Method for designing antisense oligonucleotide having reduced delayed central toxicity, and method for producing same

Abstract

Provided is a method for designing an antisense oligonucleotide having reduced delayed central toxicity, the method comprising a step for designing a second antisense oligonucleotide on the basis of a first antisense oligonucleotide. The first antisense oligonucleotide is configured such that nucleosides are bonded together via a phosphate group and / or a modified phosphate group. The first antisense oligonucleotide comprises a gap region, a 3' wing region that is bonded to the 3' end of the gap region, and a 5' wing region that is bonded to the 5' end of the gap region. The gap region has nucleic acids constituted of deoxyribose that may include a nucleic acid in which sugar is modified; the 3' wing region and the 5' wing region are modified nucleic acids having a substituent at the 2' position; and the base length is 12-30 mer. The second antisense oligonucleotide has the same base sequences for the gap region, the 3' wing region and the 5' wing region as the first antisense oligonucleotide, and has the same sugar structures of the nucleic acids in the 3' wing region and the 5' wing region as those of the first antisense oligonucleotide. The gap region contains at least one 5'-CP nucleic acid, and the delayed central toxicity is reduced as compared with the first antisense oligonucleotide.

Description

Design method of antisense oligonucleotides with reduced delayed central toxicity and method for producing the same

[0001] The present invention relates to a method for designing an antisense oligonucleotide with reduced delayed central toxicity, and a method for producing the same.

[0002] Antisense oligonucleotides exert their effects by hybridizing to target nucleic acid sequences and suppressing gene expression itself. Various modified nucleic acids have been developed and introduced to improve the nuclease resistance of antisense oligonucleotides and improve their binding affinity and specificity to target nucleic acids, and various formulations have been marketed. However, a new problem has recently emerged: how to avoid the potential toxicity of modified nucleic acids.

[0003] The toxicity of antisense oligonucleotides can be categorized into "toxicity due to hybridization with non-target RNA (narrowly defined off-target toxicity)" and "toxicity not due to hybridization with RNA (broadly defined off-target toxicity)" that results from binding to intracellular and extracellular proteins, metal ions, etc. Various approaches have been adopted to avoid these toxicities.

[0004] For example, Patent Document 1 discloses that broadly defined off-target toxicity can be avoided by appropriate chemical modifications to the base moiety or sugar moiety of a nucleic acid. Patent Document 2 discloses that narrowly defined off-target toxicity can be avoided by modifying the carbonyl group at the 2' position of the base moiety (thymine) and cross-linking the 2'-4' positions of the sugar moiety to suppress the formation of non-Watson-Crick base pairs during hybridization with off-target RNA. Furthermore, while it is generally known that phosphorothioate modifications between nucleosides cause hepatotoxicity (e.g., Non-Patent Document 1), Patent Document 3 discloses that hepatotoxicity can be reduced while maintaining activity by substituting phosphorothioate nucleic acids, which are of concern due to accumulation in specific organs (e.g., the liver), with modified nucleic acids having a cyclopropane structure at the 5' position.

[0005] International Publication No. WO 2018 / 155450, International Publication No. WO 2018 / 155451, International Publication No. WO 2020 / 158910, International Publication No. WO 2016 / 127000, International Publication No. WO 2011 / 052436, International Publication No. WO 2014 / 046212, International Publication No. WO 2015 / 125783, International Publication No. WO 99 / 14226

[0006] Migawa MT et al., Nucleic Acids Res, 20;47(11):5465-5479 (2019)Moazami MP et al., bioRxiv, doi: https: / / doi.org / 10.1101 / 2021.02.14.431096 (2021)W. Brad Wan et. Al. J. Med. Chem. (2016) 59:9645-9667. Peter H. Hagedorn et. Al. Nucleic Acid Ther. (2022) 32:151-162.

[0007] Toxicity assessments of antisense oligonucleotide toxicity reduction technologies to date have primarily focused on hepatotoxicity, given that the liver is the organ most likely to accumulate after systemic exposure, and no in-depth knowledge has been gained about toxicity assessments in other tissues. For example, central nervous system toxicity (acute central nervous system toxicity, delayed central nervous system toxicity, etc.) is an important evaluation item in pharmaceutical safety testing, but while the development of antisense oligonucleotides targeting central nervous system disorders has been actively progressing in recent years, very little is known about the central nervous system toxicity of antisense oligonucleotides.

[0008] As a finding focusing on the relationship between antisense oligonucleotides and central toxicity, particularly the reduction of central toxicity, Patent Document 4 discloses the correlation between the sequence of antisense oligonucleotides and central toxicity (oscillation of intracellular free calcium concentration in neurons). Furthermore, as far as the present inventors know, there are only two publications evaluating the central toxicity of phosphorothioate modifications in antisense oligonucleotides (Non-Patent Document 2, Non-Patent Document 4). Non-Patent Document 2 discloses that central toxicity can be reduced by replacing phosphorothioate bonds in the wing portions of phosphorothioate-modified gapmer oligonucleotides with phosphodiester bonds.

[0009] However, Non-Patent Document 2 discloses that antisense oligonucleotides (hereinafter sometimes referred to as "ASO") in which some phosphorothioate modifications have been removed and a stability-related modification has been added to the 2' position of the sugar moiety may have reduced antisense activity compared to ASOs in which the phosphorothioate modifications have not been removed. The reduced antisense activity is thought to be due to the reduced stability of the ASO resulting from the removal of the phosphorothioate modifications.

[0010] The present invention has been made in consideration of the above circumstances, and aims to provide a method for designing and producing an antisense oligonucleotide that has reduced delayed central nervous system toxicity while retaining antisense activity.

[0011] As a result of intensive research to solve the above-mentioned problems, the present inventors have found that by designing and producing an antisense oligonucleotide or a pharmaceutically acceptable salt thereof (hereinafter sometimes referred to as the "antisense oligonucleotide of the present invention") so that it contains a 5'-CP nucleic acid in the gap region, delayed central nervous system toxicity can be reduced while maintaining antisense activity, and have completed the present invention. That is, the present invention is as follows.

[0012] [1] A method for designing an antisense oligonucleotide of the present invention, comprising the step of designing a second antisense oligonucleotide based on a first antisense oligonucleotide, is a method for designing an antisense oligonucleotide having reduced delayed central toxicity, wherein the first antisense oligonucleotide is an antisense oligonucleotide in which each nucleoside is linked via a phosphate group and / or a modified phosphate group, and which comprises a gap region, a 3' wing region linked to the 3' end of the gap region, and a 5' wing region linked to the 5' end of the gap region, the gap region being a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the 3' wing region and the 5' wing region being modified nucleic acid having a substituent at the 2' position, and having a base length of 12 to 30 mer, and the second antisense oligonucleotide is an antisense oligonucleotide in which the base sequences of the gap region, 3' wing region, and 5' wing region are identical to those of the first antisense oligonucleotide, and the sugar moieties of the nucleic acids in the 3' wing region and 5' wing region have the same structures as those of the first antisense oligonucleotide, The gap region contains at least one 5'-CP nucleic acid, and the delayed central toxicity is reduced compared to the first antisense oligonucleotide.

[0013] [2] In the above [1], it is preferable that in the first antisense oligonucleotide, the gap region does not contain a 5'-CP nucleic acid, and in the second antisense oligonucleotide, the structure of the sugar moiety other than the 5'-CP nucleic acid in the gap region is the same as that of the first antisense oligonucleotide.

[0014] [3] In the above [1] or [2], in the first antisense oligonucleotide and the second antisense oligonucleotide, it is preferable that the number of bases in the gap region is 5 to 20 mer, the 3' wing region is a 3 to 5 mer modified nucleic acid having a substituent at the 2' position, and the 5' wing region is a 3 to 5 mer modified nucleic acid having a substituent at the 2' position.

[0015] [4] In any one of the above [1] to [3], the first antisense oligonucleotide and the second antisense oligonucleotide preferably have a base length of 15 to 30 mer.

[0016] [5] In any of the above [1] to [4], it is preferable that in the first antisense oligonucleotide and the second antisense oligonucleotide, the cytosine located in the gap region is 5-methylcytosine.

[0017] [6] In any of [1] to [5] above, in the first antisense oligonucleotide and the second antisense oligonucleotide, it is preferred that the modified nucleic acid having a substituent at the 2'-position in the 3'-wing region comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA, and the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

[0018] [7] In any one of the above [1] to [6], it is preferable that the GC ratio in the base sequence of the first antisense oligonucleotide and the second antisense oligonucleotide is 0.2 to 0.6.

[0019] [8] In any of the above [1] to [7], it is preferable that the first antisense oligonucleotide and the second antisense oligonucleotide are single-stranded antisense oligonucleotides.

[0020] [9] In any of [1] to [8] above, in the second antisense oligonucleotide, the 5'-CP nucleic acid is preferably positioned at least at the second position counting from the 5' side of the gap region.

[0021]

[10] In any one of [1] to [9] above, in the second antisense oligonucleotide, it is preferable that two or more of the 5'-CP nucleic acids are arranged in the gap region.

[0022]

[11] In any one of [1] to

[10] above, in the second antisense oligonucleotide, it is preferable that 2 to 5 of the 5'-CP nucleic acids are arranged in the gap region.

[0023]

[12] In any of [1] to

[11] above, in the second antisense oligonucleotide, the 5'-CP nucleic acid is preferably arranged in a continuous 2- to 4-mer configuration at least at one position in the gap region.

[0024]

[13] In any one of [1] to

[12] above, in the second antisense oligonucleotide, the 5'-CP nucleic acid is preferably arranged at one-ninth or more of the gap region.

[0025]

[14] In any one of [1] to

[13] above, in the second antisense oligonucleotide, the 5'-CP nucleic acid is preferably positioned at one-fifth or more of the gap region.

[0026]

[15] In any one of [1] to

[14] above, in the second antisense oligonucleotide, the 5'-CP nucleic acid is preferably located on the 5'-end side of the gap region.

[0027]

[16] In any of [1] to

[15] above, in the second antisense oligonucleotide, the 5'-CP nucleic acid is preferably located on the 5'-end side and the 3'-end side of the gap region.

[0028]

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

[16] , it is preferable that the proportion of phosphorothioate bonds in the internucleoside bonds constituting the second antisense oligonucleotide is 50% to 80%.

[0029]

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

[17] , it is preferable that the proportion of phosphorothioate bonds in the internucleoside bonds constituting the second antisense oligonucleotide is 50% to 70%.

[0030]

[19] In any of [1] to

[18] above, in the gap region constituting the second antisense oligonucleotide, it is preferable that the bond between the 5'-CP nucleic acid and the nucleoside adjacent to the 3' side of the 5'-CP nucleic acid is a phosphorothioate bond (excluding the case where the 5'-CP nucleic acid is positioned at the 3' end of the gap region).

[0031]

[20] In any of [1] to

[19] above, in the gap region constituting the second antisense oligonucleotide, it is preferable that the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is a phosphorothioate bond (excluding the cases where the 5'-CP nucleic acid is positioned at the 3' end of the gap region and the nucleoside adjacent on the 3' side of the 5'-CP nucleic acid is a 5'-CP nucleic acid).

[0032]

[21] In any of [1] to

[20] above, in the gap region constituting the second antisense oligonucleotide, 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.

[0033]

[22] In any of [1] to

[21] above, it is preferable that the gap region constituting the second antisense oligonucleotide contains five or more bases composed of deoxyribose (but excluding 5'-CP nucleic acid) which may contain a nucleic acid whose sugar moiety is modified.

