNA and its precursor. Examples of target RNAs include, for instance, human RPS25 mRNA with the base sequence described in Serial No. 1 (hereinafter sometimes referred to as "hRPS25"), monkey RPS25 mRNA with the base sequence described in Serial No. 2 (hereinafter sometimes referred to as "cRPS25"), mouse RPS25 mRNA with the base sequence described in Serial No. 3 (hereinafter sometimes referred to as "mRPS25"), and rat RPS25 mRNA with the base sequence described in Serial No. 4 (sometimes referred to as "rRPS25").

[0188] (Binding with target RNA)

[0189] In this embodiment, "binding to the target RNA" refers to the formation of a double-stranded nucleic acid by the nucleic acid bases of the single-stranded antisense oligonucleotide and the nucleic acid bases of the target RNA through complementarity. The double-stranded nucleic acid only needs to be formed in at least a portion of the target RNA. It should be noted that the binding strength to the target RNA can be measured, for example, by a thermal stability indicator. Examples of thermal stability indicators include, for instance, the melting temperature (Tm value) of the double-stranded nucleic acid. Preferably, the Tm value is 40–90°C, more preferably 50–70°C.

[0190] (Target area)

[0191] The aforementioned target regions refer to the regions in RPS25 mRNA and its precursor that bind to the aforementioned single-stranded antisense oligonucleotides. These target regions include the target region composed of the indicated base sequence and regions on the RPS25 mRNA precursor.

[0192] (mRNA precursor)

[0193] The aforementioned mRNA precursor refers to the primary transcript of RNA transcribed from DNA. That is, the aforementioned mRNA precursor is RNA containing exon regions, intron regions, and the untranslated region (UTR). The aforementioned mRNA precursor can also be understood as RNA that is transcribed but not yet spliced. If the aforementioned mRNA precursor is spliced, it becomes mRNA.

[0194] (Combination with the target region)

[0195] Binding to the target region refers to the formation of a double helix with the target region by the single-stranded antisense oligonucleotide of the present invention. However, the single-stranded antisense oligonucleotide of the present invention does not necessarily have to form a double helix with the entire target region; it is sufficient to form a double helix with a portion of the target region. That is, the single-stranded antisense oligonucleotide of the present invention preferably has complete complementarity with the target region, but it can also be complementary to at least a portion of the target region as long as it can bind to the target RNA of RPS25.

[0196] (Part of the target area)

[0197] The aforementioned target region refers to a region within the target region that is 10 to 15 nucleotide bases in length.

[0198] (Complementary to at least a portion of the target region)

[0199] Complementarity with at least a portion of the target region means that the bases of at least a portion of the target region on the target RNA are complementary. This also includes base complementarity with regions on the mRNA or mRNA precursor corresponding to at least a portion of the target region.

[0200] <Base sequence of a single-stranded antisense oligonucleotide>

[0201] The preferred base sequence of the single-stranded antisense oligonucleotide in this embodiment is as follows:

[0202] (A) A base sequence that has more than 90% and less than 100% sequence identity with the target region of the base sequence recorded in Serial No. 1, which consists of 14 to 22 mers consecutively starting from the bases at positions 123, 124, 185, 186, 263, 264, 324, 325, 442, 443 or 447 to 453 from the 5' end;

[0203] (B) A base sequence complementary to a base sequence that has one or more bases deleted, substituted, inserted, or added in the target region described above; or

[0204] (C) Base sequences that can hybridize with oligonucleotides having the above target regions under strict conditions.

[0205] Furthermore, in this embodiment, the base sequences shown in the sequence listing are only used to represent the sequence information of the nucleic acid base portion. The structural information of oligonucleotides that include the sugar portion and phosphate portion in addition to the above-mentioned nucleic acid base portion is shown in the format described in Tables 2-1 to 2-4, Table 3-1, Table 3-2 and Table 4 below.

[0206] In this embodiment, "sequence identity" refers to the percentage (%) of identical bases relative to the entire overlapping sequence in the best alignment of two base sequences achieved using a mathematical algorithm known in the art (preferably, the algorithm may consider introducing gaps into one or both sequences to achieve the best alignment). Those skilled in the art can readily confirm the "sequence identity" of a base sequence. For example, it can be done using NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool).

[0207] The base sequence of the single-stranded antisense oligonucleotide in this embodiment preferably has 95% or more and 100% sequence identity with the complementary base sequence of the target region specified above in the base sequence recorded in Serial No. 1, more preferably 98% or more and 100% sequence identity, and even more preferably 100% sequence identity.

[0208] In this embodiment, "a base sequence with one or more bases deleted, substituted, inserted, or added" can be exemplified by base sequences that, through 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 the original base sequence before deletion, substitution, insertion, or addition. The specific number of "one or more bases" can be 1, 2, 3, 4, or 5 instances of deletion, substitution, insertion, or addition, or a combination of several.

[0209] In this embodiment, "strict conditions" refers to incubation for 12 hours at room temperature in a solution containing 6×SSC (1×SSC composition: 0.15M NaCl, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5×Denhardt's reagent, 100 μg / mL of denatured salmon sperm DNA, and 50% (v / v) formamide, followed by washing at 50°C or higher with 0.5×SSC. Furthermore, even stricter conditions are also included, such as incubation for 12 hours at 45°C or 60°C, washing with 0.2×SSC or 0.1×SSC, or washing at 60°C or 65°C or higher during washing.

[0210] In one aspect of this embodiment, the target region is preferably a sequence of 14 to 22 bases from the bases at positions 123, 124, 185, 186, 263, 264, 324, 325, 442, 443, or 447 to 453, as recorded in Serial No. 1.

[0211] In another aspect of this embodiment, the target region is preferably a base sequence consisting of 14 to 22 consecutive bases from the 5' end, starting at positions 324, 325, or 448 to 453.

[0212] In another aspect of this embodiment, the target region is preferably a base sequence consisting of 18 to 22 consecutive bases from the 448th to 453rd position, starting from the 5' end, in the base sequence recorded in Serial No. 1.

[0213] In another aspect of this embodiment, the target region is preferably a base sequence consisting of 18 to 20 consecutive bases from the 450th to 451st or 453rd position, starting from the 5' end.

[0214] The aforementioned single-stranded antisense oligonucleotides can bind to the target region of RPS25. In this specification, the "binding to the target region of RPS25" of the single-stranded antisense oligonucleotides of the present invention includes direct binding of the single-stranded antisense oligonucleotides of the present invention to RPS25 mRNA and direct binding to RPS25 mRNA precursors.

[0215] 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 any of the base sequences listed in Table 1, wherein the single-stranded oligonucleotide is complementary to the target region in the human RPS25 mRNA listed in Table 1. It should be noted that the single-stranded oligonucleotide may extend by 1 to 5 bases on the 3' and / or 5' sides, respectively, as long as it contains the base sequences listed in Table 1. The aforementioned target region can be defined as a region in the human RPS25 mRNA, particularly one related to the regulation of human RPS25 gene expression (e.g., a region with a secondary structure of mRNA that readily binds to antisense nucleotides). For example, in Table 1, when the 5' end position is "451" and the 3' end position is "465", the base sequence from position 451 to position 465 from the 5' end in the sequence recorded in sequence number 1 becomes the target region in the human RPS25 mRNA targeted by the corresponding single-stranded antisense oligonucleotide (sequence name "h451-465").

[0216] [Table 1]

[0217]

[0218] In Table 1 above, the symbols “A’”, “C’”, “G’” and “T’” are selected from natural nucleosides (a, A, c, C, g, G, t and U, as described below) or modified nucleosides (including modified glyconucleotides). It should be noted that, as a modified glyconucleotide, the symbol "A'" is selected from A(M), A(m), A(L), A(Y), A(Gx), A(5'-CP), A(Mx) or A(S) as described below; the symbol "C'" is selected from 5(x), C(M), 5(m), 5(L), 5(Y), 5(Gx), 5(5'-CP), C(Mx) or 5(S) as described below; the symbol "G'" is selected from G(M), G(m), G(L), G(Y), G(Gx), G(5'-CP), G(Mx) or G(S) as described below; and the symbol "T'" is selected from U(M), T(m), T(L), T(Y), T(Gx), T(5'-CP), U(Mx) or T(S) as described below.

[0219] In one aspect of this embodiment, the base sequence of the single-stranded antisense oligonucleotide is preferably one base sequence selected from the group consisting of the base sequences of sequence numbers 5-47 and 49-56.

[0220] In one aspect of this embodiment, the base sequence of the single-stranded antisense oligonucleotide is more preferably one base sequence selected from the group consisting of sequence numbers 6, 9, 12, 15-18, 22, 24, 27-29, 31-36, 38, 40-42, 47 and 49-54.

[0221] In one aspect of this embodiment, the base sequence of the single-stranded antisense oligonucleotide is further preferably one base sequence selected from the group consisting of the base sequences of sequence numbers 6, 9, 12, 15-18, 22, 24, 27-29, 31-32, 34-36, 38, 40-42, 47 and 49-54.

[0222] In one aspect of this embodiment, the base sequence of the single-stranded antisense oligonucleotide is more preferably one base sequence selected from the group consisting of the base sequences of sequence numbers 41, 47, 50 and 53-55.

[0223] <Pharmacologically acceptable salts>

[0224] The single-stranded antisense oligonucleotide of this embodiment can also be in the form of a pharmacologically acceptable salt. Here, "pharmacologically acceptable salt" refers to the salt of the single-stranded antisense oligonucleotide of the present invention, meaning a physiologically acceptable salt of the single-stranded antisense oligonucleotide of the present invention, that is, a salt that maintains the desired biological activity of the single-stranded antisense oligonucleotide without maintaining undesirable toxicological effects. It should be noted that the same applies to the double-stranded antisense oligonucleotide and antisense oligonucleotide complex described later.

[0225] <Pharmaceutical-acceptable salts>

[0226] In one aspect of this embodiment, the aforementioned single-stranded antisense oligonucleotide may also be in the form of a pharmaceutically acceptable salt. Here, "pharmaceutically acceptable salt" refers to the aforementioned pharmacologically acceptable salt, and is an acid addition salt or a base addition salt. Examples of acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate, as well as organic acid salts such as citrate, oxalate, phthalate, fumarate, maleate, succinate, malate, acetate, formate, propionate, benzoate, trifluoroacetate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, and camphorsulfonate. In addition, examples of base addition salts include inorganic base salts such as sodium, potassium, calcium, magnesium, barium, and aluminum salts, as well as organic base salts such as trimethylamine, triethylamine, pyridine, methylpyridine, 2,6-dimethylpyridine, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, dicyclohexylamine, and N,N-dibenzylethylamine. Furthermore, salts (amino acid salts) with basic or acidic amino acids such as arginine, lysine, ornithine, aspartic acid, or glutamic acid can also be included. It should be noted that the same applies to the double-stranded antisense oligonucleotides and antisense oligonucleotide complexes described later.

[0227] <Structure of a single-stranded antisense oligonucleotide>

[0228] The single-stranded antisense oligonucleotide of this embodiment includes a gap region, a 3' wing region that binds to the 3' end of the gap region, and a 5' wing region that binds to the 5' end of the gap region (for example, refer to...). Figure 1 The single-stranded antisense oligonucleotide described above is preferably in a single-stranded form. In one aspect of this embodiment, the single-stranded antisense oligonucleotide can hybridize with the second-stranded oligonucleotide described later to form 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 based on a base sequence complementary to the base sequence of the single-stranded antisense oligonucleotide.

[0229] The aforementioned single-stranded antisense oligonucleotides are so-called space-body type single-stranded antisense oligonucleotides. Space-body type single-stranded antisense oligonucleotides inhibit the function of target RNA through the following mechanism: First, the single-stranded antisense oligonucleotide binds to the target region of the target RNA (from...). Figure 2 (From the upper part to the central part). Then, RNase H, as an RNA-degrading enzyme, recognizes and binds to the complex of this single-stranded antisense oligonucleotide and the target RNA ( Figure 2 The central portion). Then, the target RNA is cleaved and decomposed by the enzymatic reaction of RNase H. At this time, the above-mentioned single-stranded antisense oligonucleotides are not affected by the enzymatic reaction of RNase H ( Figure 2(The lower part). Therefore, the above-mentioned single-stranded antisense oligonucleotides can bind to other target RNAs and cleave and decompose them. In this way, the space body-type single-stranded antisense oligonucleotides function as catalysts in the above-mentioned enzymatic hydrolysis reaction using RNaseH, so it is believed that even small doses can continuously exert the expected effect.

[0230] Furthermore, in this embodiment, the aforementioned single-stranded antisense oligonucleotide can be suitable for regulating the expression of the RPS25 gene through the mechanism described above (including cases where it functions by regulating the maturation of the RPS25 mRNA precursor). Moreover, according to this embodiment, in intrathecal administration, which is a commonly used route of administration in clinical applications, the effect of regulating the expression of the RPS25 gene through the aforementioned single-stranded antisense oligonucleotide can also be achieved. Here, "regulating the expression of the RPS25 gene" at least refers to inhibiting the expression of the RPS25 gene, and consequently, at least refers to inhibiting the function of the RPS25 protein (RAN translation, etc.).

[0231] (Gap area)

[0232] The aforementioned gap region contains at least one 5'-CP nucleic acid. Preferably, the gap region is a 5-20 mer nucleic acid composed of deoxyribose, optionally containing a nucleic acid with a modified glycoside. In other words, the gap region can also be understood as a 5-20 mer nucleic acid containing a deoxyribose with an optionally modified glycoside. Alternatively, the gap region can be understood as being composed of a 5-20 mer natural nucleotide with a deoxyribose glycoside, a non-natural nucleotide, or both. Because the glycoside of the gap region is deoxyribose or modified deoxyribose, it can form a complex that can be recognized by RNase H together with mRNA such as RPS25, which is a target RNA. Here, 5'-CP nucleic acids can be cited as examples of nucleic acids containing modified deoxyribose.

[0233] The number of bases in the aforementioned interstitial region is preferably 5 to 20 mers, more preferably 6 to 17 mers, even more preferably 7 to 13 mers, and even more preferably 9 to 13 mers.

[0234] Examples of natural nucleotides whose sugar portion is deoxyribose include deoxyadenosine monophosphate, deoxyguanosine monophosphate, thymidine monophosphate, deoxycytidine monophosphate, and deoxy-5-methylcytidine monophosphate (also known as 5-methyldeoxycytidine). In other words, examples of natural nucleotides constituting the aforementioned interstitial regions include natural nucleotides containing structural formulas corresponding to the symbols a, g, t, and c described below.

[0235] Non-natural nucleotides whose sugar part is deoxyribose or modified deoxyribose include, for example, 5'-CP nucleic acid, 2-thio-thymidine monophosphate, 2-aminoadenosine monophosphate, 7-dezoguanosine monophosphate, etc.

[0236] It should be noted that, as long as the effects of the present invention are achieved, a portion of the sugar portion of the natural nucleotide in the aforementioned interstitial region, where the sugar portion is deoxyribose, can also be a modified sugar. That is, in one aspect of this embodiment, the aforementioned interstitial region can also be a nucleic acid in which a portion of the sugar portion is deoxyribose and another portion of the sugar portion is a modified sugar (e.g., modified deoxyribose).

[0237] In this embodiment, it is preferable that the 5'-CP nucleic acid is disposed at least in the second position from the 5' side of the aforementioned gap region.

[0238] In this embodiment, the 5'-CP nucleic acid is preferably arranged in two or more locations in the interstitial region, more preferably in two to five locations. Here, the concept of "arranged in two or more locations" includes both arrangements of two or more 5'-CP nucleic acids of the object being consecutively arranged and arrangements of two or more locations being dispersed in the cap region.

[0239] In this embodiment, the 5'-CP nucleic acid is preferably arranged continuously at at least one location in the interstitial region, with 2 to 4 mers.

[0240] In this embodiment, the 5'-CP nucleic acid is preferably disposed on the 5' end side of the gap region. Furthermore, it is preferable that the 5'-CP nucleic acid is disposed on both the 5' end side and the 3' end side of the gap region. Here, "disposed on the 5' end side of the gap region" means disposed closer to the 5' end side than the center of the gap region. "Disposed on the 3' end side of the gap region" means disposed closer to the 3' end side than the center of the gap region.

[0241] In this embodiment, it is preferable to configure at least 1 / 9 of the aforementioned 5'-CP nucleic acid in the aforementioned gap region, and more preferably, at least 1 / 5 of the aforementioned 5'-CP nucleic acid. In this case, the upper limit can be, for example, less than 1 / 2.

[0242] In this embodiment, in the aforementioned interstitial region, the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is preferably a phosphate thioester bond (except when the 5'-CP nucleic acid is located at the 3' end of the interstitial region). Here, "the 5'-CP nucleic acid is located at the 3' end of the interstitial region" can also be understood as "the 5'-CP nucleic acid is bound to the nucleoside in the 3' wing region".

[0243] In one aspect of this embodiment, in the aforementioned interstitial region, the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is preferably a phosphate thioester bond (except in the case where the 5'-CP nucleic acid is disposed at the 3' end of the interstitial region and the case where the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is a 5'-CP nucleic acid).

[0244] In this embodiment, in the aforementioned gap region, the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 5' side of the 5'-CP nucleic acid is preferably a phosphodiester bond.

[0245] (3' wing area)

[0246] The aforementioned 3' wing region is a modified nucleic acid with a substituent at the 2' position. In other words, it can also be understood that the aforementioned 3' wing region is composed of a modified nucleotide with a substituent at the 2' position. Preferably, the modified nucleic acid with a substituent at the 2' position in the aforementioned 3' wing region comprises at least one selected from the group consisting of 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, and MCE nucleic acid as non-bridging 2' position modified nucleic acids, and LNA, AmNA, GuNA, and scpBNA as bridging modified nucleic acids. It is believed that since the aforementioned 3' wing region and the aforementioned 5' wing region described later are composed of the aforementioned modified nucleotides, a high binding affinity to the target RNA can be expected, thereby effectively inhibiting the function of the target RNA. In one aspect of this embodiment, the aforementioned 3' wing region can be a modified nucleic acid with a modified sugar as its sugar portion. Examples of modified nucleic acids with a modified sugar portion include the substances listed above (sugar modification, modified sugar). In one aspect of this embodiment, the aforementioned modified nucleic acid with a substituent at the 2' position in the 3' wing region can be composed solely of 2'-MOE nucleic acid. It should be noted that the modified nucleic acids in the 3' wing region mentioned above can also be multiple in a single single-stranded antisense oligonucleotide.

[0247] In this embodiment, the modified nucleic acid with a substituent at the 2' position in the 3' wing region preferably comprises at least one nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA, and more preferably, it is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA. In another aspect of this embodiment, the modified nucleic acid with a substituent at the 2' position in the 3' wing region is preferably composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA.

[0248] The number of bases in the 3' wing region is preferably 3 to 5 mers, more preferably 3 to 4 mers.

[0249] (5' Wing Area)

[0250] The aforementioned 5'-wing region is a modified nucleic acid with a substituent at the 2' position. In other words, it can also be understood that the aforementioned 5'-wing region is composed of a modified nucleotide with a substituent at the 2' position. Preferably, the modified nucleic acid of the aforementioned 5'-wing region comprises at least one selected from the group consisting of 2'-O-methyl nucleic acids, 2'-MOE nucleic acids, and MCE nucleic acids as non-bridging modified nucleic acids at the 2' position, and LNA, AmNA, GuNA, and scpBNA as bridging modified nucleic acids. In one aspect of this embodiment, the aforementioned 5'-wing region may be a modified nucleic acid with a modified sugar as its sugar moiety. Examples of modified nucleic acids with a modified sugar moiety include the substances listed above (sugar modification, modified sugar). In one aspect of this embodiment, the aforementioned modified nucleic acid with a substituent at the 2' position in the 5'-wing region may be composed solely of 2'-MOE nucleic acids. It should be noted that the modified nucleic acid of the aforementioned 5'-wing region may also contain multiple types within a single-stranded antisense oligonucleotide. In another aspect of this embodiment, the modified nucleic acids in the 3' wing region and the 5' wing region may be composed only of 2'-MOE nucleic acids, or they may be composed only of bridging modified nucleic acids.

[0251] In this embodiment, the modified nucleic acid with a substituent at the 2' position in the 5' wing region preferably comprises at least one nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA, and more preferably, it is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA. In another aspect of this embodiment, the modified nucleic acid with a substituent at the 2' position in the 5' wing region is preferably composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA.

[0252] In one aspect of this embodiment, preferably, the modified nucleic acid having a substituent at the 2' position in the 3' wing region comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

[0253] The modified nucleic acid with a substituent at the 2' position in the 5' wing region mentioned above includes at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

[0254] Furthermore, more preferably, the modified nucleic acid with a substituent at the 2' position in the 3' wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

[0255] The modified nucleic acid with a substituent at the 2' position in the aforementioned 5' wing region consists of modified nucleic acids selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

[0256] In another aspect of this embodiment, more preferably, the modified nucleic acid having a substituent at the 2' position in the 3' wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA.

[0257] The modified nucleic acid with a substituent at the 2' position in the aforementioned 5' wing region consists of modified nucleic acids selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA.

[0258] The number of bases in the 5' wing region is preferably 3 to 5 mers, more preferably 3 to 4 mers.

[0259] In one aspect of this embodiment, it is preferred that the number of bases in the interstitial region is 5 to 20 mers, the number of bases in the 3' wing region is 3 to 5 mers, and the number of bases in the 5' wing region is 3 to 5 mers.

[0260] In one aspect of this embodiment, more preferably, the number of bases in the interstitial region is 7 to 13 mers, the number of bases in the 3' wing region is 3 to 5 mers, and the number of bases in the 5' wing region is 3 to 5 mers.

[0261] In one aspect of this embodiment, it is further preferred that the number of bases in the interstitial region is 9 to 13 mers, the number of bases in the 3' wing region is 3 to 4 mers, and the number of bases in the 5' wing region is 3 to 4 mers.

[0262] (Methods for describing the structure of interstitial bodies)

[0263] The single-stranded antisense oligonucleotide of this invention is of the interstitial form. When describing the structure of the interstitial form, the notation "XYZ" is sometimes used. In the above notation, "X" represents the number of bases in the 5' wing region, "Y" represents the number of bases in the interstitial region, and "Z" represents the number of bases in the 3' wing region.

[0264] As "XYZ", the following can be listed: 2-8-4, 2-8-3, 2-8-5, 2-9-2, 2-9-3, 2-9-4, 2-9-5, 2-10-3, 2-10-4, 2-10-5, 2-11-3, 2-11-4, 2-11-5, 2-12-3, 2-12-4, 2-12-5, 3-8-2, 3-8-3, 3-8-4, 3-8-5, 3-9-3, 3-9-4, 3-9-5, 3-10-3, 3-10-4, 3-10-5, 3-11-3, 3-11-4, 3-11-5, 3-12-3, 3-12-4, 3-12-5, 3 -13-3, 3-13-4, 4-8-2, 4-8-3, 4-8-4, 4-8-5, 4-9-3, 4-9-4, 4-9-5, 4-10-3, 4-10-4, 4-10-5, 4-11-2, 4-11-3, 4-11-4, 4-11-5, 4-12-4, 4-13-3, 5-8-2, 5-8-3, 5-8-4, 5-8-5, 5-9-2, 5-9-3, 5-9-4, 5-9-5, 5-10-2, 5-10-3, 5-10-4, 5-10-5, 5-11-2, 5-11-3, 5-11-4, 5-11-5, etc. For example, when expressed as "2-8-4", it refers to an oligonucleotide with a 2-mer 5' wing region, an 8-mer interstitial region, and a 4-mer 3' wing region.

[0265] The single-stranded antisense oligonucleotide of the present invention has a base length of 12-30 mere, preferably 15-20 mere, and more preferably 18-20 mere. When the base length of the single-stranded antisense oligonucleotide of the present invention is 15-20 mere or 18-20 mere, it has a particularly strong binding to RPS25 mRNA, and can more effectively regulate the expression of the RPS25 gene.

[0266] In this embodiment, the nucleosides of the above-mentioned single-stranded antisense oligonucleotides are bonded by phosphate groups and / or modified phosphate groups, preferably by phosphodiester bonds or thiophosphate bonds.

[0267] In one aspect of this embodiment, it is preferred that at least one nucleoside internucleotide bond in the aforementioned single-stranded antisense oligonucleotide is a phosphate thioester bond. In another aspect of this embodiment, it is preferred that at least one nucleoside internucleotide bond in the aforementioned single-stranded antisense oligonucleotide is a phosphodiester bond.

[0268] In one aspect of this embodiment, the proportion of thiophosphate bonds in the internucleotide bonds constituting the single-stranded antisense oligonucleotide is preferably 50% to 80%, more preferably 50% to 70%.

[0269] One embodiment of the single-stranded antisense oligonucleotide of the present invention is a single-stranded antisense oligonucleotide with a gap region consisting of 5 to 20 meristems, a 5' wing region consisting of 3 to 5 meristems, and a 3' wing region consisting of 3 to 5 meristems. Here, the gap region is located between the 5' wing region and the 3' wing region. The gap region contains at least one 5'-CP nucleic acid. Moreover, it is preferable that the 5' wing region and the 3' wing region each contain at least one 2'-MOE nucleic acid, LNA, AmNA, GuNA, or scpBNA. The 5' wing region and the 3' wing region may contain nucleotides that have been 2'-O-alkylated or 2'-F-substituted. As a 2'-O-alkylated nucleotide, a 2'-O-alkylated (e.g., 2'-O-methylated, etc.) nucleotide of D-furanose can be used. In addition, the above-described gap-shaped single-stranded antisense oligonucleotide can also form a double strand by hybridization with a second-stranded oligonucleotide.

[0270] In one aspect of this embodiment, the single-stranded antisense oligonucleotide is preferably at least one selected from the group consisting of Examples 2, 5, 8, 11-14, 18, 20, 23-25, 27-32, 34, 36-38, 43, and 45-50 in Tables 2-1 to 2-4 described below. Furthermore, the single-stranded antisense oligonucleotide is more preferably at least one selected from the group consisting of Examples 37, 43, 46, and 49-51 in Tables 2-1 to 2-4 described below.

[0271] Double-stranded antisense oligonucleotides

[0272] The double-stranded antisense oligonucleotide in this embodiment is a double-stranded antisense oligonucleotide comprising the above-described single-stranded antisense oligonucleotide and a second-stranded oligonucleotide capable of hybridizing with the above-described single-stranded antisense oligonucleotide, or a pharmaceutically acceptable salt thereof. The base sequence of the above-described second-stranded oligonucleotide is preferably a base sequence having 90% to 100% sequence identity based on a base sequence complementary to the base sequence of the above-described single-stranded antisense oligonucleotide.

[0273] The aforementioned double-stranded antisense oligonucleotide can dissociate in solution to separate into the aforementioned single-stranded antisense oligonucleotide and the aforementioned second-stranded oligonucleotide. The separated single-stranded antisense oligonucleotide can bind to the aforementioned target RNA. It should be noted that, in relation to the aforementioned second-stranded oligonucleotide, the aforementioned single-stranded antisense oligonucleotide can also be understood as a "first-stranded oligonucleotide." It should be noted that, among the oligonucleotides constituting the aforementioned double-stranded antisense oligonucleotide, the aforementioned first-stranded oligonucleotide has an antisense strand targeting the aforementioned target RNA. For convenience, the double-stranded oligonucleotide composed of the aforementioned first-stranded oligonucleotide and the aforementioned second-stranded oligonucleotide is referred to as a "double-stranded antisense oligonucleotide."

[0274] Methods for manufacturing single-stranded antisense oligonucleotides

[0275] The single-stranded antisense oligonucleotides of the present invention can be manufactured by solid-phase synthesis using the phosphoramide method. For example, firstly, single-stranded oligonucleotides having a prescribed base sequence are synthesized on a solid-phase support using a commercially available automated nucleic acid synthesizer. Next, the synthesized single-stranded oligonucleotides are cleaved from the solid-phase support using an alkaline substance, etc., and deprotected to obtain crude single-stranded oligonucleotide products. Then, the obtained crude single-stranded oligonucleotide products are purified using HPLC or the like. Not limited to the above manufacturing method, the single-stranded antisense oligonucleotides of the present invention can be manufactured by appropriately modifying the base sequence of the nucleic acid, modification sites, etc., according to methods known to those skilled in the art. Furthermore, AmNA, GuNA, and scpBNA can be manufactured 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. For 2'-MOE nucleic acids, they can be manufactured using phosphoramide (amidite), which is available as a reagent. 5'-CP nucleic acid can be manufactured by the method described in International Patent Publication No. 2020 / 158910 (Patent Document 5). For LNA, it can be manufactured by the method described in International Patent Publication No. 99 / 14226 (Patent Document 6).