[0034]

[23] In any of [1] to

[22] above, in the gap region constituting the second antisense oligonucleotide, the number of bases composed of deoxyribose (but excluding 5'-CP nucleic acid) which may contain a nucleic acid whose sugar moiety is modified is preferably 5 to 10.

[0035]

[24] In any of [1] to

[23] above, it is preferred that in the gap region constituting the second antisense oligonucleotide, 5 to 10 consecutive bases composed of deoxyribose (but excluding 5'-CP nucleic acid) which may contain a nucleic acid whose sugar moiety is modified are arranged.

[0036]

[25] A method for producing an antisense oligonucleotide or a pharmaceutically acceptable salt thereof of the present invention is a method for producing an antisense oligonucleotide or a pharmaceutically acceptable salt thereof having reduced delayed central toxicity, comprising the steps of: designing the second antisense oligonucleotide by the antisense oligonucleotide design method according to any one of [1] to

[24] above; and synthesizing the second antisense oligonucleotide.

[0037]

[26] The antisense oligonucleotide of the present invention is an antisense oligonucleotide or a pharmaceutically acceptable salt thereof having reduced delayed central toxicity, wherein each nucleoside in the antisense oligonucleotide is linked via a phosphate group and / or a modified phosphate group, the antisense oligonucleotide comprises a gap region, a 3' wing region linked to the 3' end of the gap region, and a 5' wing region linked to the 5' end of the gap region, the gap region being a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the gap region comprising at least one 5'-CP nucleic acid, the 3' wing region and the 5' wing region being modified nucleic acids having a substituent at the 2' position, and the base length of the antisense oligonucleotide is 12 to 30 mer.

[0038]

[27] The antisense oligonucleotide conjugate or pharmaceutically acceptable salt thereof of the present invention comprises the antisense oligonucleotide of

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

[0039]

[28] A therapeutic agent for a disease according to a first aspect of the present invention is a therapeutic agent for a disease, comprising the antisense oligonucleotide of

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

[27] above or a pharmaceutically acceptable salt thereof, wherein the therapeutic agent is administered so as to be exposed to the central nervous system.

[0040]

[29] A therapeutic agent for a disease according to a second aspect of the present invention is a therapeutic agent for a disease, comprising the antisense oligonucleotide of

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

[27] above or a pharmaceutically acceptable salt thereof, wherein the therapeutic agent is administered to a subject sensitive to delayed central nervous system toxicity.

[0041]

[30] A therapeutic agent for a disease in a third aspect of the present invention, comprising the antisense oligonucleotide of

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

[27] above or a pharmaceutically acceptable salt thereof, wherein the disease is a disease of the central nervous system.

[0042] According to the present invention, it is possible to provide a method for designing and producing an antisense oligonucleotide that has reduced delayed central nervous system toxicity while retaining antisense activity.

[0043] Fig. 1 is a schematic diagram showing an example of the structure of an antisense oligonucleotide according to this embodiment. Fig. 2 is a schematic diagram illustrating the mechanism by which target RNA expression is suppressed when the antisense oligonucleotide according to this embodiment is used.

[0044] An embodiment of the present invention (hereinafter referred to as "this embodiment") will be described below. However, this embodiment is not limited thereto. In this specification, the notation in the form of "I to J" means the upper and lower limits of a range (i.e., I or more and J or less), and when no unit is specified for I and a unit is specified only for J, the unit of I and the unit of J are the same.

[0045] <Method for Designing Antisense Oligonucleotide with Reduced Delayed Central Toxicity> The method for designing an antisense oligonucleotide with reduced delayed central toxicity of the present embodiment comprises the step of designing a second antisense oligonucleotide based on a first antisense oligonucleotide, wherein the first antisense oligonucleotide is an antisense oligonucleotide in which each nucleoside is linked via a phosphate group and / or a modified phosphate group, and which comprises a gap region, a 3' wing region linked to the 3' end of the gap region, and a 5' wing region linked to the 5' end of the gap region, the gap region being a nucleic acid composed of deoxyribose which may contain a nucleic acid with a modified sugar moiety, the 3' wing region and the 5' wing region being modified nucleic acid having a substituent at the 2' position, and having a base length of 12 to 30 mer, and the second antisense oligonucleotide is an antisense oligonucleotide in which the base sequences of the gap region, 3' wing region, and 5' wing region are identical to those of the first antisense oligonucleotide, The sugar moieties of the nucleic acids in the 3' wing region and 5' wing region have the same structure as those of the first antisense oligonucleotide, the gap region contains at least one 5'-CP nucleic acid, and the delayed central toxicity is reduced compared to the first antisense oligonucleotide.

[0046] <Definitions of Terms, etc.> First, definitions of terms used in this specification will be explained below.

[0047] (Single-Stranded Antisense Oligonucleotide) In this embodiment, the terms "single-stranded antisense oligonucleotide," "antisense oligonucleotide," or "ASO" refer to an oligonucleotide or a pharmacologically acceptable salt thereof that is complementary to the mRNA, pre-mRNA, or non-coding RNA (ncRNA) of a target gene (hereinafter, these three may be collectively referred to as "target RNA"). Antisense oligonucleotides are composed of DNA, RNA, and / or analogs thereof. Antisense oligonucleotides inhibit the activity of the target mRNA, pre-mRNA, or ncRNA by forming a duplex with the target mRNA, pre-mRNA, or ncRNA. Antisense oligonucleotides include those that have a base sequence that is completely complementary to the base sequence of the target mRNA, pre-mRNA, or ncRNA; those that have a base sequence in which one or more bases are deleted, substituted, inserted, or added in the complementary base sequence; and those that contain bases that form wobble base pairs in their base sequence. Furthermore, the antisense oligonucleotide of the present invention may further contain modified nucleotides known in the art other than "modified nucleic acids in which the sugar moiety is a modified sugar" (sugar-modified modified nucleotides) described below. Examples of modified nucleotides known in the art include sugar-modified modified nucleotides, as well as phosphate-modified nucleotides and nucleobase-modified nucleotides described below. The antisense oligonucleotide of this embodiment is not particularly limited in terms of the structure of both termini, and may be, for example, -OH or -OR (where R represents an alkyl chain, a phosphate ester, or an additional substance described below). The single-stranded antisense oligonucleotide of this embodiment may be in a single-stranded form, or may hybridize with a second-strand oligonucleotide described below to form a double-stranded form. A double-stranded oligonucleotide consisting of the single-stranded antisense oligonucleotide and a second-strand oligonucleotide hybridized to the single-stranded antisense oligonucleotide may be referred to as a "double-stranded antisense oligonucleotide."

[0048] (Oligonucleotide) In this embodiment, "oligonucleotide" refers to a polymer of nucleotides in which 2 to 30 identical or different nucleosides are linked together via phosphodiester bonds or other bonds. The oligonucleotide can also be understood as being composed of a nucleic acid base moiety, a phosphate moiety, and a sugar moiety, as shown in the following structural formula:

[0049]

[0050] The oligonucleotides are broadly classified into natural oligonucleotides and non-natural oligonucleotides. "Natural oligonucleotides" refer to oligonucleotides composed of naturally occurring nucleotides. "Non-natural oligonucleotides" refer to oligonucleotides containing at least one modified nucleotide as a constituent unit, as described below. "Non-natural oligonucleotides" preferably include modified sugar derivatives in which the sugar moiety is modified; phosphorothioate derivatives in which one non-bridging oxygen atom in the phosphodiester bond is replaced with a sulfur atom; phosphorodithioate derivatives in which two non-bridging oxygen atoms in the phosphodiester bond are replaced with sulfur atoms; ester derivatives in which the phosphodiester bond is triesterized; phosphoamide derivatives in which the phosphodiester bond is amidated; boranophosphate derivatives in which the phosphodiester bond is boronated; alkylphosphonate (e.g., methylphosphonate, methoxypropylphosphonate, etc.) derivatives in which the non-bridging oxygen atom in the phosphodiester bond is replaced with an alkyl group; amide derivatives in which the phosphodiester bond is replaced with an amide bond; and modified base derivatives in which the nucleobase is modified. More preferably, the unnatural oligonucleotide includes a cross-linked modified sugar derivative in which the sugar moiety is modified; a phosphorothioate derivative in which one non-bridging oxygen atom in the phosphodiester bond is replaced with a sulfur atom; an ester derivative in which the phosphodiester bond is esterified; and an alkylphosphonate derivative in which the sugar moiety is modified with a modified sugar (e.g., a cross-linked sugar) described below and one non-bridging oxygen atom in the phosphodiester bond is replaced with a sulfur atom or the non-bridging oxygen atom in the phosphodiester bond is substituted with an alkyl group.

[0051] (Nucleoside) In this embodiment, "nucleoside" refers to a compound in which a purine base or a pyrimidine base is bound to a sugar. A naturally occurring nucleoside may be referred to as a "natural nucleoside." A modified nucleoside that does not exist in nature may be referred to as a "modified nucleoside." A modified nucleoside in which the sugar moiety is particularly modified may be referred to as a "modified sugar nucleoside."

[0052] (Nucleotide) In this embodiment, "nucleotide" refers to a compound in which a phosphate group is bound to the sugar of the above-mentioned nucleoside. A naturally occurring nucleotide may be referred to as a "natural nucleotide." A non-naturally occurring modified nucleotide may be referred to as a "modified nucleotide" or "modified nucleic acid." Examples of "modified nucleotide" or "modified nucleic acid" include a compound in which a phosphate group is bound to the sugar moiety of the above-mentioned modified nucleoside, a compound in which a modified phosphate group described below is bound to the sugar moiety of the above-mentioned modified nucleoside, and a compound in which a modified phosphate group described below is bound to the sugar moiety of a natural nucleoside.

[0053] (Sugar modification, modified sugar) In this embodiment, "sugar modification" means that the sugar moiety of the nucleotide is modified. The modified sugar moiety may be specifically referred to as a "modified sugar". Modified nucleotides with sugar modifications can be used as modified nucleic acids, such as AmNA (amide-bridged nucleic acid), GuNA (guanidine-bridged nucleic acid), scpBNA (2'-O,4'-C-spirocyclopropylene bridged nucleic acid), 2'-O-alkyl (e.g., 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, etc.), 2'-F,5'-methyl-DNA, and LNA (2',4'-Bridged Nucleic Acid / Locked Nucleic Acid). acid), ENA (2'-O,4'-C-Ethylene-Bridged Nucleic Acid), S-cEt (2',4'-constrained Ethyl Nucleic Acid), 5'-CP nucleic acid (5'-cyclopropyl Nucleic Acid), etc. Examples of LNAs include those containing structures represented by the symbols "A(L)", "5(L)", "G(L)", and "T(L)" described below. Examples of AmNAs include those containing structures represented by the symbols "A(Y)", "5(Y)", "G(Y)", and "T(Y)" described below. Examples of GuNAs include those containing structures represented by the symbols "A(Gx)", "5(Gx)", "G(Gx)", and "T(Gx)" described below. Examples of scpBNAs include those containing structures represented by the symbols "A(S)", "5(S)", "G(S)", and "T(S)" described below. Examples of 2'-MOE nucleic acids include those containing structures represented by the symbols "A(m)", "5(m)", "G(m)", and "T(m)" described below. Examples of 5'-CP nucleic acids include those containing structures represented by the symbols "A(5'-CP)", "5(5'-CP)", "G(5'-CP)", and "T(5'-CP)" described below.Examples of 2'-OMe nucleic acids include those containing structures represented by the symbols "A(M)", "C(M)", "G(M)", "U(M)", and "T(M)" as described below. Examples of MCE nucleic acids include those containing structures represented by the symbols "A(Mx)", "C(Mx)", "G(Mx)", and "U(Mx)" as described below.