[0276] Methods for manufacturing double-stranded antisense oligonucleotides

[0277] Regarding the double-stranded antisense oligonucleotide of the present invention, firstly, an oligonucleotide (second-stranded oligonucleotide) with a predetermined sequence identity based on a base sequence complementary to the single-stranded antisense oligonucleotide described above is manufactured using the same manufacturing method as described above. Then, the single-stranded antisense oligonucleotide and the second-stranded oligonucleotide are hybridized, thereby enabling the manufacturing process.

[0278] Antisense oligonucleotide complex

[0279] The antisense oligonucleotide complex of this embodiment comprises: the aforementioned single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the aforementioned double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof; and an additional substance bound to the aforementioned single-stranded antisense oligonucleotide or the aforementioned second-stranded oligonucleotide. The aforementioned additional substance is selected from the group consisting of polyethylene glycol, peptides, alkyl chains (e.g., saturated aliphatic hydrocarbons), nucleic acids, ligand compounds, antibodies, proteins, and glycans (e.g., carbohydrates, polysaccharides).

[0280] In one aspect of this embodiment, the antisense oligonucleotide complex comprises: the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof; and an additional substance bound to the single-stranded antisense oligonucleotide, the additional substance being selected from the group consisting of polyethylene glycol, peptides, alkyl chains (e.g., saturated aliphatic hydrocarbons), nucleic acids, ligand compounds, antibodies, proteins, and glycans (e.g., carbohydrates, polysaccharides).

[0281] In another aspect of this embodiment, the antisense oligonucleotide complex comprises: the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof; and an additional substance bound to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide, wherein the additional substance is selected from the group consisting of polyethylene glycol, peptides, alkyl chains (e.g., saturated aliphatic hydrocarbons), nucleic acids, ligand compounds, antibodies, proteins, and glycans (e.g., carbohydrates, polysaccharides).

[0282] In this embodiment, the term "additional substance" refers to a substance that binds to the aforementioned single-stranded antisense oligonucleotide or the aforementioned second-stranded oligonucleotide, and is used to impart a specific function. The additional substance may bind to the 5' end, the 3' end, or both of the 5' and 3' ends of the single-stranded antisense oligonucleotide. Similarly, the additional substance may bind to the 5' end, the 3' end, or both of the 5' and 3' ends of the second-stranded oligonucleotide. In one aspect of this embodiment, it is preferable that the additional substance binds to either the 5' end or the 3' end of the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide. Alternatively, the additional substance may be directly covalently bonded to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide. The additional substance may also bind to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide via a linker substance. Examples of linker substances include linkers composed of alkyl groups, polyethylene glycol, peptides, disulfides, nucleic acids, and / or combinations thereof. Methods for binding the above-mentioned additional substance to the above-mentioned single-stranded antisense oligonucleotide or the above-mentioned second-stranded oligonucleotide can be exemplified by the methods described in the examples described later.

[0283] Peptides used as the aforementioned additives include, but are not limited to, peptides such as, the following: CPPs (Cell Penetrating Peptides), nuclear-targeting peptides, TAT (trans-transcription activator), polyarginine, glucagon-like peptide-1 analogs, synthetic cyclic RGD peptides, and brain-targeting peptides.

[0284] Examples of ligand compounds that can be used as the above-mentioned additives 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 (indatriline, etc.).

[0285] Antibodies used as the above-mentioned additives include, but are not limited to, the following: anti-insulin receptor antibodies, anti-transferrin receptor antibodies, anti-LDL receptor-associated protein antibodies, anti-CD22 antibodies, anti-CD30 antibodies, and anti-HER2 antibodies.

[0286] Proteins used as the aforementioned additional substances include, but are not limited to, the following proteins: albumin.

[0287] Nucleic acids used as the aforementioned additional substances include, but are not limited to, the following: natural nucleotides and aptamers.

[0288] It should be noted that the nucleic acids used as the above-mentioned additional substances are not included in the base length of the aforementioned antisense oligonucleotides.

[0289] Regulators of RPS25 gene expression

[0290] The RPS25 gene expression regulator of this embodiment comprises the single-stranded antisense oligonucleotide of the present invention, the aforementioned double-stranded antisense oligonucleotide, or the aforementioned antisense oligonucleotide complex as an active ingredient. In one aspect of this embodiment, the aforementioned expression regulator can also be understood as an inhibitor of RPS25 gene expression. In another aspect of this embodiment, the aforementioned expression regulator can also be understood as an inhibitor of RAN translation. In another aspect of this embodiment, the aforementioned expression regulator can also be understood as an inhibitor of dipeptide repeat expression achieved by inhibiting RAN translation. The single-stranded antisense oligonucleotide of the present invention inhibits the expression of the RPS25 gene by binding to RPS25 mRNA or its precursor, thereby inhibiting RAN translation caused by its translation product. The administration method and formulation of the RPS25 gene expression regulator of the present invention can be any administration method and formulation known in the art.

[0291] Pharmaceutical compositions containing single-stranded antisense oligonucleotides as active ingredients

[0292] The pharmaceutical composition of this embodiment comprises the single-stranded antisense oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof, the above-described double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the above-described antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof as an active ingredient. The administration method and formulation of the pharmaceutical composition of this embodiment can be used as long as they are administration methods and formulations known in the art. Hereinafter, the above-described pharmaceutical composition will sometimes be described as "antisense oligonucleotide and other pharmaceutical compositions".

[0293] The above-described pharmaceutical composition is used to treat or prevent diseases related to the RPS25 gene, namely diseases that may be caused by dipeptide duplications generated through RAN translation. In other words, the above-described pharmaceutical composition can be used to treat or prevent diseases in which symptom improvement can be expected by inhibiting the expression of the RPS25 gene. Such diseases are sometimes referred to as "duplicated diseases". Specific examples of duplicated diseases include various neuropsychiatric and muscular diseases such as C9orf72 ALS, C9orf72 FTLD, Huntington's disease, spinocerebellar ataxia (types 1, 2, 3, 6, 7, 8, 12, and 17), hypothalamic atrophy of the dentate nucleus, globus pallidus, and hypothalamus, spinobulbar muscular atrophy, Friedreich ataxia, fragile X-related tremor / ataxia syndrome, and myotonic dystrophy.

[0294] Treatments and preventative agents for recurrent diseases

[0295] The therapeutic agent for recurrent diseases in 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 preventive agent for recurrent diseases in 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 recurrent diseases are preferably selected from at least one of the following groups: C9orf72 ALS, C9orf72 FTLD, Huntington's disease, spinocerebellar ataxia, dentate nucleus-rubra-pallidus-thalamic atrophy, spinobulbar muscular atrophy, Friedreich-type ataxia, fragile X-related tremor / ataxia syndrome, and myotonic dystrophy.

[0296] <C9orf72 ALS>

[0297] The aforementioned C9orf72 ALS refers to ALS characterized by an abnormal repetitive 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 frequent causative gene for ALS, accounting for approximately 6% of sporadic ALS and approximately 40% of familial ALS. ALS is a neurodegenerative disease caused by selective death of motor neurons, leading to muscle atrophy. ALS is diagnosed by combining clinical or electrophysiological features of upper / lower motor neuron damage. Specifically, in the four regions of the brainstem, cervical spinal cord, thoracic spinal cord, and lumbosacral spinal cord, if signs of upper / lower motor neuron damage are located in one region and electromyography signs are confirmed in other regions, the diagnosis is laboratory-supported probable; if located in two regions, the diagnosis is probable; and if located in three regions, the diagnosis is definite.

[0298] <C9orf72 FTLD>

[0299] The aforementioned C9orf72 FTLD refers to FTLD with an abnormal repetitive extension of the GGGGCC sequence in the intron region between exon 1a and exon 1b of the C9orf72 gene. In addition to progressive abnormal behavior and cognitive impairment that impairs daily life, this type of FTLD is characterized by at least three of the following symptoms: disinhibition, apathy / lack of energy, rigid / stereotypical behavior, hyperlipsies, and changes in eating habits. Imaging studies reveal atrophy or decreased metabolism and blood flow in the prefrontal and anterior lateral temporal lobes, leading to a diagnosis of behavioral variant FTLD after ruling out specific diseases. Furthermore, in addition to object naming difficulties and word comprehension difficulties, this type of FTLD also presents with object recognition difficulties or superficial alexia / agraphia, with atrophy observed in the dominant hemisphere's lateral temporal lobe, leading to a diagnosis of semantic dementia-type FTLD after ruling out specific diseases.

[0300] Huntington's Disease

[0301] Huntington's disease, as described above, is a hereditary neurodegenerative disease characterized by an abnormal duplication and extension of the CAG sequence in exon 1 of the Huntington's gene. It is inherited in an autosomal dominant pattern. Huntington's disease presents with motor disorders characterized by involuntary movements, psychiatric symptoms, and cognitive symptoms. A diagnosis of Huntington's disease is made upon confirmation of specific neurological signs and the identification of an abnormal elongation mutation in the CAG sequence during genetic diagnosis, or upon confirmation of a family history of an autosomal dominant gene indicating a progressive course, specific neurological signs and clinical examination results, and exclusion of similar diseases in differential diagnosis.

[0302] Spinocerebellar ataxia (types 1, 2, 3, 6, 7, 8, 12, and 17) and atrophy of the dentate nucleus, rubra, globus pallidus, and hypothalamus.

[0303] The aforementioned spinocerebellar ataxia (types 1, 2, 3, 6, 7, 8, 12, and 17) and dentate nucleus-rubra-pallidus-thalamic atrophy refer to hereditary neurodegenerative diseases characterized by an autosomal dominant inheritance pattern, resulting from the abnormal repetition and extension of specific 3-base sequences (CAG or CTG) present on the responsible genes in each disease. Repetitive CAG sequences have been identified in spinocerebellar ataxia types 1, 2, 3, 6, 7, 12, and 17, as well as dentate nucleus-rubra-pallidus-thalamic atrophy. Repetitive CTG sequences have been identified in spinocerebellar ataxia type 8. Both spinocerebellar ataxia and dentate nucleus-rubra-pallidus-thalamic atrophy are characterized by cerebellar or posterior column ataxia or spastic paraplegia as the main symptoms, exhibiting a slow progression. Diagnosis is based on a combination of genetic and neuropathological diagnostic methods.

[0304] Spinal bulbar muscular atrophy

[0305] The aforementioned spinal bulbar muscular atrophy (SMA) is a hereditary disease caused by abnormal repetitive extension of the CAG sequence in the exon region of the androgen receptor gene. SMA is diagnosed through a combination of neurological signs (medullar symptoms, lower motor neuron signs, finger tremors, and diminished tendon reflexes in the limbs), clinical signs / examination results, and genetic diagnosis.

[0306] Friedreich type ataxia

[0307] Friedreich ataxia, as described above, is an autosomal recessive inherited neurodegenerative disease caused by mutations in the ataminin gene. Most cases of Friedreich ataxia are due to abnormal repetitive extension of the GAA sequence located in the first intron.

[0308] <Fragile X-related tremor / ataxia syndrome>

[0309] Fragile X-related tremor / ataxia syndrome is a hereditary neurodegenerative disease caused by abnormal repetition and extension of the CGG sequence in the 5'UTR of the FMP1 gene. It is diagnosed through a combination of clinical symptoms (cerebellar ataxia, action tremor, Parkinson's syndrome, dementia, intellectual disability), the cerebellar middle peduncle sign based on MRI, and genetic diagnosis.

[0310] Myotonic dystrophy

[0311] The aforementioned myotonic dystrophy refers to a hereditary muscle disease that occurs due to an abnormal duplication and extension of the CUG sequence in the 3'UTR of the DMPK gene, and is inherited in an autosomal dominant pattern.

[0312] <Individual>

[0313] The individuals mentioned above refer to mammals. Preferably, the individuals are humans, monkeys, marmosets, dogs, pigs, rabbits, guinea pigs, rats, and mice. Humans are more preferred.

[0314] In one aspect of this embodiment, the aforementioned single-chain antisense oligonucleotide or pharmaceutical composition thereof can be administered to individuals sensitive to delayed neurotoxicity via a suitable route of administration. Here, "delayed neurotoxicity" refers to central nervous system toxicity that occurs after recovery from the period when acute central nervous system toxicity may have occurred. Symptoms caused by delayed neurotoxicity include, for example, decreased spontaneous movement, gait abnormalities and hind limb dysfunction, tremors, weakness in the hind limbs or tail, loss of hind limb reflexes, and weight loss. Regarding this toxicity, the general condition observed on the observation day is scored according to the criteria shown below, and the scores are accumulated throughout the observation period to calculate the clinical sign score. Since the intensity of the toxicity is accumulated into the score, single-chain antisense oligonucleotides with low clinical sign scores can be considered to have high safety. Furthermore, in the pathological examination of the brain, a pathological score is calculated by scoring cases where abnormalities are confirmed and cases where no abnormalities are confirmed. The average value of each score is used for each group. In addition, "subjects (individuals) sensitive to delayed neurotoxicity" refers to subjects screened using biomarkers, etc.

[0315] Clinical signs and symptoms score;

[0316] 0 points: No abnormalities

[0317] 1 point: Hind limb dysfunction, tremor, reduced spontaneous movement

[0318] 2 points: Dragging of hind limbs, weakness in the tail or hind limbs

[0319] 3 points: Complete paralysis of the hind limbs, hind limb paralysis, lying horizontally, lying prone

[0320] 4 points: Euthanasia

[0321] Pathological score;

[0322] 0 points: No abnormalities

[0323] 1 point: Abnormalities are present (single-cell necrosis, vacuolization, etc.)

[0324] It is known that nucleic acid drugs can hybridize with RNAs (off-target candidate genes or off-target candidate RNAs) having the same or similar base sequences as the target RNA, potentially exhibiting toxicity (sometimes referred to as "narrow-sense off-target toxicity") due to their influence on the off-target candidate gene. To avoid unintended narrow-sense off-target toxicity, it is important to design the target RNA in advance in regions that do not hybridize with non-target RNAs. In one aspect of this embodiment, as a method for evaluating the effects of the aforementioned single-stranded antisense oligonucleotides or their pharmaceutical compositions on off-target candidate genes, a search for similar base sequences can be performed using a "high-speed base sequence search: GGGenome (https: / / gggenome.dbcls.jp / ja / )" to predict narrow-sense off-target candidate genes. When there are few off-target candidate genes, the risk of narrow-sense off-target toxicity can be determined to be low.

[0325] When administering the single-chain antisense oligonucleotide or its pharmaceutical composition (including therapeutic and preventative agents against C9orf72 ALS) of the present invention, there are no particular limitations on the method of administration and dosage form. That is, any administration method and formulation known in the art can be used as an administration method and formulation for the antisense oligonucleotide, etc., of the present invention. Examples of administration methods include oral administration and non-oral administration. Examples of non-oral administration include ophthalmic drops, vaginal administration, rectal administration, nasal administration, percutaneous administration, intravenous injection, intravenous infusion, subcutaneous injection, intraperitoneal or intramuscular injection, pulmonary administration based on aspiration or inhalation, intrathecal administration, intraventricular administration, etc. In one aspect of this embodiment, it is preferable to administer the above-mentioned single-chain antisense oligonucleotide or its pharmaceutical composition by means of exposure to the central nervous system. Examples of administration methods involving exposure to the central nervous system include intrathecal administration and intraventricular administration.

[0326] In the formulations of antisense oligonucleotides of the present invention, various pharmaceutical additives such as excipients, binders, wetting agents, disintegrants, lubricants, diluents, flavoring agents, fragrances, solubilizers, suspending agents, emulsifiers, stabilizers, preservatives, and isotonic agents can be mixed as needed.

[0327] When administering the antisense oligonucleotide and other pharmaceutical compositions of the present invention topically, preparations such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders can be used.

[0328] When administering the antisense oligonucleotide and other pharmaceutical compositions of the present invention orally, formulations such as powders, granules, suspensions or solutions dissolved in water or non-aqueous media, capsules, powders, tablets, etc., can be used.

[0329] When administering the antisense oligonucleotide and other pharmaceutical compositions of the present invention to non-oral, intrathecal, or intraventricular administration, a formulation such as a sterile aqueous solution may be used.

[0330] The effective dosage of the single-stranded antisense oligonucleotide of the present invention can be arbitrarily determined according to the individual's sex, age, weight, symptoms, etc. Furthermore, it can also be arbitrarily determined according to the method, route, frequency, etc. of administration. For example, dosages of 0.01 to 100 mg / kg can be listed. Preferably, it is 0.1 to 50 mg / kg, more preferably 0.1 to 10 mg / kg.

[0331] Methods for regulating RPS25 gene expression

[0332] The method for regulating the expression of the RPS25 gene in this embodiment includes the following steps: administering the above-mentioned single-stranded antisense oligonucleotide or its pharmaceutically acceptable salt, or the above-mentioned antisense oligonucleotide complex or its pharmaceutically acceptable salt, as an active ingredient to cells, tissues, or individuals expressing the above-mentioned RPS25 gene.

[0333] In this embodiment, the method of administering the above-mentioned single-stranded antisense oligonucleotides to cells, tissues, or individuals can be performed in vitro or in vivo. In the case of in vivo administration, the administration route described above is used.

[0334] In this embodiment, "cells expressing the RPS25 gene" can include, for example, nerve cells that constitute the central nervous system, nerve cells that constitute the peripheral nervous system, and other cells that constitute skin tissue.

[0335] The treatment or prevention method for recurrent disease in this embodiment includes the following steps: administering the above-mentioned single-stranded antisense oligonucleotide or its pharmaceutically acceptable salt, or the above-mentioned antisense oligonucleotide complex or its pharmaceutically acceptable salt, as an active ingredient to an individual suffering from the above-mentioned recurrent disease. The aforementioned individual is preferably a subject (individual) sensitive to delayed neurotoxicity.

[0336] As the above-mentioned repetitive diseases, the above-mentioned mental and neurological diseases, muscle diseases, etc. can be cited. When administering to an individual, the dosage form, administration route, and dosage can be appropriately adopted from the above-mentioned dosage form, administration route, and dosage.

[0337] Above, the antisense oligonucleotides of the present embodiment have been described. The above-mentioned single-stranded antisense oligonucleotide having the above-mentioned structure can regulate the expression of the RPS25 gene. Here, the inhibitory activity (knockdown activity) against the expression of the RPS25 gene can be measured by a known method. As a method for measuring the knockdown activity, for example, the methods described in Nature (2015) 518(7539): 409-12 (Non-Patent Document 12), International Publication No. 2022 / 097727 (Patent Document 7), etc. can be cited. In addition, it can also be measured by transfecting the antisense oligonucleotide into HEK293T cells described later. In addition, since the above-mentioned single-stranded antisense oligonucleotide contains at least one 5'-CP nucleic acid in the gap region, the delayed neurotoxicity is reduced.

[0338] 《Evaluation Method for Inhibitory Activity against RPS25 Gene Expression》

[0339] <Introduction of Antisense Oligonucleotide into Cells>

[0340] The "cells expressing the RPS25 gene" are treated with an antisense oligonucleotide for 6 hours to 3 days by methods such as lipofection, electroporation, or introduction based on direct addition. The cells used only need to be cells expressing the RPS25 gene. For example, HEK293T cells can be cited, more preferably nerve cells, and further preferably human-derived nerve cells. The cells treated with the antisense oligonucleotide can be recovered immediately after the treatment, or the antisense oligonucleotide can be removed and the cells can be continued to be cultured.

[0341] <Evaluation of RPS25 mRNA Amount>

[0342] A reverse transcription reaction is performed on the total RNA extracted from the recovered cells, and real-time PCR or the like is performed on the obtained complementary DNA using an RPS25 gene-specific probe, whereby the amount of RPS25 mRNA is measured. As a probe for real-time PCR, for example, a Taqman probe can be cited. As a reaction method, for example, a method of repeating any number of times the three steps of "(denaturation of cDNA)-(annealing)-(elongation reaction)" or the two steps of "(denaturation of cDNA)-(annealing and elongation reaction)" can be cited. The number of repetitions of the two or three steps is, for example, 25 to 45 times, preferably 35 to 40 times. The temperature of (denaturation of cDNA) is, for example, 90°C to 98°C, preferably 92°C to 95°C. The temperature of (annealing) is, for example, 40°C to 70°C, preferably 50°C to 60°C. The temperature of (elongation reaction) is, for example, 65°C to 75°C, preferably the optimal temperature of the polymerase used in the reaction. The temperature of (annealing and elongation reaction) is, for example, 55°C to 70°C.

[0343] <Evaluation of the amount of RPS25 protein>

[0344] The recovered cells are lysed to obtain an extract. The amount of RPS25 protein contained in the above extract is evaluated by an immunochemical method such as Western blotting, ELISA (Enzyme-Linked Immuno Sorbent Assay), etc. 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 set arbitrarily, for example, overnight at 4°C or for 1 to 3 hours at room temperature.

[0345] It should be noted that the present invention is not limited to the above embodiments. For example, the above single-stranded antisense oligonucleotides include the following embodiments.

[0346] The antisense oligonucleotide of the first aspect of the present invention is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that regulates the expression and / or function of the RPS25 gene,

[0347] The respective nucleotides of the above single-stranded antisense oligonucleotide are bonded through a phosphate group and / or a modified phosphate group,

[0348] The above single-stranded antisense oligonucleotide includes a gap region, a 3' wing region that binds to the 3' end of the gap region, and a 5' wing region that binds to the 5' end of the gap region,

[0349] The above gap region is a nucleic acid composed of deoxyribose that optionally contains a nucleic acid with a modified sugar moiety,

[0350] The above gap region contains at least one 5'-CP nucleic acid,

[0351] The aforementioned 3' wing region and 5' wing region are modified nucleic acids with substituents at the 2' position.

[0352] The base length of the aforementioned single-stranded antisense oligonucleotides is 12–30 meters.

[0353] The base sequence of the above single-stranded antisense oligonucleotide is as follows:

[0354] Based on a base sequence that is complementary to at least one target region consisting of the same base length as the single-stranded antisense oligonucleotide described in sequence number 1, a base sequence having more than 90% and less than 100% sequence identity.

[0355] A base sequence complementary to a base sequence that has one or more bases missing, substituted, inserted, or added in the target region described above; or

[0356] A base sequence that can hybridize with oligonucleotides having the above-mentioned target regions under strict conditions.

[0357] The antisense oligonucleotide of the second aspect of the present invention is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that regulates the expression and / or function of the RPS25 gene, except for the single-stranded antisense oligonucleotides of Reference Examples 1-31.

[0358] The nucleosides of the aforementioned single-stranded antisense oligonucleotides are bonded together via phosphate groups and / or modified phosphate groups.

[0359] The aforementioned single-stranded antisense oligonucleotide includes a gap region, a 3' wing region that binds to the 3' end of the gap region, and a 5' wing region that binds to the 5' end of the gap region.

[0360] The aforementioned interstitial regions are nucleic acids composed of deoxyribose, which may contain glycosidic modified nucleic acids.

[0361] The aforementioned interstitial region contains at least one 5'-CP nucleic acid.

[0362] The aforementioned 3' wing region and 5' wing region are modified nucleic acids with substituents at the 2' position.

[0363] The base length of the aforementioned single-stranded antisense oligonucleotides is 12–30 meters.

[0364] The base sequence of the above single-stranded antisense oligonucleotide is as follows:

[0365] Based on a base sequence that is complementary to at least one target region consisting of the same base length as the single-stranded antisense oligonucleotide described in sequence number 1, a base sequence having more than 90% and less than 100% sequence identity.

[0366] A base sequence complementary to a base sequence that has one or more bases missing, substituted, inserted, or added in the target region described above; or

[0367] A base sequence that can hybridize with oligonucleotides having the above-mentioned target regions under strict conditions.

[0368] The third-party antisense oligonucleotide of the present invention is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that regulates the expression and / or function of the RPS25 gene, except for the single-stranded antisense oligonucleotides of Reference Examples 1-31 and Design Examples 1-18.

[0369] The nucleosides of the aforementioned single-stranded antisense oligonucleotides are bonded together via phosphate groups and / or modified phosphate groups.

[0370] The aforementioned single-stranded antisense oligonucleotide includes a gap region, a 3' wing region that binds to the 3' end of the gap region, and a 5' wing region that binds to the 5' end of the gap region.

[0371] The aforementioned interstitial regions are nucleic acids composed of deoxyribose, which may contain glycosidic modified nucleic acids.

[0372] The aforementioned interstitial region contains at least one 5'-CP nucleic acid.

[0373] The aforementioned 3' wing region and 5' wing region are modified nucleic acids with substituents at the 2' position.

[0374] The base length of the aforementioned single-stranded antisense oligonucleotides is 12–30 meters.

[0375] The base sequence of the above single-stranded antisense oligonucleotide is as follows:

[0376] Based on a base sequence that is complementary to at least one target region consisting of the same base length as the single-stranded antisense oligonucleotide described in sequence number 1, a base sequence having more than 90% and less than 100% sequence identity.

[0377] A base sequence complementary to a base sequence that has one or more bases missing, substituted, inserted, or added in the target region described above; or

[0378] A base sequence that can hybridize with oligonucleotides having the above-mentioned target regions under strict conditions.

[0379] In one aspect of the present invention, the base sequence of the single-stranded antisense oligonucleotide is preferably a base sequence having 95% or more and 100% or less sequence identity with 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 Serial No. 1.

[0380] In one embodiment of the present invention, the base sequence of the single-stranded antisense oligonucleotide is preferably a base sequence complementary to at least one target region in the base sequence described in Serial No. 1, which consists of the same base length as the single-stranded antisense oligonucleotide.

[0381] In one embodiment of the present invention, preferably, the number of bases in the interstitial region is 5 to 20.

[0382] The aforementioned 3' wing region refers to modified nucleic acids with substituents at the 2' position, which are 3-5 mers.

[0383] The aforementioned 5' wing region refers to modified nucleic acids with substituents at the 2' position of the 3-5mer.

[0384] In one aspect of the invention, the 5'-CP nucleic acid is preferably disposed at least in the second position from the 5' side of the aforementioned gap region.

[0385] In one embodiment of the present invention, preferably, two or more of the 5'-CP nucleic acids are configured in the aforementioned interstitial region.

[0386] In one embodiment of the present invention, preferably, 2 to 5 of the above-mentioned 5'-CP nucleic acids are arranged in the above-mentioned interstitial region.

[0387] In one aspect of the invention, it is preferable to configure more than 1 / 9 of the aforementioned 5'-CP nucleic acid in the aforementioned gap region.

[0388] In one aspect of the invention, it is preferable that more than 1 / 5 of the 5'-CP nucleic acid is disposed in the aforementioned gap region.

[0389] In one aspect of the invention, it is preferable to continuously arrange 2 to 4 mers of the above-mentioned 5'-CP nucleic acid at at least one location in the aforementioned gap region.

[0390] In one embodiment of the invention, the 5'-CP nucleic acid is preferably disposed on the 5' end side of the gap region.

[0391] In one embodiment of the invention, the 5'-CP nucleic acid is preferably disposed on the 5' end side and the 3' end side of the gap region.

[0392] In one embodiment of the present invention, the base length of the single-stranded antisense oligonucleotide is preferably 15 to 20 meters.

[0393] In one embodiment of the present invention, the base length of the single-stranded antisense oligonucleotide is preferably 18 to 20 meters.