[0054] (Modified Nucleic Acid Having a Substituent at the 2'-Position) In this embodiment, the term "modified nucleic acid having a substituent at the 2'-position" refers to a modified nucleic acid having a substituent at the 2'-position of the sugar moiety of the nucleotide. For example, non-crosslinked modified nucleic acids include 2'-O-alkyl (e.g., 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, MCE nucleic acid, etc.) and 2'-F, while crosslinked modified nucleic acids include LNA, AmNA, GuNA, scpBNA, ENA, S-cEt, etc.

[0055] (Nucleotide Modifications Known in the Art Other Than Sugar Modifications) Nucleotide modifications known in the art other than the sugar modifications described above can be used as modified nucleic acids for producing the antisense oligonucleotides of the present invention. Known nucleotide modifications include phosphate group modifications and nucleobase modifications, which will be described later. Examples of such nucleotide modifications include the nucleotide modifications described in W. Brad Wan et. al. J. Med. Chem. (2016) 59:9645-9667. (Non-Patent Document 3) and the like. These nucleotide modifications can be carried out based on methods known in the art described in the literature cited in the above literature.

[0056] (Phosphate Group) In this embodiment, the term "phosphate group" refers to a nucleotide in which the bond at the phosphate moiety is a naturally occurring phosphodiester bond (a bond indicated by the symbol "-" described below).

[0057] (Phosphate group modification, modified phosphate group) In this embodiment, "phosphate group modification" means that the phosphate moiety of the nucleotide is modified. The modified phosphate moiety may be particularly referred to as a "modified phosphate group." Examples of bond types containing the modified phosphate group include a phosphorothioate bond (a bond indicated by the symbol "∧" described below), a phosphorodithioate bond, a phosphoamidate bond (a bond indicated by the symbol "=" described below), a boranophosphate bond (a bond indicated by the symbol "x" described below), and an alkylphosphonate.

[0058] (Nucleobase modification, modified nucleobase) In this embodiment, "nucleobase modification" means that the nucleobase portion of the nucleotide is modified. The modified nucleobase portion may be particularly referred to as a "modified nucleobase". Examples of modified nucleobases include 5-methylcytosine, 5-hydroxymethylcytosine, and 5-propynylcytosine.

[0059] (DNA or RNA Analogs) The DNA or RNA analogs mentioned above refer to molecules having a structure similar to that of DNA or RNA, such as peptide nucleic acids (pNA) and morpholino nucleic acids.

[0060] (ncRNA) In this embodiment, "ncRNA" refers to a general term for RNA that is not involved in protein translation. Examples of the ncRNA include ribosomal RNA, transfer RNA, miRNA, and Natural Antisense Transcript (NAT).

[0061] (Nucleobase Moiety of Oligonucleotide) Examples of the nucleobase moiety of the oligonucleotide include a thyminyl group, a cytosinyl group, an adeninyl group, a guaninyl group, a 5-methylcytosinyl group, a uracilyl group, a 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl group, a 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl group, a 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl group, and a 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl group. Preferred examples of the nucleobase moiety include a thyminyl group, a cytosinyl group, an adeninyl group, a guaninyl group, a 5-methylcytosinyl group, and a uracilyl group. Among the nucleic acid bases, uracil (U) and thymine (T) are interchangeable, and both can form base pairs with adenine (A) in a complementary strand. Furthermore, cytosine (C) and 5-methylcytosine (5(x)) are interchangeable, and both can form base pairs with guanine (G) in a complementary strand. The same is true for the nucleic acid base portion of antisense oligonucleotides.

[0062] (Target RNA) In this embodiment, the term "target RNA" refers to RNA whose function is controlled by binding of the antisense oligonucleotide.

[0063] (Binding to target RNA) In this embodiment, "binding to target RNA" means that the nucleobases of the antisense oligonucleotide form a double-stranded nucleic acid together with the nucleobases of the target RNA due to complementarity with the target RNA. The double-stranded nucleic acid may be formed in at least a portion of the target RNA. The strength of binding to the target RNA can be measured, for example, by an index of thermal stability. An example of the index of thermal stability is the melting temperature (Tm value) of the double-stranded nucleic acid. The Tm value is preferably 40 to 90°C, more preferably 50 to 70°C.

[0064] (Target Region) The target region refers to a region in the target RNA that binds to the antisense oligonucleotide.

[0065] (Binding with target region) The binding with target region means that the antisense oligonucleotide of the present invention forms a double strand with target region.However, the antisense oligonucleotide of the present invention does not necessarily need to form a double strand with the whole target region, but can only form a double strand with a part of the target region.That is, the antisense oligonucleotide of the present invention is preferably one that has complete complementarity with target region, but as long as it binds with target RNA, it can only be complementary to at least a part of the target region.

[0066] (Part of the target region) The part of the target region means a region of the target region having a base length of 10 to 15 mer.

[0067] (Complementary to at least a portion of the target region) "Complementary to at least a portion of the target region" means complementary to the bases of at least a portion of the target region on the target RNA, which also includes complementary to the bases of a region on the mRNA or pre-mRNA corresponding to at least a portion of the region.

[0068] (Design of antisense oligonucleotide) In this embodiment, "designing antisense oligonucleotide" does not require that antisense oligonucleotide be actually synthesized, but can be sufficiently imagined in mind.However, typically, the imagined antisense oligonucleotide is embodied in a program (e.g., oligonucleotide design software, graphic design tool, office software, etc.) that runs on a computer or on paper.Therefore, for example, as described in the examples below, designing an antisense oligonucleotide that is expected to reduce delayed central nervous system toxicity, and summarizing this antisense oligonucleotide in table format also falls under the implementation of the design method of the present invention.

[0069] (Central Toxicity) In this embodiment, "central toxicity" is also referred to as "central neurotoxicity" and refers to toxic findings derived from the central nervous system identified by changes in general condition and histopathological changes in the brain in rodents, non-human primates, and humans. Central toxicity is classified into acute central toxicity and delayed central toxicity. Toxic findings derived from the central nervous system include, for example, in rodents, changes in general condition ranging from reversible and mild symptoms such as irritability, decreased locomotor activity, ataxic gait, and ptosis to severe symptoms such as convulsions and death. Specifically, "central toxicity" can be evaluated by behavioral scoring using a modified Irwin method (Irwin S., Psychopharmacologia. 1968; 13(3): 222-257).

[0070] (Delayed central toxicity) In this embodiment, "delayed central toxicity" refers to central toxicity that appears after the period during which acute central toxicity can appear has passed and recovery has occurred. Symptoms observed due to delayed central toxicity include, for example, decreased spontaneous movement, abnormal gait and hind limb function, tremor, weakness of the hind limbs or tail, loss of hind limb reflexes, weight loss, etc.

[0071] (Reduction of Delayed Central Toxicity) In this embodiment, "delayed central toxicity is reduced" means that the delayed central toxicity of the second antisense oligonucleotide is lower or expected to be lower than the delayed central toxicity of the first antisense oligonucleotide described below. To evaluate this toxicity, general condition findings observed on the observation day are scored according to the criteria exemplified below, and a clinical sign score is calculated by adding up the scores throughout the observation period, although the scoring criteria are not limited thereto. Since scores are added up depending on the intensity of toxic findings, antisense oligonucleotides for which a low clinical sign score is calculated can be determined to be highly safe. Furthermore, the pathological score is calculated by scoring cases in which abnormal findings were observed and cases in which they were not observed in brain pathology tests, although the scoring criteria are not limited thereto. The average value for each group is used for each score. For example, when a group of three animals is used and pathological findings are observed in only one animal at the time of sampling, the pathological score is calculated as 1 / 3 = 0.33. Furthermore, "subjects (individuals) sensitive to delayed central toxicity" refers to subjects selected using biomarkers, etc. Clinical sign score; 0 points: no abnormalities 1 point: abnormal hind limb function, tremors, decreased spontaneous movement 2 points: dragging of hind limbs, weakness of tail or hind limbs 3 points: complete hind limb dysfunction, paralysis of hind limbs, recumbency, prone position 4 points: euthanasia Pathological score; 0 points: no abnormalities 1 point: abnormalities (single cell necrosis, vacuolation, etc.)

[0072] <First Antisense Oligonucleotide> In this embodiment, the first antisense oligonucleotide is an antisense oligonucleotide in which each nucleoside is linked via a phosphate group and / or a modified phosphate group, and which includes a gap region, a 3' wing region linked to the 3' end of the gap region, and a 5' wing region linked to the 5' end of the gap region, the gap region being a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the 3' wing region and the 5' wing region being modified nucleic acids having a substituent at the 2' position, and the base length is 12 to 30 mer. The first antisense oligonucleotide can also be understood as the "base antisense oligonucleotide" or the "antisense oligonucleotide before improvement" in the design method according to this embodiment.

[0073] <Second Antisense Oligonucleotide> In this embodiment, the second antisense oligonucleotide has the same base sequences in the gap region, 3' wing region, and 5' wing region as the first antisense oligonucleotide, the sugar moieties of the nucleic acids in the 3' wing region and 5' wing region have the same structures as the first antisense oligonucleotide, and contains at least one 5'-CP nucleic acid in the gap region, and has reduced delayed central nervous system toxicity compared to the first antisense oligonucleotide. The second antisense oligonucleotide can also be understood as an "improved antisense oligonucleotide" in the design method according to this embodiment.

[0074] In this embodiment, "having the same base sequence" does not only mean that when two oligonucleotides are compared, their base sequences are completely identical, but also means that the corresponding nucleic acid bases are replaced with modified versions of the nucleic acid bases (e.g., modified versions with the same core structure but different substituents). For example, cytosine and 5-methylcytosine can be considered to be identical.

[0075] In this embodiment, "the sugar moieties of the nucleic acids have the same structure" means that when two oligonucleotides are compared, the sugar moieties of the nucleotides located at the same positions in both oligonucleotides have the same structure.

[0076] <Base Sequence of Antisense Oligonucleotide> The base sequence of the antisense oligonucleotide according to this embodiment is not particularly limited as long as it is identical between the first antisense oligonucleotide and the second antisense oligonucleotide, but examples include the base sequences described in the Examples below. In one aspect of this embodiment, the base sequence of the antisense oligonucleotide is preferably: (A) a base sequence having 90% to 100% sequence identity with respect to the base sequence set forth in any one of SEQ ID NOS: 1 to 27; (B) a base sequence in which one or several bases have been deleted, substituted, inserted, or added in the base sequence set forth in any one of SEQ ID NOS: 1 to 27; or (C) a base sequence that hybridizes under stringent conditions to an oligonucleotide having the base sequence set forth in any one of SEQ ID NOS: 1 to 27. Furthermore, in this embodiment, each base sequence shown in the sequence listing is used to indicate only the sequence information of the nucleobase portion. The structural information of the oligonucleotide, including the sugar portion and phosphate portion in addition to the nucleobase portion, is shown in the format shown in Table 1 in the Examples below.

[0077] In this embodiment, "sequence identity" refers to the percentage (%) of identical bases in the total overlapping base sequence in the optimal alignment when two base sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm can take into account the introduction of gaps into one or both of the sequences for optimal alignment). The "sequence identity" of a base sequence can be easily confirmed by those skilled in the art. For example, NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) can be used.

[0078] The base sequence of the single-stranded antisense oligonucleotide according to this embodiment preferably has 95% to 100% sequence identity to the base sequence set forth in any one of SEQ ID NOs: 1 to 27, more preferably 98% to 100% sequence identity, and even more preferably 100% sequence identity.

[0079] In this embodiment, examples of "a base sequence in which one or several bases have been deleted, substituted, inserted or added" include a base sequence that has, due to the deletion, substitution, insertion or addition, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the base sequence before the deletion, substitution, insertion or addition. The specific number of "one or several bases" may be one, two, three, four, or five of the above-mentioned deletions, substitutions, insertions or additions, each independently, or a combination of multiple deletions, substitutions, insertions or additions.

[0080] In this embodiment, "stringent conditions" refers to, for example, conditions in which the sample is incubated for 12 hours at room temperature in a solution containing 6xSSC (1xSSC has the composition: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5x Denhardt's solution, 100 μg / mL denatured salmon sperm DNA, and 50% (v / v) formamide, followed by washing in 0.5xSSC at a temperature of 50°C or higher. More stringent conditions are also encompassed, such as incubation for 12 hours at 45°C or 60°C, washing with 0.2xSSC or 0.1xSSC, or washing at a temperature of 60°C or 65°C or higher.

[0081] <Pharmacologically acceptable salt> The antisense oligonucleotide according to this embodiment may be in the form of a pharmacologically acceptable salt. Here, "pharmacologically acceptable salt" refers to a salt of the single-stranded antisense oligonucleotide of the present invention, which is a physiologically acceptable salt of the antisense oligonucleotide of the present invention, that is, a salt that retains the desired biological activity of the antisense oligonucleotide and does not retain undesired toxicological effects. The same applies to the double-stranded antisense oligonucleotide and antisense oligonucleotide complex described below.

[0082] Pharmaceutically Acceptable Salts In one aspect of this embodiment, the antisense oligonucleotide may be in the form of a pharmaceutically acceptable salt. Here, "pharmaceutically acceptable salts" refers to the pharmacologically acceptable salts described above that are acid addition salts or base addition salts. Examples of acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate, as well as organic acid salts such as citrate, oxalate, phthalate, fumarate, maleate, succinate, malate, acetate, formate, propionate, benzoate, trifluoroacetate, methanesulfonate, benzenesulfonate, para-toluenesulfonate, and camphorsulfonate. Examples of base addition salts include inorganic base salts such as sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, and aluminum salts, as well as organic base salts such as trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, dicyclohexylamine, and N,N-dibenzylethylamine. Further examples include salts with basic or acidic amino acids such as arginine, lysine, ornithine, aspartic acid, and glutamic acid (amino acid salts). The same applies to the double-stranded antisense oligonucleotides and antisense oligonucleotide complexes described below.

[0083] <Structure of Antisense Oligonucleotide> The antisense oligonucleotide according to this embodiment comprises a gap region, a 3' wing region attached to the 3' end of the gap region, and a 5' wing region attached to the 5' end of the gap region (see, for example, FIG. 1). The antisense oligonucleotide is preferably in a single-stranded form (single-stranded antisense oligonucleotide). In one aspect of this embodiment, the single-stranded antisense oligonucleotide may hybridize with a second-strand oligonucleotide described below to form a double-stranded form (double-stranded antisense oligonucleotide). The base sequence of the second-strand oligonucleotide preferably has a sequence identity of 90% to 100% based on a base sequence complementary to the base sequence of the single-stranded antisense oligonucleotide.

[0084] The single-stranded antisense oligonucleotide is a so-called gapmer-type single-stranded antisense oligonucleotide. Gapmer-type single-stranded antisense oligonucleotides inhibit the function of target RNA through the following mechanism. First, the single-stranded antisense oligonucleotide binds to the target region of the target RNA (top to center of Figure 2). Next, RNase H, an RNase, recognizes and binds to the complex of the single-stranded antisense oligonucleotide and the target RNA (center of Figure 2). The target RNA is then cleaved and degraded by an enzymatic degradation reaction caused by RNase H. At this time, the single-stranded antisense oligonucleotide is not affected by the enzymatic degradation by RNase H (bottom of Figure 2). Therefore, the single-stranded antisense oligonucleotide can bind to another target RNA and cleave and degrade that RNA. In this way, gapmer-type single-stranded antisense oligonucleotides function as a catalyst in the enzymatic degradation reaction caused by RNase H, and are therefore thought to have a sustained effect even when administered in small amounts.

[0085] (Gap Region) In the second antisense oligonucleotide, the gap region contains at least one 5'-CP nucleic acid. Furthermore, in the first antisense oligonucleotide and the second antisense oligonucleotide, the gap region is preferably a 5- to 20-mer nucleic acid composed of deoxyribose, which may contain a nucleic acid whose sugar moiety is modified. In other words, the gap region can also be understood as a 5- to 20-mer nucleic acid containing deoxyribose, which may contain a nucleic acid whose sugar moiety is modified. The gap region can also be understood as being composed of 5- to 20-mer natural nucleotides, non-natural nucleotides, or both, whose sugar moiety is deoxyribose. By having a sugar moiety of deoxyribose or modified deoxyribose, the gap region can form a complex with the target RNA that can be recognized by RNase H. Here, an example of a nucleic acid containing modified deoxyribose is a 5'-CP nucleic acid.

[0086] The number of bases in the gap region is preferably 5 to 20 mer, more preferably 6 to 17 mer, even more preferably 7 to 13 mer, and even more preferably 9 to 13 mer.

[0087] Examples of natural nucleotides in which the sugar moiety is deoxyribose include deoxyadenosine monophosphate, deoxyguanosine monophosphate, thymidine monophosphate, deoxycytidine monophosphate, deoxy-5-methylcytidine monophosphate (also referred to as 5-methyldeoxycytidine), etc. In other words, natural nucleotides constituting the gap region include those containing structural formulas corresponding to the symbols a, g, t, and c described below.

[0088] Examples of non-natural nucleotides in which the sugar moiety is deoxyribose or modified deoxyribose include 5'-CP nucleic acid, 2-thio-thymidine monophosphate, 2-aminoadenosine monophosphate, and 7-deazaguanosine monophosphate.

[0089] In addition, the gap region may be a nucleic acid in which some of the sugar moieties of a natural nucleotide whose sugar moiety is deoxyribose are modified sugars, as long as the effects of the present invention are achieved. That is, in one aspect of this embodiment, the gap region may be a nucleic acid in which some of the sugar moieties are deoxyribose and other sugar moieties are modified sugars (e.g., modified deoxyribose).

[0090] In the second antisense oligonucleotide, the 5'-CP nucleic acid is preferably positioned at least at the second position counting from the 5' side of the gap region.

[0091] In the second antisense oligonucleotide, the 5'-CP nucleic acid is preferably located in the gap region by two or more units, more preferably by 2 to 5 units. Here, the term "located by two or more units" encompasses both an embodiment in which two or more of the target 5'-CP nucleic acid are located contiguous with each other, and an embodiment in which two or more are located dispersedly in the cap region.

[0092] In the second antisense oligonucleotide, the 5'-CP nucleic acid is preferably arranged in a continuous 2- to 4-mer sequence at least at one position in the gap region.

[0093] In the second antisense oligonucleotide, the 5'-CP nucleic acid is preferably located on the 5'-end side of the gap region. Furthermore, the 5'-CP nucleic acid is preferably located on the 5'-end side and the 3'-end side of the gap region. Here, "located on the 5'-end side of the gap region" means located closer to the 5'-end than the center of the gap region. "located on the 3'-end side of the gap region" means located closer to the 3'-end than the center of the gap region.

[0094] In the second antisense oligonucleotide, the 5'-CP nucleic acid is preferably located in at least one-ninth of the gap region, more preferably at least one-fifth of the gap region, and the upper limit may be, for example, one-half or less.

[0095] In the second antisense oligonucleotide, the 5'-CP nucleic acid may be located at least at the second position counting from the 5' side of the gap region, and two or more 5'-CP nucleic acids may be located. Furthermore, in the second antisense oligonucleotide, the 5'-CP nucleic acid may be located at least at the second position counting from the 5' side of the gap region, and may be located consecutively in a 2- to 4-mer configuration at at least one location. Furthermore, in the second antisense oligonucleotide, the 5'-CP nucleic acid may be located at the 5'-end side of the gap region, and may be located consecutively in a 2- to 4-mer configuration at at least one location. Furthermore, in the second antisense oligonucleotide, the 5'-CP nucleic acid may be located at both the 5'-end and 3'-end sides of the gap region, and may be located consecutively in a 2- to 4-mer configuration at at least one location.

[0096] In the gap region constituting the second antisense oligonucleotide, the bond between the 5'-CP nucleic acid and the nucleoside adjacent to it on the 3' side is preferably a phosphorothioate bond (excluding cases where the 5'-CP nucleic acid is positioned at the 3' end of the gap region). Here, "the 5'-CP nucleic acid is positioned at the 3' end of the gap region" can also be understood as "the 5'-CP nucleic acid is bonded to a nucleoside in the 3' wing region." In one aspect of this embodiment, the bond between the 5'-CP nucleic acid and the nucleoside adjacent to it on the 3' side is preferably a phosphorothioate bond (excluding cases where the 5'-CP nucleic acid is positioned at the 3' end of the gap region or where the nucleoside adjacent to it on the 3' side is a 5'-CP nucleic acid).

[0097] In the gap region constituting the second antisense oligonucleotide, 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.

[0098] In the gap region constituting the second antisense oligonucleotide, the number of bases composed of deoxyribose (but excluding 5'-CP nucleic acid) which may contain nucleic acid whose sugar moiety is modified is preferably 5 or more, and more preferably 5 to 10.

[0099] The gap region constituting the second antisense oligonucleotide may contain two or more of the 5'-CP nucleic acids, and the number of bases composed of deoxyribose (but excluding 5'-CP nucleic acids) which may contain a nucleic acid whose sugar moiety is modified may be five or more. Alternatively, the gap region constituting the second antisense oligonucleotide may contain 2 to 5 of the 5'-CP nucleic acids, and the number of bases composed of deoxyribose (but excluding 5'-CP nucleic acids) which may contain a nucleic acid whose sugar moiety is modified may be 5 to 10.

[0100] In one aspect of this embodiment, it is preferred that in the first antisense oligonucleotide, the gap region does not contain a 5'-CP nucleic acid, and that in the second antisense oligonucleotide, the structure of the sugar moiety other than the 5'-CP nucleic acid in the gap region is the same as that of the first antisense oligonucleotide.

[0101] In another aspect of this embodiment, the cytosine located in the gap region in the first antisense oligonucleotide and the second antisense oligonucleotide may be 5-methylcytosine.

[0102] (3' Wing Region) In the first antisense oligonucleotide and the second antisense oligonucleotide, the 3' wing region is a modified nucleic acid having a substituent at the 2' position. In other words, the 3' wing region can also be understood to be composed of modified nucleotides having a substituent at the 2' position. The modified nucleic acid having a substituent at the 2' position in the 3' wing region preferably includes, as non-crosslinked 2'-modified nucleic acids, 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, and MCE nucleic acid, and as crosslinked modified nucleic acids, at least one selected from the group consisting of LNA, AmNA, GuNA, and scpBNA. By comprising the 3' wing region and the 5' wing region described below with the specified modified nucleotides, high binding affinity to the target RNA can be expected, and ultimately, the function of the target RNA can be effectively controlled. In one aspect of this embodiment, the 3' wing region may be a modified nucleic acid in which the sugar moiety is a modified sugar. Examples of modified nucleic acids in which the sugar moiety is a modified sugar include those listed above (Sugar modification, modified sugar). In one aspect of this embodiment, the modified nucleic acid having a substituent at the 2'-position in the 3'-wing region may consist solely of 2'-MOE nucleic acids. Note that multiple types of modified nucleic acids in the 3'-wing region may be contained in one single-stranded antisense oligonucleotide.