[0394] In one embodiment of the invention, preferably, the modified nucleic acid having a substituent at the 2' position in the 3' wing region comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

[0395] The modified nucleic acid with a substituent at the 2' position in the 5' wing region mentioned above includes at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

[0396] In one embodiment of the invention, preferably, the modified nucleic acid having a substituent at the 2' position in the 3' wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

[0397] The modified nucleic acid with a substituent at the 2' position in the aforementioned 5' wing region consists of modified nucleic acids selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

[0398] In one embodiment of the invention, preferably, the modified nucleic acid having a substituent at the 2' position in the 3' wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA.

[0399] The modified nucleic acid with a substituent at the 2' position in the aforementioned 5' wing region consists of modified nucleic acids selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA.

[0400] In one embodiment of the present invention, it is preferred that at least one internucleotide bond of the single-stranded antisense oligonucleotide is a phosphate thioester bond.

[0401] In one embodiment of the present invention, it is preferred that at least one internucleotide bond of the above-mentioned single-stranded antisense oligonucleotide is a phosphodiester bond.

[0402] In one embodiment of the present invention, the proportion of thiophosphate bonds in the internucleotide bonds constituting the above-mentioned single-stranded antisense oligonucleotide is preferably 50% to 80%.

[0403] In one embodiment of the present invention, the proportion of thiophosphate bonds in the internucleotide bonds constituting the above-mentioned single-stranded antisense oligonucleotide is preferably 50% to 70%.

[0404] In one embodiment of the invention, in the aforementioned interstitial region, the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is preferably a phosphate thioester bond (except where the 5'-CP nucleic acid is located at the 3' end of the interstitial region).

[0405] In one embodiment of the present invention, in the aforementioned interstitial region, the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is preferably a phosphate thioester bond (except in the case where the 5'-CP nucleic acid is disposed at the 3' end of the interstitial region and the case where the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is a 5'-CP nucleic acid).

[0406] In one embodiment of the invention, in the aforementioned gap region, the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 5' side of the 5'-CP nucleic acid is preferably a phosphodiester bond.

[0407] In one embodiment of the present invention, the preferred base sequence of the above-mentioned single-stranded antisense oligonucleotide is as follows:

[0408] Based on the base sequence complementary to the target region consisting of 14 to 22 mers consecutively starting from the 5' end at positions 123, 124, 185, 186, 263, 264, 324, 325, 442, 443, or 447 to 453, the base sequence having a sequence identity of more than 90% and less than 100%;

[0409] A base sequence complementary to a base sequence that has one or more bases missing, substituted, inserted, or added in the target region described above; or

[0410] A base sequence that can hybridize with oligonucleotides having the above-mentioned target regions under strict conditions.

[0411] In one embodiment of the present invention, the preferred base sequence of the above-mentioned single-stranded antisense oligonucleotide is as follows:

[0412] Based on the base sequence complementary to the target region consisting of 14 to 22 consecutive bases starting from the 324th, 325th, or 448th to 453rd positions from the 5' end, the base sequence having more than 90% and less than 100% sequence identity.

[0413] A base sequence complementary to a base sequence that has one or more bases missing, substituted, inserted, or added in the target region described above; or

[0414] A base sequence that can hybridize with oligonucleotides having the above-mentioned target regions under strict conditions.

[0415] In one embodiment of the present invention, preferably, the base sequence of the aforementioned single-stranded antisense oligonucleotide is a base sequence having 90% to 100% sequence identity with the target region consisting of 18 to 22 consecutive bases starting from positions 448 to 453 from the 5' end in the base sequence recorded in Serial No. 1.

[0416] The aforementioned 3' wing region is 3–5mer.

[0417] The aforementioned 5' wing region is 3 to 5mer.

[0418] In one aspect of the present invention, the base sequence of the above-mentioned single-stranded antisense oligonucleotide is preferably a base sequence having 90% or more and 100% or less sequence identity with the target region consisting of 18 to 20 consecutive bases starting from the 450th to 451st or 453rd position from the 5' end in the base sequence recorded in Serial No. 1.

[0419] In one embodiment of the present invention, the base sequence of the above-mentioned single-stranded antisense oligonucleotide is preferably one base sequence selected from the group consisting of the base sequences of sequence numbers 5-47 and 49-56.

[0420] In one embodiment of the present invention, the base sequence of the above-mentioned single-stranded antisense oligonucleotide is preferably one base sequence selected from the group consisting of the base sequences of sequence numbers 6, 9, 12, 15-18, 22, 24, 27-29, 31-36, 38, 40-42, 47 and 49-54.

[0421] In one embodiment of the present invention, the base sequence of the single-stranded antisense oligonucleotide is preferably one base sequence selected from the group consisting of the base sequences of sequence numbers 41, 47, 50 and 53-55.

[0422] An antisense oligonucleotide complex of one aspect of the present invention is an antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof having the above-described single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, and an additional substance bound to the above-described single-stranded antisense oligonucleotide, wherein the additional substance is selected from the group consisting of polyethylene glycol, peptides, alkyl chains, nucleic acids, ligand compounds, antibodies, proteins, and sugar chains.

[0423] One aspect of the present invention comprises the above-described single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the above-described antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0424] In one manner of administering the above-mentioned drug, it is preferred that the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof, be administered by means of exposure to the central nervous system.

[0425] In one embodiment of the aforementioned drug, the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof, is preferably administered to a subject sensitive to delayed neurotoxicity.

[0426] One embodiment of the present invention is a regulator of RPS25 gene expression and / or function that comprises the above-mentioned single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the above-mentioned antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof as an active ingredient.

[0427] One aspect of the present invention is an inhibitor of dipeptide repeat generation caused by RAN translation, comprising the above-described single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the above-described antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof as an active ingredient.

[0428] One aspect of the present invention provides a therapeutic agent for recurrent diseases comprising the above-described single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the above-described antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0429] One aspect of the present invention provides a preventative agent for recurrent diseases comprising the above-described single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the above-described antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof, as an active ingredient.

[0430] In one of the above-mentioned therapeutic agents and the above-mentioned preventive agents, the above-mentioned recurrent disease is preferably selected from at least one of the following groups: C9orf72ALS, C9orf72 FTLD, Huntington's disease, spinocerebellar ataxia, dentate nucleus-rubra-pallidus-thalamic atrophy, spinobulbar muscular atrophy, Friedreich ataxia, fragile X-related tremor / ataxia syndrome, and myotonic dystrophy.

[0431] One aspect of the present invention provides a method for treating or preventing recurrent diseases, comprising the following steps: administering the above-mentioned single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the above-mentioned antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof, to an individual.

[0432] In one of the methods of treatment or prevention of the aforementioned recurrent disease, the aforementioned recurrent disease is preferably selected from at least one of the following groups: C9orf72 ALS, C9orf72 FTLD, Huntington's disease, spinocerebellar ataxia, dentate nucleus-rubra-pallidus-thalamic atrophy, spinobulbar muscular atrophy, Friedreich ataxia, fragile X-related tremor / ataxia syndrome, and myotonic dystrophy.

[0433] One aspect of the present invention provides the above-described single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the above-described antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof, for the treatment or prevention of C9orf72 ALS.

[0434] One aspect of the present invention provides the above-described single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the above-described antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof, for the preparation of therapeutic or preventative agents of C9orf72 ALS.

[0435] Example

[0436] The following describes embodiments of the present invention, but the present invention is not limited to these embodiments.

[0437] Production of single-stranded antisense oligonucleotides targeting the RPS25 gene

[0438] First, design the single-stranded antisense oligonucleotides shown in Tables 2-1 to 2-4, 3-1, 3-2, and 4. For a portion of the designed oligonucleotides, fabricate single-stranded antisense oligonucleotides targeting the RPS25 gene according to the following steps.

[0439] Single-stranded antisense oligonucleotides containing 2'-O-methyl nucleic acids, 2'-MOE nucleic acids, AmNA, scpBNA, 5'-CP nucleic acids and / or GuNA, and / or nucleic acids with a 5-methylcytosine base, as modified nucleic acids, were synthesized at a scale of 0.2 μmol using an automated nucleic acid synthesizer (nS-8, GeneDesign). Chain length elongation was performed using a standard phosphoramidite protocol. Specifically, in the case of monomers with a levulinoyl protecting group at the 5' position, the solid support was deprotected by treating it at room temperature for 1 hour outside the synthesizer with 0.5 M hydrazine hydrate in pyridine-acetic acid (1:1 v / v), and then the solid support was mounted in the synthesis apparatus, and synthesis was restarted. CPG resin was used as the solid support. Additionally, sulfidation for forming the thiophosphoryl esterification (PS) backbone was performed using DDTT (((dimethylaminomethylene)amino)-3H-1,2,4-dithiazoline-3-thione), etc. Single-stranded antisense oligonucleotides containing 2'-MOE nucleic acid, AmNA, and / or scpBNA were obtained with the 5'-terminal hydroxyl group unprotected by a DMTr (4,4'-dimethoxytriphenylmethyl) group and the 3'-terminal hydroxyl group unsupported on a solid phase. The single-stranded antisense oligonucleotides were then cleaved from the solid-phase support by alkali treatment and recovered in solution. The solvent was then distilled off from the recovered solution to obtain the crude product. The crude product was purified by reversed-phase HPLC to obtain the purified single-stranded antisense oligonucleotides. The purity and structure of each obtained single-stranded antisense oligonucleotide were confirmed by LC-MS (Waters Corporation).

[0440] The single-stranded antisense oligonucleotides produced by the above method are listed in Tables 2-1 to 2-4, 3-1, and 3-2 below. The single-stranded antisense oligonucleotides shown in Tables 2-1 to 2-4, 3-1, and 3-2 are single-stranded antisense oligonucleotides targeting human RPS25 mRNA (Sequence No. 1).

[0441] [Table 2-1]

[0442]

[0443] [Table 2-2]

[0444]

[0445] [Table 2-3]

[0446]

[0447] [Table 2-4]

[0448]

[0449] [Table 3-1]

[0450]

[0451] [Table 3-2]

[0452]

[0453] [Table 4]

[0454]

[0455] In this specification, the following symbols or expressions are sometimes used to represent the corresponding structures.

[0456]

[0457]

[0458] In the structural formula shown above, R 1 and R 2 Each of these can independently represent a hydrogen atom or an alkyl group with 1 to 3 carbon atoms, either straight or branched. Here, the R in the bonds indicated by "=" above... 1 and R 2 Both indicate methyl. R 3 R 4 and R 5 Each of these can independently represent a hydrogen atom, a straight-chain 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 "Gx" above, R... 3 and R 5 All are hydrogen atoms and R 4 In the case of methyl, it is represented as "Gm", and in R 3 It is a hydrogen atom and R 4 and R 5 When all components are methyl, it is represented as "Gdm". In R... 3 and R 5 It is a hydrogen atom and R 4 In the case of tert-butyl, it is represented as "GtB".

[0459] Evaluation of RPS25 gene expression

[0460] The expression evaluation of the RPS25 gene was performed using human fetal kidney cells based on the produced single-stranded antisense oligonucleotides. Alternatively, the expression evaluation of the RPS25 gene can also be performed using human iPS cell-derived neural cells. In this embodiment, gene expression evaluation refers to assessing mRNA levels by measuring the amount of complementary DNA (cDNA) obtained from reverse transcription. The specific steps for each expression evaluation are described below.

[0461] <Expression Evaluation Using Human Fetal Kidney Cells>

[0462] Human fetal kidney cells HEK293T (ATCC (registered trademark) CRL-3216 (trademark)) were cultured in culture medium at 37°C and 5% CO2. The culture medium for HEK293T cells used had the following composition.

[0463] The culture medium for HEK293T cells consisted of...

[0464] Dalberke Modified Eagle Medium (DMEM): manufactured by SIGMA, Cat#D6429

[0465] 10% Fetal Bovine Serum (FBS): Biowest, Cat#S1820

[0466] 100-fold dilution of penicillin-streptomycin mixed solution: Nacalai Tesque, Cat#09367-34 (containing 10,000 units / ml of penicillin, 10,000 μg / ml of streptomycin, and stabilizer).

[0467] First, the day before the human RPS25 gene expression assay, HEK293T cells (12,000 cells / well) were seeded in 96-well plates and cultured overnight at 37°C and 5% CO2. Then, single-stranded antisense oligonucleotides diluted with phosphate-buffered saline (PBS) (final concentrations of 0.5 nM, 5 nM, 15 nM, or 50 nM) were transfected into these cells using lipid transfection. As a negative control, cells transfected with PBS containing undissolved single-stranded antisense oligonucleotides were used. The transfected cells were cultured in proliferation medium at 37°C and 5% CO2 for 48 hours. Then, the proliferation medium was removed, and 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 a Taqman gene expression detection kit (Applied Biosystems) with pre-designed gene-specific probes (see below) (40 cycles of 95°C for 3 seconds and 60°C for 30 seconds).

[0468] A list of gene-specific probes used in evaluating human RPS25 gene expression

[0469] Human RPS25: Hs01568661_g1

[0470] Human GAPDH: 4326317E (Internal Reference)

[0471] The expression ratios of human RPS25 mRNA for each single-stranded antisense oligonucleotide targeting human RPS25 mRNA, obtained using the above method, are shown in Tables 5-1 to 5-3 and Tables 6-1 to 6-2 (in the "hRPS25 Expression Ratio" column). The expression ratio of human RPS25 mRNA obtained from the negative control group was set to 1.00. Single-stranded antisense oligonucleotides with an expression ratio below 0.80 were considered to inhibit the expression of human RPS25 mRNA. It should be noted that in the tables, "-" indicates that no measurement was performed. It is generally believed that if mRNA expression is inhibited, subsequent processes such as protein translation are also inhibited. Therefore, single-stranded antisense oligonucleotides with an expression ratio below 0.80 can be identified as those capable of regulating the function of the human RPS25 gene.

[0472] [Table 5-1]

[0473]

[0474] [Table 5-2]

[0475]

[0476] [Table 5-3]

[0477]

[0478] [Table 6-1]

[0479]

[0480] [Table 6-2]

[0481]

[0482] <Expression Evaluation Using Primary Cultured Mouse Nerve Cells>

[0483] Primary cultured mouse neural cells were cultured in culture medium at 37°C and 5% CO2. The culture medium used for primary cultured mouse neural cells had the following composition.