[0103] In the first antisense oligonucleotide and the second antisense oligonucleotide, the modified nucleic acid having a substituent at the 2'-position in the 3'-wing region preferably comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA, and more preferably is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

[0104] In the first antisense oligonucleotide and the second antisense oligonucleotide, the number of bases in the 3' wing region is preferably 3 to 5 mer.

[0105] (5' Wing Region) In the first antisense oligonucleotide and the second antisense oligonucleotide, the 5' wing region is a modified nucleic acid having a substituent at the 2' position. In other words, the 5' wing region can also be understood to be composed of modified nucleotides having a substituent at the 2' position. The modified nucleic acid in the 5' wing region preferably includes, as non-crosslinked 2'-position modified nucleic acids, 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, and MCE nucleic acid, and as crosslinked modified nucleic acids, at least one selected from the group consisting of LNA, AmNA, GuNA, and scpBNA. In one aspect of this embodiment, the 5' wing region may be a modified nucleic acid in which the sugar moiety is a modified sugar. Examples of modified nucleic acids in which the sugar moiety is a modified sugar include those listed above (Sugar modification, modified sugar). In one aspect of this embodiment, the modified nucleic acid in the 5' wing region having a substituent at the 2' position may be composed solely of 2'-MOE nucleic acid. In addition, multiple types of modified nucleic acids in the 5' wing region may be contained in one 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.

[0106] In the first antisense oligonucleotide and the second antisense oligonucleotide, the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region preferably comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA, and more preferably is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

[0107] In the first antisense oligonucleotide and the second antisense oligonucleotide, it is preferred that the modified nucleic acid having a substituent at the 2'-position in the 3'-wing region comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA, and that the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA. It is also more preferred that the modified nucleic acid having a substituent at the 2'-position in the 3'-wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA, and that the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

[0108] In the first antisense oligonucleotide and the second antisense oligonucleotide, the number of bases in the 5' wing region is preferably 3 to 5 mer.

[0109] In the first antisense oligonucleotide and the second antisense oligonucleotide, it is preferable that the number of bases in the gap region is 5 to 20 mer, the number of bases in the 3' wing region is 3 to 5 mer, and the number of bases in the 5' wing region is 3 to 5 mer.

[0110] In the first antisense oligonucleotide and the second antisense oligonucleotide, it is more preferable that the number of bases in the gap region is 7 to 13 mer, the number of bases in the 3' wing region is 3 to 5 mer, and the number of bases in the 5' wing region is 3 to 5 mer.

[0111] In the first antisense oligonucleotide and the second antisense oligonucleotide, it is more preferable that the number of bases in the gap region is 9 to 13 mer, the number of bases in the 3' wing region is 3 to 5 mer, and the number of bases in the 5' wing region is 3 to 5 mer.

[0112] (Sugar moiety structure of nucleic acid) The sugar moiety structure of the nucleic acid is not particularly limited as long as it has the configurations described above in the (gap region), (3' wing region), and (5' wing region) columns, and examples include those shown in the following Table 1. In Table 1, structural examples 1, 5, 9, 12, 15, 20, 22, and 25 correspond to the sugar moiety structures in the first antisense oligonucleotide, and the others correspond to the sugar moiety structures in the second antisense oligonucleotide.

[0113]

[0114] (Other Configurations) In one aspect of this embodiment, the single-stranded antisense oligonucleotide may further comprise a natural nucleotide linked to the 3'-end of the 3'-wing region. The number of bases of the natural nucleotide linked to the 3'-end of the 3'-wing region may be one or several, or may be one.

[0115] (Method of Representing Gapmer-Type Structures) The single-stranded antisense oligonucleotides of the present invention are gapmer-type. When representing gapmer-type structures, the notation method of "X-Y-Z" may be used. In the above notation method, "X" represents the number of bases in the 5' wing region, "Y" represents the number of bases in the gap region, and "Z" represents the number of bases in the 3' wing region.

[0116] "X-Y-Z" includes 2-8-4, 2-8-3, 2-8-5, 2-9-2, 2-9-3, 2-9-4, 2-9-5, 2-10-3, 2-10-4, 2-10-5, 2-11-3, 2-11-4, 2-11-5, 2-12-3, 2-12-4, 2-12-5, 3-8-2, 3-8-3, 3-8-4, 3-8-5, 3-9-3, 3-9-4, 3-9-5, 3-10-3, 3-10-4, 3-10-5, 3-11-3, 3-11-4, 3-11-5, 3-12-3, 3-12-4, 3-12-5, 3-13 -3, 3-13-4, 4-8-2, 4-8-3, 4-8-4, 4-8-5, 4-9-3, 4-9-4, 4-9-5, 4-10-3, 4-10-4, 4-10-5, 4-11-2, 4-11-3, 4-11-4, 4-11-5, 4-12-4, 4-13-3, 5-8-2, 5-8-3, 5-8-4, 5-8-5, 5-9-2, 5-9-3, 5-9-4, 5-9-5, 5-10-2, 5-10-3, 5-10-4, 5-10-5, 5-11-2, 5-11-3, 5-11-4, 5-11-5, etc. For example, the notation "2-8-4" means that the 5' wing region is a 2-mer oligonucleotide, the gap region is an 8-mer oligonucleotide, and the 3' wing region is a 4-mer oligonucleotide.

[0117] In the first antisense oligonucleotide and the second antisense oligonucleotide, the base length of the antisense oligonucleotide is 15 to 30 mer, preferably 15 to 20 mer, and more preferably 18 to 20 mer. When the base length of the single-stranded antisense oligonucleotide of the present invention is 15 to 20 mer or 18 to 20 mer, binding to the target RNA is particularly strong, and the expression of the target RNA can be more effectively regulated.

[0118] In this embodiment, the single-stranded antisense oligonucleotide has each nucleoside linked via a phosphate group and / or a modified phosphate group, and is preferably linked via a phosphodiester bond or a phosphorothioate bond.

[0119] In one aspect of this embodiment, at least one internucleoside linkage of the single-stranded antisense oligonucleotide is preferably a phosphorothioate linkage. In another aspect of this embodiment, at least one internucleoside linkage of the single-stranded antisense oligonucleotide is preferably a phosphodiester linkage.

[0120] In the internucleoside linkages constituting the second antisense oligonucleotide, the proportion of phosphorothioate linkages is preferably 50% to 80%, more preferably 50% to 70%.

[0121] In the first antisense oligonucleotide and the second antisense oligonucleotide, the GC ratio in the base sequence is preferably 0.2 to 0.6. The "GC ratio" refers to the ratio of the number of guanines and cytosines (including 5-methylcytosines) to the total number of guanines and cytosines in the base sequence of the antisense oligonucleotide.

[0122] One embodiment of the single-stranded antisense oligonucleotide of the present invention (the second antisense oligonucleotide) is a gapmer-type single-stranded antisense oligonucleotide having a gap region of 5-20 mer, a 5' wing region of 3-5 mer, and a 3' wing region of 3-5 mer. The gap region is positioned between the 5' wing region and the 3' wing region. The gap region contains at least one 5'-CP nucleic acid. Preferably, the 5' wing region and the 3' wing region each contain at least one 2'-MOE nucleic acid, LNA, AmNA, GuNA, or scpBNA. The 5' wing region and the 3' wing region may contain 2'-O-alkylated or 2'-F-substituted nucleotides. As the 2'-O-alkylated nucleotide, a 2'-O-alkylated (e.g., 2'-O-methylated) D-ribofuranose nucleotide may be used. The gapmer-type single-stranded antisense oligonucleotide may also form a double strand by hybridizing with a second strand oligonucleotide.

[0123] In one aspect of this embodiment, the antisense oligonucleotide is an antisense oligonucleotide or a pharmaceutically acceptable salt thereof with reduced delayed central toxicity, wherein each nucleotide of the antisense oligonucleotide is linked via a phosphate group and / or a modified phosphate group, the antisense oligonucleotide comprises a gap region, a 3' wing region linked to the 3' end of the gap region, and a 5' wing region linked to the 5' end of the gap region, the gap region is a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the gap region comprises at least one 5'-CP nucleic acid, the 3' wing region and the 5' wing region are modified nucleic acids having a substituent at the 2' position, and the base length of the antisense oligonucleotide is 12 to 30 mer.

[0124] In one aspect of this embodiment, the single-stranded antisense oligonucleotide (the second antisense oligonucleotide) is preferably at least one selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 7, 8, 10, 12, 13, 15, 16, 18, 19, 20, 21, 23, 24, 26, and 27 in Tables 2-1 to 2-8 described below. Here, "the single-stranded antisense oligonucleotide of SEQ ID NO: 2 in Table 2-1" refers to the single-stranded antisense oligonucleotide whose sequence name in Table 2-1 is "h451-465-N." The same applies to other cases.

[0125] <Double-stranded antisense oligonucleotide> The double-stranded antisense oligonucleotide of this embodiment is a double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, comprising the single-stranded antisense oligonucleotide (the second antisense oligonucleotide) and a second-strand oligonucleotide hybridized to the single-stranded antisense oligonucleotide. The base sequence of the second-strand oligonucleotide preferably has a sequence identity of 90% to 100% with respect to a base sequence complementary to the base sequence of the single-stranded antisense oligonucleotide.

[0126] The double-stranded antisense oligonucleotide can be dissociated in solution and separated into the single-stranded antisense oligonucleotide and the second-stranded oligonucleotide.The separated single-stranded antisense oligonucleotide can bind to the target RNA.The single-stranded antisense oligonucleotide can also be understood as a "first-stranded oligonucleotide" in relation to the second-stranded oligonucleotide.In addition, among the oligonucleotides constituting the double-stranded antisense oligonucleotide, the first-stranded oligonucleotide has an antisense strand to the target RNA, but for convenience, the double-stranded oligonucleotide consisting of the first-stranded oligonucleotide and the second-stranded oligonucleotide will be referred to as a "double-stranded antisense oligonucleotide".

[0127] <Method for Producing Single-Stranded Antisense Oligonucleotide> The method for producing an antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to this embodiment is a method for producing an antisense oligonucleotide or a pharmaceutically acceptable salt thereof having reduced delayed central toxicity, and includes the steps of: designing the second antisense oligonucleotide by the antisense oligonucleotide design method; and synthesizing the second antisense oligonucleotide.

[0128] The single-stranded antisense oligonucleotide (second antisense oligonucleotide) of the present invention can be produced by solid-phase synthesis using the phosphoramidite method. For example, a single-stranded oligonucleotide having a predetermined base sequence is first synthesized on a solid support using a commercially available automated nucleic acid synthesizer. Next, the synthesized single-stranded oligonucleotide is cleaved from the solid support using a basic substance or the like, and deprotected to obtain a crude single-stranded oligonucleotide. The crude single-stranded oligonucleotide obtained is then purified using HPLC or the like. In addition to the above-mentioned production method, the single-stranded antisense oligonucleotide of the present invention can also be produced by appropriately modifying the base sequence, modification site, etc. of the nucleic acid according to methods known to those skilled in the art. Furthermore, AmNA, GuNA, and scpBNA can be produced by the methods described in WO 2011 / 052436 (Patent Document 5), WO 2014 / 046212 (Patent Document 6), and WO 2015 / 125783 (Patent Document 7), respectively. 2'-MOE nucleic acids can be produced using commercially available amidites as reagents. 5'-CP nucleic acids can be produced by the method described in International Publication No. 2020 / 158910 (Patent Document 3). LNAs can be produced by the method described in International Publication No. 99 / 14226 (Patent Document 8).