[0484] Composition of culture medium for primary cultured mouse nerve cells

[0485] B-27 Electrophysiology Kit: Gibco, Cat#A1413701

[0486] 100-fold dilution of 200 mM L-glutamine solution: Prepared by Nacalai Tesque: Cat#16948-04

[0487] 100-fold dilution of penicillin-streptomycin mixed solution: Nacalai Tesque, Cat#09367-34 (containing 10,000 units / ml of penicillin, 10,000 μg / ml of streptomycin, and stabilizer).

[0488] First, primary cultured mouse neural cells (derived from mouse fetal brain) were seeded at 40,000 cells / well in 96-well plates and cultured for 5 days at 37°C and 5% CO2. Then, single-stranded antisense oligonucleotides diluted with phosphate-buffered saline (PBS) (final concentrations of 0.01 μM, 0.1 μM, or 1 μM) were added to the culture medium. As a negative control, PBS containing undissolved single-stranded antisense oligonucleotides was added to the culture medium. Cells were cultured in the culture medium at 37°C and 5% CO2 for 48 hours. Then, the culture medium was removed, and total RNA was reverse transcribed using a Taqman FastCells-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 a Taqman gene expression detection kit (Applied Biosystems) with pre-designed gene-specific probes (see below) (40 cycles of 95°C for 3 seconds and 60°C for 30 seconds).

[0489] A list of gene-specific probes used in evaluating RPS25 gene expression in mice

[0490] Mouse Rps25: Mm02342783_g1

[0491] Mouse GAPDH: 4352339E (Internal Reference)

[0492] Evaluation of the use of motor neurons derived from human iPS cells

[0493] Motor neurons were induced from human iPS cells and used for evaluation. Cell maintenance and differentiation induction were performed in the culture media described below at 37°C and 5% CO2.

[0494] Culture medium composition overview

[0495] (SNL cell culture medium)

[0496] DMEM (Sigma-Aldrich, Cat#D6429)

[0497] 100-fold dilution of penicillin-streptomycin mixture (ThermoFisher Scientific, Cat#15140-122)

[0498] 10-fold dilution of fetal bovine serum (ThermoFisher Scientific, Cat#10437-028)

[0499] (iPS cell culture medium)

[0500] Culture medium for primate ES / iPS cells (manufactured by Reprocell, Cat#RCHEMD001B)

[0501] 100-fold dilution of penicillin-streptomycin mixed solution (ThermoFisher Scientific, CAT#15140-122)

[0502] (Mixed medium A)

[0503] DMEM / Ham'sF12 GlutaMAX (ThermoFisher Scientific, Cat#10565-018)

[0504] 2 mM L-glutamine (ThermoFisher Scientific, Cat#25030-081)

[0505] Non-essential amino acids (NEAA) (ThermoFisher Scientific, Cat#11140-050)

[0506] 100-fold dilution of penicillin-streptomycin mixed solution (ThermoFisher Scientific, CAT#15140-122)

[0507] 2 μg / mL heparin (Sigma-Aldrich, H-4784)

[0508] N2 additive (manufactured by ThermoFisher Scientific, Cat#17502-048)

[0509] (Mixed medium B)

[0510] Neurobasal medium (manufactured by ThermoFisher Scientific, CAT #21103-049)

[0511] 2 mM L-glutamine (ThermoFisher Scientific, Cat#25030-081)

[0512] Non-essential amino acids (NEAA) (ThermoFisher Scientific, Cat#11140-050)

[0513] Antibiotic-antifungal agent (manufactured by ThermoFisher Scientific, CAT #15240-062)

[0514] 2 μg / mL heparin (Sigma-Aldrich, H-4784)

[0515] N2 additive (manufactured by ThermoFisher Scientific, Cat#17502-048)

[0516] 10 ng / mL IGF-1 (manufactured by PeproTech, cat#100-11)

[0517] 10 ng / mL Human CNTF (manufactured by PeproTech, CAT#450-13) 10 ng / mL Human GDNF (manufactured by R&D Systems, CAT#212-GD-050)

[0518] Additive B27 (manufactured by ThermoFisher Scientific, CAT#12587010)

[0519] 200μM ascorbic acid (Sigma-Aldrich, Cat#A5960)

[0520] 10 ng / mL human BDNF (Peprotech, Cat#450-02)

[0521] (Culture medium for nerve cells)

[0522] Neurobasal medium Electro (ThermoFisher Scientific, CAT#A14098-01)

[0523] 2 mM L-glutamine (ThermoFisher Scientific, Cat#25030-081)

[0524] Non-essential amino acids (NEAA) (ThermoFisher Scientific, Cat#11140-050)

[0525] Antibiotic-antifungal agent (manufactured by ThermoFisher Scientific, CAT #15240-062)

[0526] 2 μg / mL heparin (Sigma-Aldrich, H-4784)

[0527] N2 additive (manufactured by ThermoFisher Scientific, Cat#17502-048)

[0528] 10 ng / mL IGF-1 (manufactured by PeproTech, cat#100-11)

[0529] 10 ng / mL human CNTF (PeproTech, cat#450-13)

[0530] 10 ng / mL human GDNF (manufactured by R&D Systems, CAT#212-GD-050)

[0531] Additive B27, Electro (manufactured by ThermoFisher Scientific, CAT#A14097-01)

[0532] 200μM ascorbic acid (Sigma-Aldrich, Cat#A5960)

[0533] 10 ng / mL human BDNF (Peprotech, Cat#450-02)

[0534] 25 μM 2-mercaptoethanol (ThermoFisher Scientific, CAT#21985-0123)

[0535] 0.1% bovine serum albumin (Sigma-Aldrich, CAT#A9576)

[0536] Mitomycin Cure Treatment of Feeder Cells

[0537] As feeder cells for inoculating human iPS cells, mitomycin C-cells (CellBiolabs, Cat#CBA-316) were prepared. The mitomycin C treatment of SNL cells was performed as follows: First, 0.1% gelatin (FUJIFILM Wako Pure Chemical, Cat#190-15805) was added to a 10 cm culture dish (IWAKI, Cat#3020-100), and the dish was incubated at 37°C and 5% CO2 for at least 1 hour (hereinafter, this operation is sometimes referred to as "gelatin treatment"). Then, excess gelatin was aspirated from the culture dish, and the thawed SNL cells were cultured in SNL cell culture medium at 1–2 × 10⁶ cells per culture dish. 6 Seedling is done in individual cell lines. Cells are diluted 8-16 times and passaged every 3-4 days to allow proliferation to the desired cell number. Then, 2-4 × 10⁶ cells are seeded per 15cm culture dish (IWAKI, Cat#3030-150) treated with 0.1% gelatin. 6 SNL cells were cultured until they reached 80-90% confluence. Mitomycin C (Kyowa Kirin, YJ No. 4231400D1031), diluted to 0.4 mg / mL with culture medium, was added to the culture dishes to a final concentration of 6.2 μg / mL. The culture dishes were incubated at 37°C and 5% CO2 for 2 hours and 15 minutes. The culture medium was then removed from the dishes, and the SNL cells were washed once with PBS. 2.5% trypsin / EDTA (Thermo Fisher Scientific, Cat#15090-046), diluted with PBS (final concentration 0.25%), was added to the SNL cells and incubated at room temperature for 1 minute. The SNL cells were then collected in a tube, centrifuged, resuspended in cryopreservation tubes (R) (ZENOAQ Resource, Cat#CB011), and frozen.

[0538] Maintenance of human iPS cells

[0539] Add 0.1% gelatin to a 10cm culture dish and incubate at 37°C and 5% CO2 for at least 1 hour. Suspend the SNL cells treated with mitomycin C using SNL cell culture medium. Then, add 1.5 × 10⁻⁶ cells... 6The above-mentioned SNL cells were seeded in 10 cm culture dishes and cultured for 2–3 days. Next, the SNL cell culture medium was removed from the culture dishes, and the SNL cells were washed with PBS. Then, human iPS cells (strain 201B7, obtained from iPS Academia Japan, AJ-H1-01) suspended in iPS cell culture medium containing 1 / 1000 of Y-27632 (manufactured by Tocris, Cat#1254) were seeded into the above-mentioned culture dishes. Culture medium changes were performed daily from the third day after seeding until differentiation induction began.

[0540] <Differentiation Induction from Human iPS Cells to Motor Neurons>

[0541] The iPS cells were exposed to Y-27632 (final concentration 10 μM) for at least 1 hour by adding Y-27632 to the cell culture medium. After removing the culture supernatant, the cells were washed with PBS, and then cell dissociation buffer (CTK solution) (Reprocell, Cat#RCHETP002) was added. The mixture was reacted at room temperature for 1 minute. The CTK solution was then removed, and the cells were washed twice with PBS. 1 mL of iPS cell culture medium was added. The cells were dissected using a cell scraper and dispersed through a cell filter (Becton, Dickinson, Cat#352350) to obtain a cell suspension. The resulting suspension was transferred to 6-well plates (Corning, Cat#3471). The culture medium was replaced with mixed medium A containing LDN193189 (Stemgent, Cat#04-0074) (final concentration 0.3 μM), SB431542 (Tocris Bioscience, Cat#1614) (final concentration 2 μM), CHIR-99021 (Stemgent, Cat#04-0004-10) (final concentration 3 μM) and Y-27632 (final concentration 10 μM). The cells were cultured in an incubator at 37°C and 5% CO2 (day 0 of culture).

[0542] On days 2 and 4 of culture, the culture medium was removed by pipette and replaced with fresh medium containing LDN193189 (final concentration 0.3 μM), SB431542 (final concentration 2 μM), and CHIR-99021 (final concentration 3 μM) added to mixed medium A.

[0543] On days 7, 9, and 11 of culture, the culture medium was removed by pipette and replaced with fresh medium containing LDN193189 (final concentration 0.3 μM), SB431542 (final concentration 2 μM), CHIR-99021 (final concentration 3 μM), purmorphamine (FUJIFILM Wako Pure Chemical, Cat#166-23991) (final concentration 0.5 μM), and retinoic acid (Sigma-Aldrich, Cat#R2625) (final concentration 0.1 μM).

[0544] On days 14 and 16 of culture, the culture medium was removed by pipette and replaced with fresh medium containing puremorphine (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).

[0545] On day 18 of culture, the medium was replaced with fresh medium containing puremorphine (final concentration 0.5 μM), retinoic acid (final concentration 0.1 μM), and compound E (Calbiochem, Cat#565790) (final concentration 0.1 μM) added to mixed medium B.

[0546] On day 21 of culture, cell clumps were washed with PBS, centrifuged, and the supernatant was removed. Accutase (Innovative Cell Technologies, Cat#AT104) and Y27632 (final concentration 10 μM) were added to the cell clumps, and the mixture was incubated at 37°C for 10 minutes. After chilling, the cell clumps were dispersed by pipetting. The centrifugation process was repeated twice (300×g, 5 minutes, 4°C), and the precipitated cells were recovered and resuspended in mixed medium B. Through these steps, motor neurons derived from iPS cells were obtained. The obtained motor neurons were aliquoted into cryovials (R) and cryopreserved.

[0547] <Cultivation of motor neurons derived from human iPS cells and evaluation of single-stranded antisense nucleotides>

[0548] Add a 6.66-fold diluted poly-L-ornithine (PLO) solution (Sigma-Aldrich, Cat#P4957) to 96-well Optical Btm Plt Polybase Black w / Lid Cell Culture Sterile PS (ThermoFisher Scientific, Cat#165305) and let it stand at room temperature for at least 2 hours. After washing three times with PBS, add iMatrix diluted with PBS to a concentration of 0.5 μg / cm³. 2 The cells were added to wells and incubated overnight at 4°C. Next, the frozen human iPS cell-derived motor neurons from 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 of Culture One additive (A3320201, ThermoFisher Scientific) and compound E (final concentration: 0.1 μM). The cells were seeded at 30,000 cells / well in coated 96-well plates and cultured for 28 days at 37°C with 5% CO2. Half of the neuronal cell culture medium was replaced every 2–3 days. From the start of culture until day 7, the neuronal cell culture medium contained Culture One additive and compound E was used.

[0549] Following inoculation, on days 1, 11, 18, and 26 (denoted as D1, D11, D18, and D26, respectively), single-stranded antisense oligonucleotides diluted with PBS (final concentrations of 0.01 μM, 0.1 μM, and 1 μM) were added to the culture medium. As a negative control, cells were cultured in PBS with undiluted single-stranded antisense oligonucleotides added. After culturing the cells in the culture medium at 37°C and 5% CO2 for 48 or 72 hours, the medium containing the single-stranded antisense oligonucleotides was removed, and the cells were cultured in neural cell culture medium (half the volume of medium was replaced every 2–3 days). Then, 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 the reverse transcription reaction, real-time PCR was performed using a Taqman gene expression detection kit (Applied Biosystems) with pre-designed gene-specific probes (see below) (40 cycles of 95°C for 3 seconds and 60°C for 30 seconds). The "expression inhibition rate" was calculated using the following formula (1).

[0550] (Expression inhibition rate) = 1 - (Expression ratio) (Equation 1)

[0551] The higher the expression inhibition rate value, the more effectively the single-stranded antisense oligonucleotide can inhibit the expression of human RPS25 mRNA.

[0552] A list of gene-specific probes used in evaluating human RPS25 gene expression

[0553] Human RPS25: Hs01568661_g1

[0554] Human GAPDH: 4326317E (Internal Reference)

[0555] Evaluation of RPS25 protein expression

[0556] The expression of RPS25 protein was evaluated using human fetal kidney cells based on the manufactured single-stranded antisense oligonucleotide. In this example, protein expression level evaluation refers to assessing the amount of protein translated from mRNA. The specific steps for expression evaluation are described below.

[0557] <Expression Evaluation Using Human Fetal Kidney Cells>

[0558] Human fetal kidney cells HEK293T (ATCC (registered trademark) CRL-3216 (trademark)) were cultured in culture medium at 37°C and 5% CO2. The culture medium for HEK293T cells used had the following composition.

[0559] The culture medium for HEK293T cells consisted of...

[0560] Dalberke Modified Eagle Medium (DMEM): manufactured by SIGMA, Cat#D6429

[0561] 10% Fetal Bovine Serum (FBS): Biowest, Cat#S1820

[0562] 100-fold dilution of penicillin-streptomycin mixed solution: Nacalai Tesque, Cat#09367-34 (containing 10,000 units / ml of penicillin, 10,000 μg / ml of streptomycin, and stabilizer).