[0129] <<Method for Producing Double-Stranded Antisense Oligonucleotide>> The double-stranded antisense oligonucleotide of the present invention can be produced by first producing an oligonucleotide (second strand oligonucleotide) having a predetermined sequence identity based on a base sequence complementary to the single-stranded antisense oligonucleotide using a production method similar to that of the single-stranded antisense oligonucleotide, and then hybridizing the single-stranded antisense oligonucleotide and the second strand oligonucleotide.

[0130] <<Antisense Oligonucleotide Conjugate>> The antisense oligonucleotide conjugate of this embodiment comprises the single-stranded antisense oligonucleotide (second antisense oligonucleotide) or a pharmaceutically acceptable salt thereof, or the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, and an additional substance bound to the single-stranded antisense oligonucleotide or the second strand oligonucleotide. The additional substance is selected from the group consisting of polyethylene glycol, peptides, alkyl chains (e.g., saturated aliphatic hydrocarbons, etc.), nucleic acids, ligand compounds, antibodies, proteins, and sugar chains (e.g., carbohydrates, polysaccharides, etc.).

[0131] In one aspect of this embodiment, the antisense oligonucleotide complex comprises the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, and an additional substance bound to the single-stranded antisense oligonucleotide, wherein the additional substance is selected from the group consisting of polyethylene glycol, peptides, alkyl chains (e.g., saturated aliphatic hydrocarbons, etc.), nucleic acids, ligand compounds, antibodies, proteins, and sugar chains (e.g., carbohydrates, polysaccharides, etc.).

[0132] In another aspect of this embodiment, the antisense oligonucleotide complex comprises the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, and an additional substance bound to the single-stranded antisense oligonucleotide or the second strand oligonucleotide, wherein the additional substance is selected from the group consisting of polyethylene glycol, peptides, alkyl chains (e.g., saturated aliphatic hydrocarbons, etc.), nucleic acids, ligand compounds, antibodies, proteins, and sugar chains (e.g., carbohydrates, polysaccharides, etc.).

[0133] In this embodiment, the term "additional substance" refers to a substance bound to the single-stranded antisense oligonucleotide or the second-strand oligonucleotide and used to impart a predetermined effect. The additional substance may be bound to the 5'-end, the 3'-end, or both the 5'-end and the 3'-end of the single-stranded antisense oligonucleotide. The additional substance may be bound to the 5'-end, the 3'-end, or both the 5'-end and the 3'-end of the second-strand oligonucleotide. In one aspect of this embodiment, the additional substance is preferably bound to either the 5'-end or the 3'-end of the single-stranded antisense oligonucleotide or the second-strand oligonucleotide. The additional substance may be directly bound to the single-stranded antisense oligonucleotide or the second-strand oligonucleotide via a covalent bond. The additional substance may be bound to the single-stranded antisense oligonucleotide or the second-strand oligonucleotide via a linker substance. Examples of the linker substance include linkers composed of alkyl, polyethylene glycol, peptide, disulfide, nucleic acid, etc., and / or combinations thereof. Examples of methods for binding the additional substance to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide include the methods described in the Examples below.

[0134] Examples of peptides that can be used as the additional substance include, but are not limited to, CPPs (Cell Penetrating Peptides), nuclear transport peptides, TAT (Trans-Activator of Transcription Protein), polyarginine, glucagon-like peptide-1 analogue peptides, synthetic cyclic RGD peptides, and brain-translocating peptides.

[0135] Examples of the ligand compound used as the additional substance include, but are not limited to, N-acetylgalactosamine (GalNAc), sugars (glucose, mannose, etc.), lipids (cholesterol, palmitic acid, docosahexaenoic acid, etc.), vitamins (folic acid, vitamin A, vitamin E (tocopherol), etc.), amino acids, and monoamine receptor ligands (indatraline, etc.).

[0136] Examples of antibodies that can be used as the additional substance include, but are not limited to, the following: anti-insulin receptor antibody, anti-transferrin receptor antibody, anti-LDL receptor-related protein antibody, anti-CD22 antibody, anti-CD30 antibody, and anti-HER2 antibody.

[0137] Examples of proteins that can be used as the additional substance include, but are not limited to, the following: albumin

[0138] Examples of nucleic acids used as the additional substance include the following: natural nucleotides, aptamers. The nucleic acids used as the additional substance are not counted in the base length of the antisense oligonucleotide.

[0139] Pharmaceutical Compositions Comprising Single-Stranded Antisense Oligonucleotides or the Like as Active Ingredients The pharmaceutical compositions of this embodiment comprise, as active ingredients, the single-stranded antisense oligonucleotides (second antisense oligonucleotides) of the present invention or pharmaceutically acceptable salts thereof, the double-stranded antisense oligonucleotides or pharmaceutically acceptable salts thereof, or the antisense oligonucleotide complexes or pharmaceutically acceptable salts thereof. Examples of the pharmaceutical compositions include therapeutic agents for diseases, preventive agents for diseases, etc. Any administration method and formulation known in the art can be used for the pharmaceutical composition of this embodiment. Hereinafter, the pharmaceutical composition may be referred to as a "pharmaceutical composition of antisense oligonucleotides or the like."

[0140] The pharmaceutical compositions (therapeutic agents, prophylactic agents, etc.) are used for the treatment or prevention of central nervous system diseases. Examples of central nervous system diseases include diseases associated with the RPS25 gene, i.e., diseases that can be caused by dipeptide repeats produced by RNA translation (sometimes referred to as "repeat diseases"). Specific examples of repeat 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), dentatorubral-pallidoluysian atrophy, spinal-bulbar muscular atrophy, Friedreich ataxia, fragile X-associated tremor ataxia syndrome, and myotonic dystrophy.

[0141] <Individual> The individual refers to a mammal. The individual is preferably a human, monkey, marmoset, dog, pig, rabbit, guinea pig, rat, or mouse. The individual is more preferably a human.

[0142] In one aspect of this embodiment, the single-stranded antisense oligonucleotide (second antisense oligonucleotide) or a pharmaceutical composition thereof can be administered via a suitable administration route to a subject (individual) susceptible to delayed central toxicity. Here, "delayed central toxicity" refers to central toxicity that appears after recovery from a period during which acute central toxicity may appear. Symptoms of delayed central toxicity include, for example, decreased locomotor activity, abnormal gait and hindlimb function, tremors, hindlimb or tail weakness, loss of hindlimb reflexes, and weight loss.

[0143] When administering the single-stranded antisense oligonucleotide of the present invention or a pharmaceutical composition thereof, the administration method and dosage form are not particularly limited. That is, any administration method and formulation known in the art can be used as the administration method and formulation of the antisense oligonucleotide of the present invention. Examples of administration methods include oral administration and parenteral administration. Examples of parenteral administration include ophthalmic administration, intravaginal administration, rectal administration, intranasal administration, transdermal administration, intravenous injection, infusion, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration by aspiration or inhalation, intrathecal administration, and intraventricular administration. In one aspect of this embodiment, the single-stranded antisense oligonucleotide or a pharmaceutical composition thereof is preferably administered so as to be exposed to the central nervous system. Examples of administration methods that result in exposure to the central nervous system include intrathecal administration and intraventricular administration.

[0144] Various pharmaceutical additives such as excipients, binders, wetting agents, disintegrants, lubricants, diluents, flavoring agents, fragrances, solubilizers, suspending agents, emulsifiers, stabilizers, preservatives, and isotonic agents may be mixed into the formulations of the antisense oligonucleotides of the present invention as needed.

[0145] When pharmaceutical compositions such as the antisense oligonucleotides of the present invention are administered locally, formulations such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders can be used.

[0146] When pharmaceutical compositions of the present invention, such as antisense oligonucleotides, are orally administered, formulations such as powders, granules, suspensions or solutions dissolved in water or non-aqueous media, capsules, powders, tablets, etc. can be used.

[0147] When pharmaceutical compositions such as antisense oligonucleotides of the present invention are administered parenterally, intrathecally, or intracerebroventricularly, formulations such as sterile aqueous solutions can be used.

[0148] The effective dose of the single-stranded antisense oligonucleotide of the present invention can be determined arbitrarily depending on the sex, age, weight, symptoms, etc. of the individual to be administered. Furthermore, it can also be determined arbitrarily depending on the method, route, frequency, etc. of administration. For example, the dose may be 0.01 to 100 mg / kg, preferably 0.1 to 50 mg / kg, and more preferably 0.1 to 10 mg / kg.

[0149] The above describes the method for designing and producing an antisense oligonucleotide according to the present embodiment. The single-stranded antisense oligonucleotide obtained by the above-described design and production method contains at least one 5'-CP nucleic acid in the gap region, thereby reducing delayed central nervous system toxicity.

[0150] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0151] <<Design of Single-Stranded Antisense Oligonucleotides>> First, the single-stranded antisense oligonucleotides shown in Tables 2-1 to 2-8 were designed. In each table, the sequence listed in the first row is the parent sequence (the sequence corresponding to the first antisense oligonucleotide), and the second and subsequent rows are sequences into which a 5'-CP nucleic acid was introduced based on the parent sequence (the sequence corresponding to the second antisense oligonucleotide).

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160] In this specification, the following symbols or notations may be used to represent corresponding structures:

[0161]

[0162]

[0163] In the structural formula shown above, R １ and R ２ are each independently a hydrogen atom or a linear or branched alkyl group having 1 to 3 carbon atoms. １ and R ２ Both represent a methyl group. ３ , R ４ , and R ５ are each independently a hydrogen atom, a linear or branched alkyl group having 1 to 7 carbon atoms, or a cycloalkyl group having 3 to 7 carbon atoms. ３ and R ５ are both hydrogen atoms, and R ４ When R is a methyl group, it is represented as "Gm" and R ３ is a hydrogen atom, and R ４ and R ５ When both are methyl groups, it is represented as "Gdm", and R ３ and R ５ is a hydrogen atom, and R ４ When is a tert-butyl group, it is represented as "GtB".

[0164] <<Production of Single-Stranded Antisense Oligonucleotides>> Single-stranded antisense oligonucleotides were prepared for some of the designs using the following procedure. Single-stranded antisense oligonucleotides containing modified nucleic acids such as 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, AmNA, scpBNA, 5'-CP nucleic acid, and / or GuNA, and / or nucleic acids whose nucleobase moiety is 5-methylcytosine, were synthesized on a 0.2 μmol scale using an automated nucleic acid synthesizer (nS-8 model, manufactured by Gene Design Co., Ltd.). Chain elongation was carried out using a standard phosphoramidite protocol. However, when adding a monomer bearing a levunilyl protecting group at the 5' position, the solid support was deprotected by treating it with 0.5 M hydrazine hydrate in pyridine-acetic acid (1:1 v / v) at room temperature outside the synthesizer for 1 hour, after which the solid support was attached to the synthesizer and synthesis resumed. CPG resin was used as the solid support. Furthermore, DDTT (((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazaoline-3-thione) or the like was used for sulfurization to form the phosphorothioate (PS) backbone. Single-stranded antisense oligonucleotides containing 2'-MOE nucleic acid, AmNA, and / or scpBNA were obtained in such a way that the terminal 5'-position hydroxyl group was not protected with a DMTr (4,4'-dimethoxytrityl) group and the 3'-position was supported on a solid phase. Subsequently, the single-stranded antisense oligonucleotide was cleaved from the solid phase support by alkali treatment and recovered in solution. Thereafter, the solvent was evaporated from the recovered solution to obtain a crude product. The resulting crude product was purified by reverse-phase HPLC to obtain a purified single-stranded antisense oligonucleotide. The purity and structure of each single-stranded antisense oligonucleotide obtained were confirmed by LC-MS (Waters). The measurement results are shown in Tables 2-1 to 2-8.

[0165] Evaluation of Central Toxicity of Antisense Oligonucleotides (1) Delayed central toxicity of single-stranded antisense oligonucleotides was evaluated by intracerebroventricular administration to FVB / NJcl mice or ICR mice, which were kept for up to 28 days after administration, and by observing their general condition and body weight and by performing pathological examinations (hematoxylin-eosin staining) of tissue samples at the time of collection. The "score" column was calculated based on the following evaluation criteria. Each score was calculated as the average value for each group. Clinical sign score: 0 points: no abnormalities; 1 point: abnormal hind limb function, tremors, decreased spontaneous movement; 2 points: dragging of hind limbs, weakness of tail or hind limbs; 3 points: complete hind limb dysfunction, paralysis of hind limbs, recumbency, prone position; 4 points: euthanasia; Pathological score: 0 points: no abnormalities; 1 point: abnormalities (single cell necrosis, vacuolation, etc.)

[0166] As representative examples, the evaluation results of delayed central nervous system toxicity of the single-stranded antisense oligonucleotides described in Tables 2-1 to 2-7 are shown in Tables 3-1 to 3-7. In Tables 3-1 to 3-8, the symbols "-" indicate that no measurement or evaluation was performed. The "Observation Date" and "Clinical Sign Score" columns in Table 3-7 represent the evaluation results obtained from the experiment described in "Evaluation of Central Neurotoxicity of Antisense Oligonucleotides (2)" below. The results in Tables 3-1 to 3-7 confirmed that delayed central nervous system toxicity was reduced in the single-stranded antisense oligonucleotides (second antisense oligonucleotides) with 5'-CP introduced, compared to the single-stranded antisense oligonucleotides (first antisense oligonucleotides) with the parent sequence.

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173] Evaluation of Central Toxicity of Antisense Oligonucleotides (2) Delayed central toxicity of the single-stranded antisense oligonucleotides listed in Tables 2-7 and 2-8 was evaluated by intracerebroventricular administration to FVB / NJcl or ICR mice, followed by evaluation up to 28 days after administration. Evaluation items included behavioral assessment, observation of general condition and body weight, and pathological examination of the brain (hematoxylin-eosin staining) at the time of sampling. Behavioral evaluation of mice was performed using a Functional Observational Battery (FOB). Evaluation items included 1) posture, 2) external abnormalities, 3) stereotypic behavior, 4) responsiveness to stimuli, 5) grip strength, 6) respiration, and 7) tremors / convulsions. Each was scored as 0 for normal, 1 for slightly abnormal, and 2 for severely abnormal (see the Clinical sign score column in Table 3-7). Each histopathological finding was scored according to the degree of 1) single-cell necrosis, 2) vacuolization, or 3) foam cell transformation of neurons in two locations on the cerebral cortex, with 0 representing no findings, 1 representing mild, 2 representing moderate, and 3 representing severe (the column for histopathological findings in Table 3-8).

[0174] As representative examples, the evaluation results of delayed central nervous system toxicity of the single-stranded antisense oligonucleotides described in Tables 2-7 to 2-8 are shown in Tables 3-7 to 3-8. The "Sampling Date" and "Pathological Score" columns in Table 3-7 represent the evaluation results obtained from the experiment described in the above-mentioned "Evaluation of Central Nervous System Toxicity of Antisense Oligonucleotides (1)." The results in Tables 3-7 to 3-8 confirmed that delayed central nervous system toxicity was reduced in the single-stranded antisense oligonucleotide (second antisense oligonucleotide) with a 5'-CP-introduced sequence compared to the single-stranded antisense oligonucleotide (first antisense oligonucleotide) with the parent sequence.

[0175]

[0176]

[0177] <<Evaluation of RPS25 Gene Expression (In Vitro Evaluation)>> RPS25 gene expression was evaluated using human fetal kidney cells according to the single-stranded antisense oligonucleotides produced. The RPS25 gene expression evaluation can also be performed using human iPS cell-derived neurons. In this example, gene expression evaluation refers to assessing the amount of mRNA by measuring the amount of complementary DNA (cDNA) obtained by reverse transcription. The specific procedures for each expression evaluation are described below.

[0178] <Expression Evaluation Using Human Embryonic Kidney Cells> Human embryonic kidney cells HEK293T (ATCC® CRL-3216™) were cultured in a culture medium at 37°C and 5% CO ２ The HEK293T cells were cultured under the following conditions. The culture medium used had the following composition:

[0179] Composition of culture medium for HEK293T cells Dulbecco's modified Eagle's medium (DMEM): Sigma, Cat# D6429 10% Fetal bovine serum (FBS): BioWest, Cat# S1820 100-fold diluted penicillin-streptomycin mixed solution: Nacalai Tesque, Cat# 09367-34 (penicillin 10,000 units / ml, streptomycin 10,000 μg / ml, stabilizer included)

[0180] First, on the day before measuring the expression level of the human RPS25 gene, HEK293T cells (12,000 cells / well) were seeded in a 96-well plate and incubated at 37°C, 5% CO ２ The cells were cultured overnight under the conditions of 0.5 nM, 5 nM, 15 nM, or 50 nM. Then, each single-stranded antisense oligonucleotide diluted in phosphate-buffered saline (PBS) (final concentration: 0.5 nM, 5 nM, 15 nM, or 50 nM) was transfected into the above cells by lipofection. As a negative control, cells transfected with PBS containing no single-stranded antisense oligonucleotide were used. The transfected cells were incubated in growth medium at 37°C and 5% CO. ２The cells were cultured for 48 hours under the conditions of 95°C for 3 seconds and 60°C for 30 seconds. The growth medium was then removed, and the extracted total RNA was reverse-transcribed using the Taqman Fast Cells-to-CT Kit (Thermo Fisher Scientific, Cat# 4399003). The complementary DNA (cDNA) obtained from this reverse transcription reaction was used to perform real-time PCR using pre-designed gene-specific probes (see below) in Taqman gene expression assays (Applied Biosystems) (40 cycles of 95°C for 3 seconds, 60°C for 30 seconds).

[0181] List of gene-specific probes used in the evaluation of human RPS25 gene expression: human RPS25: Hs01568661_g1 human GAPDH: 4326317E (internal control)

[0182] The human RPS25 mRNA expression ratios for each single-stranded antisense oligonucleotide relative to human RPS25 mRNA, determined by the above-described method, are shown in Tables 4-1 to 4-7 (column "In Vitro Evaluation"). The human RPS25 mRNA expression ratio determined in the negative control group was set to 1.00. Single-stranded antisense oligonucleotides with an expression ratio of 0.80 or less were determined to be capable of suppressing human RPS25 mRNA expression. Note that "-" in the tables indicates that no measurement was performed. It is generally believed that suppression of mRNA expression also suppresses subsequent translation into protein. Therefore, single-stranded antisense oligonucleotides with an expression ratio of 0.80 or less can be determined to be capable of modulating the function of the human RPS25 gene.

[0183] <<In vivo expression evaluation of RPS25 gene>> Expression evaluation of the RPS25 gene was performed by intracerebroventricular administration to mice and measuring the amount of mRNA in each region of the prefrontal cortex. In this example, gene expression evaluation refers to evaluating the amount of mRNA by measuring the amount of complementary DNA (cDNA) obtained by reverse transcription. Specific procedures for each expression evaluation are described below.

[0184] FVB mice (CLEA Japan) were anesthetized with isoflurane (Pfizer, Cat# 114133403). Next, antisense oligonucleotides dissolved in artificial cerebrospinal fluid (Tocris Bioscience, Cat# 3525 / 25 mL) were administered to the anesthetized FVB mice at 10 μL / individual using a two-stage needle (Top, Medical Device Approval Number 15800BZZ01460000) attached to a 50 μL Hamilton syringe (Hamilton, Cat# 705LT). Negative control mice received only artificial cerebrospinal fluid at 10 μL / individual.

[0185] After a certain period of rearing following administration, the FVB mice were euthanized, and three sites were collected: the brain, cervical spinal cord, and lumbar spinal cord. The collected tissues were immersed in RNA Later (Applied Biosystems, Cat# AM7024) overnight and then stored at -80°C. RNA was extracted from the stored tissue samples using an RNeasy Mini Kit (QIAGEN, Cat# 74106). Reverse transcription of the extracted mRNA was performed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cat# 4368814). 1 μg of mRNA was diluted to 20 μL and used for the reverse transcription reaction. The complementary DNA (cDNA) obtained from this reverse transcription reaction was used to perform real-time PCR (40 cycles of 95°C for 3 seconds, 60°C for 30 seconds) using predesigned gene-specific probes (see below) in TaqManExpression Assays (Applied Biosystems).

[0186] List of gene-specific probes used in evaluating the expression of the mouse RPS25 gene: Mouse Rps25: Mm02342783_g1 Mouse GAPDH: 4352339E (internal control)

[0187] As representative examples, Tables 4-1 to 4-7 show the expression ratios of mouse RPS25 mRNA determined by the above-described method for the single-stranded antisense oligonucleotides listed in Tables 2-1 to 2-7 (in the "In Vivo Evaluation" column). The expression ratio of mouse RPS25 RNA determined in the negative control group was set to 1.00. The "Sampling Date" in Tables 4-1 to 4-7 indicates the number of days between administration of the single-stranded antisense oligonucleotide and collection of the specified tissue. Since inhibition of mRNA expression is generally considered to also inhibit subsequent protein translation, an expression ratio of 0.80 or less can be determined to be a single-stranded antisense oligonucleotide capable of modulating the function of the mouse RPS25 gene. The target regions to which the single-stranded antisense oligonucleotides listed in Tables 4-1 to 4-7 bind are regions whose sequences are conserved between the human RPS25 gene and the mouse RPS25 gene.

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195] <<Suppression of mRNA Expression by Antisense Oligonucleotides in Human Neuroblastoma Cell Lines>> Antisense oligonucleotides were used to evaluate the effect of suppressing UBE3A-ATS mRNA expression in human neuroblastoma cells. A cell line not treated with antisense oligonucleotides served as a control. Kelly cells (ECACC, EC92110411-F0) were used as the human neuroblastoma cell line. Each antisense oligonucleotide was introduced into Kelly cells using a commercially available transfection reagent (ThermoFisher Scientific, Lipofectamine 3000), and the mRNA expression level was measured by qRT-PCR to examine knockdown activity (mRNA expression suppression). The specific procedure is described below.

[0196] 2.0 × 10 Kelly cells in the logarithmic growth phase ４ The cells were seeded at 1000 cells / well in a 96-well plate in Roswell Park Memorial Institute medium (RPMI-1640) containing 10% fetal bovine serum (FBS). After 24 hours, each antisense oligonucleotide was added to the wells and incubated for 24 hours (final concentration of antisense oligonucleotide in the medium: 1 nM or 10 nM).

[0197] After incubation, the cells were collected and total RNA was extracted using an RNA extraction reagent (ThermoFisher Scientific, MagMAX mirVana Total RNA Isolation Kit). Using the total RNA as a template, reverse transcription and PCR amplification reactions were performed using a nucleic acid amplification reaction reagent (QIAGEN, QuantiNova Probe RT-PCR kit). The nucleic acid amplification reaction was performed using temperature cycling: 45 ° C, 10 minutes → 95 ° C, 5 minutes → [(95 ° C, 5 seconds → 60 ° C, 30 seconds) x 40 cycles]. In real-time PCR, the mRNA amount of the housekeeping gene β-actin (ACTB) was also quantified simultaneously, and the mRNA amount of UBE3A-ATS relative to the mRNA amount of ACTB was evaluated using the ΔΔCt method. The amount of mRNA induced by each antisense oligonucleotide is shown as a relative value, with the amount of mRNA in cells not treated with the oligonucleotide set at 1.