[0563] <Quantitative Analysis of Proteins Based on Western Blotting>

[0564] First, HEK293T cells (500,000 cells / well) were seeded in 6-well plates and cultured overnight at 37°C and 5% CO2. Then, single-stranded antisense oligonucleotides diluted in phosphate-buffered saline (PBS) (final concentration 50 nM) were transfected into these cells using lipid transfection. Cells transfected with PBS containing undissolved single-stranded antisense oligonucleotides were used as a negative control. The transfected cells were cultured in proliferation medium at 37°C and 5% CO2 for 48 hours. Then, the proliferation medium was removed, and the cells were washed with PBS and recovered using a cell scraper. The recovered 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 (ThemoFisher Scientific, Cat#89900) containing 1 / 100 of a protease inhibitor (ThermoFisher Scientific, Cat#1860932) was added, and the cells were lysed using an ultrasonic homogenizer. The cells were then centrifuged at 15000×g for 10 minutes at 4°C, and the supernatant was used as the sample. Protein quantification of the recovered samples was performed using the Pierce BCA Protein Quantification Kit (ThermoScientific, Cat#23225). After adjusting the concentration of each sample to a constant level, Pierce Lane Marker Reducing Sample Buffer (ThermoFisher Scientifier, Cat#39000) was added, and the samples were heated at 95°C for 5 minutes. The prepared samples were loaded onto the electrophoresis tank at a protein content of 10 μg–20 μg / lane. Electrophoresis was performed using Criterion TDX pre-prepared gels at 4–15% concentration (BIO-RAD, Cat#5671085J10) at a constant voltage of 200V for 30 minutes. The electrophoresis buffer used was SDS-PAGE electrophoresis buffer (10×) diluted to a 1× concentration (Nacalai Tesque, Cat#30329-61).

[0565] After electrophoresis, the membranes were transferred semi-dry. The membranes were transferred using the Trans-Blot Turbo Transfer Pack (BIO-RAD, Cat#1704157) and the standard procedure (30 minutes) of the BIO-RAD Trans-Blot Turbo Transfer System was followed. After transcription, the membranes were washed with TBST. It should be noted that the TBST consisted of 0.06% polyoxyethylene sorbitan monolaurate (Tween-20) (Nacalai Tesque, Cat#28353-85) diluted to a 1× concentration in Tris-buffered saline (pH 7.4) (Nacalai Tesque, Cat#35438-81). After washing, the membranes were blocked by shaking at room temperature for 1 hour with Blocking One (Nacalai Tesque, Cat#03953-95) or PVDF blocking agent (Toyobo Co., Ltd., Cat#NYPBR01). After blocking, wash with TBST, add diluted primary antibody, and shake overnight at 4°C. The primary antibody and dilution solvent are described below.

[0566] ·RPS25

[0567] Antibody: Anti-RPS25 antibody (manufactured by Abcam, Cat#ab102940)

[0568] Solvent: Canget Signal Solution 1 (manufactured by Toyobo Co., Ltd., Cat#NKB-101)

[0569] β-actin

[0570] Antibody: β-actin (13E5) rabbit mAb (HRP conjugate) (Cell Signaling, Cat#5125)

[0571] Solvent: Blocking One

[0572] After shaking the antibody once, wash with TBST, add diluted secondary antibody, and shake at room temperature for 1 hour. The secondary antibody and dilution solvent are as follows.

[0573] ·RPS25

[0574] Antibody: Rabbit IgG (H+L) cross-adsorption secondary antibody (Cat#A24537, manufactured by Invitrogen)

[0575] Solvent: Canget Signal Solution 2 (manufactured by Toyobo Co., Ltd., Cat#NKB-101)

[0576] After vortexing the secondary antibody, wash with TBST and perform detection using ECL Prime (Amersham, Cat#RPN2232). It should be noted that the Amersham Imager 680 was used for both detection and analysis.

[0577] The expression ratio of human RPS25 protein for each single-stranded antisense oligonucleotide obtained using the above method was calculated. The expression ratio of human RPS25 protein obtained from the negative control group was set to 1.00. A protein expression ratio below 0.80 indicates that the oligonucleotide is capable of regulating the function of the RPS25 gene.

[0578] Cytotoxicity Evaluation of Single-Stranded Antisense Oligonucleotides

[0579] HeLa-S3 cells, representing human cervical cancer cells, were cultured in proliferation medium at 37°C and 5% CO2. The proliferation medium used had the following composition.

[0580] Composition of proliferation medium used in cytotoxicity evaluation

[0581] 10% Fetal Bovine Serum (FBS): manufactured by GIBCO, CAT#10437028

[0582] 1% Non-Essential Amino Acids (NEAA): Manufactured by GIBCO, Cat#11140050

[0583] Durbek Modified Eagle Medium Low Glucose (Containing L-Glutamine and Phenol Red) (FUJIFILM WakoPure Chemical Co., Cat#041-29775)

[0584] The cells described above were seeded in 96-well plates one day before the experiment (1.0 × 10⁶ cells per well). 4Cells were cultured overnight at 37°C and 5% CO2. Then, single-stranded antisense oligonucleotides (final concentration: 1–200 nM) forming a complex with Lipofectamine 3000 (Thermo Fisher Scientific, Cat#31985070) were added to Opti-Minimum Essential Medium (Thermo Fisher Scientific, Cat#L3000-015), and the cells were cultured for 24 hours at 37°C and 5% CO2. Caspase-Glo 3 / 7 assay (Promega, Cat#G8093) or CellTiter-Blue cell viability assay (Promega, Cat#G8081) were added to the proliferation medium to evaluate caspase activity and cell viability. The cytotoxicity evaluation results (cell viability) of each single-stranded antisense oligonucleotide obtained by the above method are shown in Tables 7-1 to 7-3.

[0585] [Table 7-1]

[0586]

[0587] [Table 7-2]

[0588]

[0589] [Table 7-3]

[0590]

[0591] Serum stability evaluation of single-stranded antisense oligonucleotides

[0592] Mouse serum (20 μL) or human serum (20 μL) was mixed into 4 μL of Tris-EDTA buffer (pH=8.0) containing 400 pmol of single-stranded antisense oligonucleotides, followed by the addition of mineral oil (15 μL). The solution was incubated at 37°C, and then 8 mol / L urea solution (10 μL) was added to inactivate the nucleases in the serum. After adding 10 μL of ultrapure water, the solution was centrifuged to separate an aqueous layer containing single-stranded antisense oligonucleotides and a mineral oil layer. The aqueous layer was analyzed by LC-MS (Waters Corporation), and the residual single-stranded antisense oligonucleotides were calculated based on the area intensity of the UV-chromatogram of the obtained single-stranded antisense oligonucleotides. "Residual Oligonucleotides (%)" represents the percentage of undigested single-stranded antisense oligonucleotides remaining after 72 hours relative to the percentage remaining immediately after mixing with serum.

[0593] Single-stranded antisense oligonucleotides with a residual rate of more than 50% after 72 hours are considered stable.

[0594] In vivo expression evaluation of the RPS25 gene

[0595] RPS25 gene expression was evaluated by administering the drug intracerebrospinally to mice and measuring the mRNA levels in various regions of the prefrontal cortex. In this embodiment, gene expression evaluation refers to assessing mRNA levels by measuring the amount of complementary DNA (cDNA) obtained from reverse transcription. The specific steps for each expression evaluation are described below.

[0596] FVB mice (CLEA Japan, Inc.) were anesthetized using isoflurane (Pfizer, Cat#114133403). Then, using a double-jointed needle (Top Biomedical Device Approval No. 15800BZZ01460000) fitted into a 50 μL Hamilton syringe (Hamilton, Cat#705LT), 10 μL of antisense oligonucleotide dissolved in artificial cerebrospinal fluid (Tocris Bioscience, Cat#3525 / 25 mL) was administered to the anesthetized FVB mice. Mice in the negative control group were administered artificial cerebrospinal fluid only at a dose of 10 μL per mouse.

[0597] After administration and a set feeding period, the FVB mice were euthanized, and brain, cervical spinal cord, and lumbar spinal cord samples were collected. 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 a high-capacity cDNA reverse transcription kit (Applied Biosystems, Cat#4368814). In the reverse transcription reaction, 1 μg of mRNA was diluted to 20 μL. Using the complementary DNA (cDNA) obtained from this reverse transcription reaction, real-time PCR was performed using the Taqman gene expression detection kit (Applied Biosystems) with pre-designed gene-specific probes (see below) (40 cycles of 95°C for 3 seconds and 60°C for 30 seconds).

[0598] A list of gene-specific probes used in evaluating RPS25 gene expression in mice

[0599] Mouse Rps25: Mm02342783_g1

[0600] Mouse GAPDH: 4352339E (Internal Reference)

[0601] The expression ratios of mouse RPS25 mRNA for each single-stranded antisense oligonucleotide, determined using the above method, are shown in Tables 8-1 and 8-2. In this case, the expression ratio of mouse RPS25 and RNA obtained in the negative control group was set to 1.00. It is generally believed that if mRNA expression is inhibited, subsequent protein translation is also inhibited. Therefore, if the expression ratio is below 0.80, it can be determined that the single-stranded antisense oligonucleotide is capable of regulating the function of the mouse RPS25 gene. It should be noted that the target regions bound by the single-stranded antisense oligonucleotides listed in Tables 8-1 and 8-2 are conserved regions between the human and mouse RPS25 genes.

[0602] [Table 8-1]

[0603]

[0604] [Table 8-2]

[0605]

[0606] In vivo evaluation of the central nervous system toxicity of single-stranded antisense oligonucleotides

[0607] The central nervous system toxicity of each single-stranded antisense oligonucleotide was evaluated by administering the drug intracerebrospinally to mice, feeding them immediately after administration to 1 hour after administration and for up to 28 days after administration, observing their general condition / body weight, and collecting tissue samples for pathological examination (hematoxylin-eosin staining).

[0608] The results are shown in Tables 9-1 and 9-2. The "Score" column in Tables 9-1 and 9-2, under "Acute Neurotoxicity in Vivo," was calculated based on the following evaluation criteria. For items 1-5, the scores for each item were summed up based on the highest score among the observed signs recorded for each individual. For example, if ptosis, lateral decubitus position, bradyventricular breathing, and convulsions were observed in an individual, the total score would be 1 + 4 + 4 = 9. Higher scores indicate more severe symptoms; therefore, a higher total score indicates more severe acute central nervous system toxicity symptoms. It should be noted that the scores are averaged for each group. It should also be noted that in the tables, "-" indicates that no measurement was performed.

[0609] 1. Hyperactivity (1 point), stereotyped behaviors (grooming, circling) (1 point), upright behavior (1 point), vocalization

[0610] (1 point) Allergy / Tail Raising (1 point)

[0611] 2. Ptosis, decreased (or absent) spontaneous eye movements when closing the eyes (1 point)

[0612] 3. Unsteady gait (1 point), ataxia gait (2 points)

[0613] 4. Abnormal posture / irregular breathing (1 point), lateral / prone position (2 points), slow / abnormal breathing (4 points)

[0614] 5. Jumping, trembling, muscle spasms (2 points), spasms (4 points)

[0615] Additionally, the "Score" column in "Delayed Neurotoxicity in Vivo" is calculated based on the following evaluation criteria. Each score uses the average value of each group. "ICR" refers to Cr1:CD1 mice, and "FVB" refers to FVB / NJcl mice.

[0616] Clinical signs and symptoms score;

[0617] 0 points: No abnormalities

[0618] 1 point: Hind limb dysfunction, tremor, reduced spontaneous movement

[0619] 2 points: Dragging of hind limbs, weakness in the tail or hind limbs

[0620] 3 points: Complete paralysis of the hind limbs, hind limb paralysis, lying horizontally, lying prone

[0621] 4 points: Euthanasia

[0622] Pathological score;

[0623] 0 points: No abnormalities

[0624] 1 point: Abnormalities are present (single-cell necrosis, vacuolization, etc.)

[0625] It should be noted that, for Example 51 in Table 9-1 and Reference Example 31 in Table 9-2, the central nervous system toxicity was evaluated using the following method.

[0626] In vivo evaluation of the central nervous system toxicity of single-stranded antisense oligonucleotides (2)

[0627] The acute central nervous system toxicity (in vivo acute neurotoxicity) of the single-stranded antisense oligonucleotides of Example 51 in Table 9-1 and Reference Example 31 in Table 9-2 was assessed by intracerebroventricular administration to ICR mice, with evaluation performed one day after administration. The assessment item was behavioral evaluation, which was conducted using the Functional Observational Battery (FOB) test. As assessment items, scores were assigned for 1) posture, 2) abnormal appearance, 3) stereotyped behavior, 4) responsiveness to stimuli, 5) gripping force, 6) respiration, and 7) tremors / convulsions: 0 for normal, 1 for slight abnormality, and 2 for severe abnormality. The total scores for each item were recorded in the "Score" column of "Acute Neurotoxicity in Vivo" in Tables 9-1 and 9-2.

[0628] In vivo evaluation of the central nervous system toxicity of single-stranded antisense oligonucleotides (3)

[0629] The delayed neurotoxicity (in vivo clinical signs score of delayed neurotoxicity) of the single-stranded antisense oligonucleotides of Example 51 in Table 9-1 and Reference Example 31 in Table 9-2 was assessed by intracerebroventricular administration to ICR mice, with evaluations conducted within 28 days post-administration. The assessment was behavioral, performed using the Functional Observational Bundle (FOB) test. As assessment items, scores were assigned for 1) posture, 2) abnormal appearance, 3) stereotyped behaviors, 4) responsiveness to stimuli, 5) gripping strength, 6) respiration, and 7) tremors / spasticity: 0 for normal, 1 for slight abnormality, and 2 for severe abnormality. The total scores for each item were recorded in the "Clinical Signs Score" column of "In vivo Delayed Neurotoxicity" in Tables 9-1 and 9-2.

[0630] [Table 9-1]

[0631]

[0632] [Table 9-2]

[0633]

[0634] Evaluation of off-target toxicity in silico (narrowly defined)

[0635] The high-speed base sequence retrieval system (GGGenome) described above was used to evaluate the narrow-sense off-target toxicity of each single-stranded antisense oligonucleotide. The sequence information of each single-stranded antisense oligonucleotide was input into GGGenome. Using registered human spliced ​​RNA and human pre-spliced ​​RNA from the database as targets, the number of narrow-sense non-target RNAs was counted based on the inconsistency number during hybridization. If mismatches, deletions, or insertions occurred during hybridization, the inconsistency number was calculated by adding the corresponding base counts.

[0636] For example, in the case where the base sequence of the RNA that becomes the target RNA in the single-stranded antisense oligonucleotide sequence is "AGCTGTAC", it has " The mismatch (underlined base) of RNA with the base sequence “” is “1”, and the inconsistency count is “1”. Additionally, when the target RNA's base sequence is “AGCTGTAC”, the sequence containing “” is considered mismatched. The mismatch (underlined base) of the RNA sequence with the base sequence “A” is counted as “1”, the deletion (base enclosed in angle brackets) is counted as “1”, and the inconsistency count is “2”. Additionally, when the target RNA sequence is “AGCTGTAC”, the presence of “A” indicates a mismatch. T The mismatch (underlined base) of the RNA with the base sequence "CT*AC" is "1", the insertion (site indicated by *) is "1", and the inconsistency number is calculated to be "2". Thus, the inconsistency number is counted when hybridizing with non-target RNA (off-target RNA), and the number of RNAs with completely identical, inconsistency numbers below 1, and inconsistency numbers below 2 are calculated for each single-stranded antisense oligonucleotide. The fewer the number of RNAs with inconsistency numbers below 0, 1, or 2, the lower the risk of off-target toxicity in the narrow sense; conversely, the more RNAs with inconsistency numbers below 0, 1, or 2, the higher the risk of off-target toxicity in the narrow sense. The results are shown in Tables 10-1 to 10-4. If the base length is 18 or more, the number of RNAs hybridizing with an inconsistency number below 1 is less than 10, which can be considered a low risk of off-target toxicity in the narrow sense, and can be considered a more preferred single-stranded antisense oligonucleotide.

[0637] [Table 10-1]

[0638]

[0639] [Table 10-2]

[0640]

[0641] [Table 10-3]

[0642]

[0643] [Table 10-4]

[0644]

[0645] The embodiments and examples of the present invention have been described above, but it was intended from the outset that the above embodiments and examples would be appropriately combined.

[0646] The embodiments and examples disclosed herein are illustrative in all respects and should not be considered limiting. The scope of the invention is not defined by the above-described embodiments and examples, but rather by the claims, which are intended to include all modifications within the meaning and scope equivalent to the claims.

Claims

1. A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof regulates the expression and / or function of the RPS25 gene. The nucleosides of the single-stranded antisense oligonucleotides are bonded together via phosphate groups and / or modified phosphate groups. The single-stranded antisense oligonucleotide comprises a gap region, a 3' wing region that binds to the 3' end of the gap region, and a 5' wing region that binds to the 5' end of the gap region. The interstitial region is a nucleic acid composed of deoxyribose, which may optionally contain a glycosidic modified nucleic acid. The interstitial region contains at least one 5'-CP nucleic acid. The 3' wing region and the 5' wing region are modified nucleic acids with substituents at the 2' position. The single-stranded antisense oligonucleotide has a base length of 12–30 meters. The base sequence of the single-stranded antisense oligonucleotide is as follows: Based on a base sequence that is 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 Serial No. 1, a base sequence having sequence identity of more than 90% and less than 100%. A base sequence complementary to a base sequence that has one or more bases missing, substituted, inserted, or added in the target region; or A base sequence that can hybridize with oligonucleotides having the target region under strict conditions.

2. A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof regulates the expression and / or function of the RPS25 gene, wherein, Except for the single-stranded antisense oligonucleotides in Examples 1-31, The nucleosides of the single-stranded antisense oligonucleotides are bonded together via phosphate groups and / or modified phosphate groups. The single-stranded antisense oligonucleotide comprises a gap region, a 3' wing region that binds to the 3' end of the gap region, and a 5' wing region that binds to the 5' end of the gap region. The interstitial region is a nucleic acid composed of deoxyribose, which may optionally contain a glycosidic modified nucleic acid. The interstitial region contains at least one 5'-CP nucleic acid. The 3' wing region and the 5' wing region are modified nucleic acids with substituents at the 2' position. The single-stranded antisense oligonucleotide has a base length of 12–30 meters. The base sequence of the single-stranded antisense oligonucleotide is as follows: Based on a base sequence that is 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 Serial No. 1, a base sequence having sequence identity of more than 90% and less than 100%. A base sequence complementary to a base sequence that has one or more bases missing, substituted, inserted, or added in the target region; or A base sequence that can hybridize with oligonucleotides having the target region under strict conditions.

3. A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, wherein the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof regulates the expression and / or function of the RPS25 gene, wherein, Except for the single-stranded antisense oligonucleotides in Examples 1-31 and Design Examples 1-18, The nucleosides of the single-stranded antisense oligonucleotides are bonded together via phosphate groups and / or modified phosphate groups. The single-stranded antisense oligonucleotide comprises a gap region, a 3' wing region that binds to the 3' end of the gap region, and a 5' wing region that binds to the 5' end of the gap region. The interstitial region is a nucleic acid composed of deoxyribose, which may optionally contain a glycosidic modified nucleic acid. The interstitial region contains at least one 5'-CP nucleic acid. The 3' wing region and the 5' wing region are modified nucleic acids with substituents at the 2' position. The single-stranded antisense oligonucleotide has a base length of 12–30 meters. The base sequence of the single-stranded antisense oligonucleotide is as follows: Based on a base sequence that is 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 Serial No. 1, a base sequence having sequence identity of more than 90% and less than 100%. A base sequence complementary to a base sequence that has one or more bases missing, substituted, inserted, or added in the target region; or A base sequence that can hybridize with oligonucleotides having the target region under strict conditions.

4. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein, The base sequence of the single-stranded antisense oligonucleotide is a base sequence with more than 95% and less than 100% sequence identity as 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 recorded in Serial No.

1.

5. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein, The base sequence of the single-stranded antisense oligonucleotide is a base sequence complementary to at least one target region in the base sequence recorded in Serial No. 1, which consists of the same base length as the single-stranded antisense oligonucleotide.

6. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein, The number of bases in the interstitial region is 5 to 20. The 3' wing region is a modified nucleic acid with a substituent at the 2' position, consisting of 3-5 medullars. The 5' wing region is a modified nucleic acid with a substituent at the 2' position of a 3-5mer.

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

8. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein, Two or more of the 5'-CP nucleic acids are configured in the interstitial region.

9. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, wherein, Two to five 5'-CP nucleic acids are configured in the interstitial region.

10. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein, More than 1 / 9 of the 5'-CP nucleic acid was configured in the gap region.

11. The single-stranded antisense oligonucleotide of claim 10, or a pharmaceutically acceptable salt thereof, wherein, More than 1 / 5 of the 5'-CP nucleic acid was configured in the gap region.

12. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, wherein, The 5'-CP nucleic acid is continuously configured with 2 to 4 meristems at at least one location in the interstitial region.

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

14. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, wherein, The 5'-CP nucleic acid is disposed on the 5' end side and the 3' end side of the gap region.

15. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 14, wherein, The single-stranded antisense oligonucleotide has a base length of 15–20 meters.

16. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 15, wherein, The single-stranded antisense oligonucleotide has a base length of 18–20 meters.

17. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 16, wherein, The modified nucleic acid with a substituent at the 2' position in the 3' wing region comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA. The modified nucleic acid with a substituent at the 2' position in the 5' wing region comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

18. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 17, wherein, The modified nucleic acid with a substituent at the 2' position in the 3'-wing region is composed of modified nucleic acids selected from the group consisting of 2'-MOE nucleic acids, LNA, AmNA, GuNA, and scpBNA. The modified nucleic acid with a substituent at the 2' position in the 5'-wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

19. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 18, wherein, The modified nucleic acid with a substituent at the 2' position in the 3' wing region is composed of modified nucleic acids selected from the group consisting of 2'-MOE nucleic acids, AmNA, GuNA, and scpBNA. The modified nucleic acid with a substituent at the 2' position in the 5' wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, AmNA, GuNA, and scpBNA.

20. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 19, wherein, At least one nucleoside bond in the single-stranded antisense oligonucleotide is a thiophosphate bond.

21. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 20, wherein, At least one nucleoside bond in the single-stranded antisense oligonucleotide is a phosphodiester bond.

22. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 21, wherein, In the internucleotide bonds constituting the single-stranded antisense oligonucleotide, the proportion of phosphate thioester bonds is 50% to 80%.

23. The single-stranded antisense oligonucleotide of claim 22 or a pharmaceutically acceptable salt thereof, wherein, In the internucleotide bonds constituting the single-stranded antisense oligonucleotide, the proportion of phosphate thioester bonds is 50% to 70%.

24. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 23, wherein, In the interstitial region, the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is a phosphate thioester bond, except when the 5'-CP nucleic acid is located at the 3' end of the interstitial region.

25. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 24, wherein, In the interstitial region, the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is a phosphate thioester bond, except in the case where the 5'-CP nucleic acid is located at the 3' end of the interstitial region and the case where the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is a 5'-CP nucleic acid.

26. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 25, wherein, In the interstitial region, the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 5' side of the 5'-CP nucleic acid is a phosphodiester bond.

27. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 26, wherein, The base sequence of the single-stranded antisense oligonucleotide is as follows: Based on the base sequence complementary to the target region consisting of 14 to 22 mers consecutively starting from the 5' end at positions 123, 124, 185, 186, 263, 264, 324, 325, 442, 443, or 447 to 453, the base sequence having a sequence identity of more than 90% and less than 100%; A base sequence complementary to a base sequence that has one or more bases missing, substituted, inserted, or added in the target region; or A base sequence that can hybridize with oligonucleotides having the target region under strict conditions.

28. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 27, wherein, The base sequence of the single-stranded antisense oligonucleotide is as follows: Based on the base sequence complementary to the target region consisting of 14 to 22 consecutive bases starting from the 324th, 325th, or 448th to 453rd positions from the 5' end, the base sequence having more than 90% and less than 100% sequence identity. A base sequence complementary to a base sequence that has one or more bases missing, substituted, inserted, or added in the target region; or A base sequence that can hybridize with oligonucleotides having the target region under strict conditions.

29. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 28, wherein, The single-stranded antisense oligonucleotide has a base sequence that is complementary to the target region consisting of 18 to 22 consecutive bases starting from positions 448 to 453 (counting from the 5' end) in the base sequence described in Serial No. 1, and has a sequence identity of more than 90% and less than 100%. The 3' wing region is 3 to 5 mer. The 5' wing region is 3 to 5mer.

30. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 29, wherein, The single-stranded antisense oligonucleotide has a base sequence that is complementary to the target region consisting of 18 to 20 consecutive bases starting from the 450th to 451st or 453rd position from the 5' end, and has a sequence identity of more than 90% and less than 100% with respect to the base sequence recorded in Serial No.

1.

31. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 30, wherein, The single-stranded antisense oligonucleotide has a base sequence selected from the group consisting of base sequences of sequence numbers 5-47 and 49-56.

32. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 31, wherein, The single-stranded antisense oligonucleotide has a base sequence selected from the group consisting of base sequences of sequence numbers 6, 9, 12, 15–18, 22, 24, 27–29, 31–36, 38, 40–42, 47, and 49–54.

33. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 32, wherein, The single-stranded antisense oligonucleotide has a base sequence selected from the group consisting of base sequences of sequence numbers 41, 47, 50, and 53-55.

34. An antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof, having: The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 33; as well as Additional substances that bind to the single-stranded antisense oligonucleotide, The additional substance is selected from the group consisting of polyethylene glycol, peptides, alkyl chains, nucleic acids, ligand compounds, antibodies, proteins, and sugar chains.

35. A medicament comprising, as an active ingredient, any one of claims 1 to 33, a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof as claimed in claim 34.

36. The medicament according to claim 35, wherein, The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof, is administered via exposure to the central nervous system.

37. The medicament according to claim 35 or 36, wherein, The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof, is administered to a subject sensitive to delayed neurotoxicity.

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

39. An inhibitor of dipeptide repeat generation caused by RAN translation, comprising, as an active ingredient, any one of claims 1 to 33, a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex of claim 34 or a pharmaceutically acceptable salt thereof.

40. A therapeutic agent for recurrent diseases, comprising, as an active ingredient, any single-stranded antisense oligonucleotide of claims 1 to 33 or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex of claim 34 or a pharmaceutically acceptable salt thereof.

41. A preventive agent for recurrent diseases, comprising, as an active ingredient, any single-stranded antisense oligonucleotide of claims 1 to 33 or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex of claim 34 or a pharmaceutically acceptable salt thereof.

42. The therapeutic agent for recurrent diseases according to claim 40, or the preventive agent for recurrent diseases according to claim 41, wherein, The recurring disease is selected from at least one of the following groups: C9orf72 ALS, C9orf72 FTLD, Huntington's disease, spinocerebellar ataxia, dentate nucleus-rubra-pallidus-thalamic atrophy, spinobulbar muscular atrophy, Friedreich ataxia, fragile X-related tremor / ataxia syndrome, and myotonic dystrophy.

43. A treatment or prevention method for a recurring disease, comprising the following steps: The single-stranded antisense oligonucleotide of any one of claims 1 to 33 or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex of claim 34 or a pharmaceutically acceptable salt thereof, is administered to an individual.

44. The treatment or prevention method for recurrent diseases according to claim 43, wherein, The recurring disease is selected from at least one of the following groups: C9orf72 ALS, C9orf72 FTLD, Huntington's disease, spinocerebellar ataxia, dentate nucleus-rubra-pallidus-thalamic atrophy, spinobulbar muscular atrophy, Friedreich ataxia, fragile X-related tremor / ataxia syndrome, and myotonic dystrophy.

45. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 33, or the antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof according to claim 34, for the treatment or prevention of C9orf72 ALS.

46. ​​The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 33, or the antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof according to claim 34, for use in the preparation of a C9orf72ALS therapeutic or preventative agent.

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