[0198] The primer sets used were as follows: (PTEN detection primer set) TaqMan Gene Expression Assay Hs01372957_m1 (ThermoFisher Scientific) (ACTB detection primer set) TaqMan Gene Expression Assay Hs99999903_m1 (ThermoFisher Scientific).

[0199] The results are shown in Table 4-8. When the value for cells that did not incorporate the antisense oligonucleotide (untreated cells, "control") was set to 1, it was confirmed that the antisense oligonucleotide-treated cells showed knockdown of the UBE3A-ATS gene (inhibition of mRNA expression).

[0200]

[0201] <<Evaluation of Caspase 3 / 7 Activity by Antisense Nucleotides in the Human Cervical Cancer-Derived Hela S3 Cell Line>> Caspase 3 / 7 activity was evaluated using the following procedure. Hela S3 cells (ATCC, CCL-2.2) were used as the human cervical cancer-derived Hela cell line. Each antisense oligonucleotide was introduced into Hela S3 cells using a commercially available transfection reagent (ThermoFisher Scientific, Lipofectamine 3000), and caspase 3 / 7 activity was examined using the Caspase-Glo 3 / 7 Assay System (Promega). The specific procedure is described below.

[0202] First, 2.0 × 10 HeLaS3 cells in the logarithmic growth phase were ４The cells were seeded at 100 cells / well in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). After 24 hours, each antisense oligonucleotide was added to the wells (final concentration of antisense oligonucleotide in the medium: 3, 10, 30, 100, or 300 nM), and the cells were incubated for 24 hours. After incubation, the cells were harvested, and caspase 3 / 7 activity was assayed using a kit. The activity value of each antisense oligonucleotide was expressed as a relative value, with the activity value of cells that had not taken up the antisense oligonucleotide (untreated cells, "control") set at 100.

[0203] The results are shown in Table 5. The results in Table 5 confirmed that, compared to the single-stranded antisense oligonucleotide of the parent sequence (first antisense oligonucleotide), the single-stranded antisense oligonucleotide of the sequence into which 5'-CP was introduced (second antisense oligonucleotide) suppressed the increase in caspase 3 / 7 activity and reduced cytotoxicity.

[0204]

[0205] Although the embodiments and examples of the present invention have been described above, it is also planned from the beginning that the configurations of the above-described embodiments and examples may be appropriately combined.

[0206] The embodiments and examples disclosed herein are illustrative in all respects and should not be considered limiting. The scope of the present invention is defined by the claims, not by the embodiments and examples described above, and is intended to include meanings equivalent to the claims and all modifications within the scope of the claims.

Claims

1. A method for designing an antisense oligonucleotide having reduced delayed central toxicity, comprising the step of designing a second antisense oligonucleotide based on a first antisense oligonucleotide, wherein the first antisense oligonucleotide is an antisense oligonucleotide in which each nucleoside is linked via a phosphate group and / or a modified phosphate group, the first antisense oligonucleotide comprises a gap region, a 3' wing region linked to the 3' end of the gap region, and a 5' wing region linked to the 5' end of the gap region, the gap region being a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the 3' wing region and the 5' wing region being modified nucleic acid having a substituent at the 2' position, and the base length is 12 to 30 mer, and the second antisense oligonucleotide is an antisense oligonucleotide in which the base sequences of the gap region, 3' wing region and 5' wing region are identical to those of the first antisense oligonucleotide, the sugar moiety structures of the nucleic acid in the 3' wing region and 5' wing region are identical to those of the first antisense oligonucleotide, The method for designing an antisense oligonucleotide, comprising: comprising at least one 5'-CP nucleic acid in the gap region; and the antisense oligonucleotide has reduced delayed central toxicity compared to the first antisense oligonucleotide.

2. The method for designing an antisense oligonucleotide according to claim 1, wherein in the first antisense oligonucleotide, the gap region does not contain a 5'-CP nucleic acid, and in the second antisense oligonucleotide, the structure of the sugar moiety other than the 5'-CP nucleic acid in the gap region is identical to that of the first antisense oligonucleotide.

3. The method for designing an antisense oligonucleotide according to claim 1 or 2, wherein in the first antisense oligonucleotide and the second antisense oligonucleotide, the number of bases in the gap region is 5 to 20 mer, the 3' wing region is a 3 to 5 mer modified nucleic acid having a substituent at the 2' position, and the 5' wing region is a 3 to 5 mer modified nucleic acid having a substituent at the 2' position.

4. A method for designing an antisense oligonucleotide according to any one of claims 1 to 3, wherein the base length of the first antisense oligonucleotide and the second antisense oligonucleotide is 15 to 30 mer.

5. A method for designing an antisense oligonucleotide according to any one of claims 1 to 4, wherein in the first antisense oligonucleotide and the second antisense oligonucleotide, the cytosine located in the gap region is 5-methylcytosine.

6. A method for designing an antisense oligonucleotide according to any one of claims 1 to 5, wherein in the first antisense oligonucleotide and the second antisense oligonucleotide, the modified nucleic acid having a substituent at the 2' position in the 3' wing region comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA, and the modified nucleic acid having a substituent at the 2' position in the 5' wing region comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.

7. A method for designing an antisense oligonucleotide according to any one of claims 1 to 6, wherein the GC ratio in the base sequence of the first antisense oligonucleotide and the second antisense oligonucleotide is 0.2 to 0.

6.

8. A method for designing an antisense oligonucleotide according to any one of claims 1 to 7, wherein the first antisense oligonucleotide and the second antisense oligonucleotide are single-stranded antisense oligonucleotides.

9. A method for designing an antisense oligonucleotide according to any one of claims 1 to 8, wherein in the second antisense oligonucleotide, the 5'-CP nucleic acid is positioned at least at the second position counting from the 5' side of the gap region.

10. A method for designing an antisense oligonucleotide according to any one of claims 1 to 9, wherein in the second antisense oligonucleotide, two or more of the 5'-CP nucleic acids are positioned in the gap region.

11. A method for designing an antisense oligonucleotide according to any one of claims 1 to 10, wherein in the second antisense oligonucleotide, 2 to 5 of the 5'-CP nucleic acids are positioned in the gap region.

12. A method for designing an antisense oligonucleotide according to any one of claims 1 to 11, wherein in the second antisense oligonucleotide, the 5'-CP nucleic acid is arranged in a continuous 2-4 mer configuration at least at one location in the gap region.

13. A method for designing an antisense oligonucleotide according to any one of claims 1 to 12, wherein in the second antisense oligonucleotide, the 5'-CP nucleic acid is positioned at least one-ninth of the gap region.

14. A method for designing an antisense oligonucleotide according to any one of claims 1 to 13, wherein in the second antisense oligonucleotide, the 5'-CP nucleic acid is positioned at least one-fifth of the gap region.

15. A method for designing an antisense oligonucleotide according to any one of claims 1 to 14, wherein in the second antisense oligonucleotide, the 5'-CP nucleic acid is positioned on the 5'-end side of the gap region.

16. A method for designing an antisense oligonucleotide according to any one of claims 1 to 15, wherein in the second antisense oligonucleotide, the 5'-CP nucleic acid is positioned on the 5'-end and 3'-end sides of the gap region.

17. A method for designing an antisense oligonucleotide according to any one of claims 1 to 16, wherein the proportion of phosphorothioate bonds in the internucleoside linkages constituting the second antisense oligonucleotide is 50% to 80%.

18. A method for designing an antisense oligonucleotide according to any one of claims 1 to 17, wherein the proportion of phosphorothioate bonds in the internucleoside linkages constituting the second antisense oligonucleotide is 50% to 70%.

19. A method for designing an antisense oligonucleotide according to any one of claims 1 to 18, wherein in the gap region constituting the second antisense oligonucleotide, the bond between the 5'-CP nucleic acid and the nucleoside adjacent to the 3' side of the 5'-CP nucleic acid is a phosphorothioate bond (except when the 5'-CP nucleic acid is positioned at the 3' end of the gap region).

20. A method for designing an antisense oligonucleotide according to any one of claims 1 to 19, wherein in the gap region constituting the second antisense oligonucleotide, the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is a phosphorothioate bond (excluding the cases where the 5'-CP nucleic acid is positioned at the 3' end of the gap region and where the nucleoside adjacent on the 3' side of the 5'-CP nucleic acid is a 5'-CP nucleic acid).

21. A method for designing an antisense oligonucleotide according to any one of claims 1 to 20, wherein in the gap region constituting the second antisense oligonucleotide, 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.

22. A method for designing an antisense oligonucleotide according to any one of claims 1 to 21, wherein the number of bases composed of deoxyribose (but excluding 5'-CP nucleic acid) which may contain a nucleic acid whose sugar moiety is modified in the gap region constituting the second antisense oligonucleotide is 5 or more.

23. A method for designing an antisense oligonucleotide according to any one of claims 1 to 22, wherein the number of bases composed of deoxyribose (but excluding 5'-CP nucleic acid) which may contain a nucleic acid whose sugar moiety is modified in the gap region constituting the second antisense oligonucleotide is 5 to 10.

24. A method for designing an antisense oligonucleotide according to any one of claims 1 to 23, wherein in the gap region constituting the second antisense oligonucleotide, 5 to 10 bases composed of deoxyribose (but excluding 5'-CP nucleic acid) which may contain a nucleic acid whose sugar moiety is modified are arranged in succession.

25. A method for producing an antisense oligonucleotide or a pharma- ceutical acceptable salt thereof having reduced delayed central toxicity, comprising the steps of: designing the second antisense oligonucleotide by the method for designing an antisense oligonucleotide according to any one of claims 1 to 24; and synthesizing the second antisense oligonucleotide.

26. An antisense oligonucleotide or a pharma- ceutically acceptable salt thereof having reduced delayed central toxicity, wherein each nucleoside in the antisense oligonucleotide is linked via a phosphate group and / or a modified phosphate group, the antisense oligonucleotide comprises a gap region, a 3' wing region linked to the 3' end of the gap region, and a 5' wing region linked to the 5' end of the gap region, the gap region being a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the gap region comprises at least one 5'-CP nucleic acid, the 3' wing region and the 5' wing region are modified nucleic acids having a substituent at the 2' position, and the base length of the antisense oligonucleotide is 12 to 30 mer, or a pharma-ceutically acceptable salt thereof.

27. An antisense oligonucleotide complex or a pharma- ceutically acceptable salt thereof, comprising the antisense oligonucleotide according to claim 26 or a pharma- ceutically acceptable salt thereof, and an additional substance bound to the antisense oligonucleotide, wherein the additional substance is selected from the group consisting of polyethylene glycol, a peptide, an alkyl chain, a nucleic acid, a ligand compound, an antibody, a protein, and a sugar chain.

28. A therapeutic agent for a disease comprising the antisense oligonucleotide according to claim 26 or a pharma- ceutically acceptable salt thereof, or the antisense oligonucleotide complex according to claim 27 or a pharma- ceutically acceptable salt thereof, the therapeutic agent being administered so as to be exposed to the central nervous system.

29. A therapeutic agent for a disease comprising the antisense oligonucleotide of claim 26 or a pharma- ceutically acceptable salt thereof, or the antisense oligonucleotide conjugate of claim 27 or a pharma- ceutically acceptable salt thereof, said therapeutic agent being administered to a subject susceptible to delayed central nervous system toxicity.

30. A therapeutic agent for a disease comprising the antisense oligonucleotide according to claim 26 or a pharma- ceutically acceptable salt thereof, or the antisense oligonucleotide complex according to claim 27 or a pharma- ceutically acceptable salt thereof, wherein the disease is a disease of the central nervous system.