WO2024257848A1 - Procédé de conception d'oligonucléotide antisens ayant une toxicité centrale retardée réduite, et son procédé de production - Google Patents

Procédé de conception d'oligonucléotide antisens ayant une toxicité centrale retardée réduite, et son procédé de production Download PDF

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WO2024257848A1
WO2024257848A1 PCT/JP2024/021614 JP2024021614W WO2024257848A1 WO 2024257848 A1 WO2024257848 A1 WO 2024257848A1 JP 2024021614 W JP2024021614 W JP 2024021614W WO 2024257848 A1 WO2024257848 A1 WO 2024257848A1
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antisense oligonucleotide
nucleic acid
gap region
designing
region
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智彦 青山
成宏 浅野
友美 角谷
峻哲 川野邊
アジャヤラム セレスタ
高尾 鈴木
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Luxna Biotech Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing

Definitions

  • the present invention relates to a method for designing and producing an antisense oligonucleotide with reduced delayed central nervous system toxicity.
  • Antisense oligonucleotides exert their effects by hybridizing to the target nucleic acid sequence and suppressing gene expression itself.
  • a variety of modified nucleic acids have been developed and introduced with the aim of improving the nuclease resistance of antisense oligonucleotides and improving their binding affinity and specificity to the target nucleic acid, and various formulations have been brought to the market.
  • a new problem that has recently emerged is how to avoid the potential toxicity of modified nucleic acids.
  • toxicity of antisense oligonucleotides can be categorized into "toxicity caused by hybridization with non-target RNA (off-target toxicity in the narrow sense)” and “toxicity not caused by hybridization with RNA (off-target toxicity in the broad sense)” caused by binding to proteins and metal ions inside and outside the cell. Various approaches are being adopted to avoid these toxicities.
  • Patent Document 1 discloses that broad off-target toxicity can be avoided by performing appropriate chemical modifications on the base and sugar portions of nucleic acids.
  • Patent Document 2 discloses that narrow off-target toxicity can be avoided by modifying the 2' carbonyl group of the base portion (thymine) and bridging the 2'-4' positions of the sugar portion to suppress the formation of non-Watson-Crick base pairs in hybridization with off-target RNA.
  • Non-Patent Document 1 discloses that phosphorothioate modifications between nucleosides are the cause of liver toxicity
  • Patent Document 3 discloses that liver toxicity can be reduced while maintaining activity by replacing 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.
  • toxicity assessments of antisense oligonucleotide toxicity reduction technologies have been conducted mainly using hepatotoxicity as an indicator, taking into account that the organ most likely to accumulate after systemic exposure is the liver, and no in-depth knowledge has been gained about toxicity assessments in other tissues.
  • central toxicity acute central toxicity, delayed central toxicity, etc.
  • central toxicity is an important evaluation item in pharmaceutical safety testing, but while there has been active development of antisense oligonucleotides targeting central nervous system disorders in recent years, there is almost no knowledge about the central toxicity of antisense oligonucleotides.
  • Patent Document 4 discloses a correlation between the sequence of antisense oligonucleotides and central toxicity (oscillation of intracellular free calcium concentration in nerve cells).
  • 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.
  • Non-Patent Document 2 discloses that antisense oligonucleotides (hereinafter sometimes referred to as "ASO") from which some phosphorothioate modifications have been removed and from which a modification that contributes to stability has been added to the 2' position of the sugar moiety may have reduced antisense activity compared to ASOs from which the phosphorothioate modifications have not been removed.
  • ASO antisense oligonucleotides
  • the present invention was 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.
  • an antisense oligonucleotide or a pharma- ceutical acceptable salt thereof (hereinafter sometimes referred to as the "antisense oligonucleotide of the present invention") so that the gap region contains a 5'-CP nucleic acid, delayed central nervous system toxicity can be reduced while maintaining antisense activity, and thus completed the present invention. That is, the present invention is as follows.
  • the method for designing an antisense oligonucleotide of the present invention comprises: Designing a second antisense oligonucleotide based on the first antisense oligonucleotide;
  • a method for designing an antisense oligonucleotide having reduced delayed central toxicity comprising the steps of:
  • the first antisense oligonucleotide is each nucleoside is linked by a phosphate group and/or a modified phosphate group; a gap region, a 3' wing region attached to the 3' end of the gap region, and a 5' wing region attached to the 5' end of the gap region;
  • the gap region is a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the 3' wing region and the 5' wing region are modified nucleic acids having a substitution at the 2'position;
  • the base length is 12 to 30 mer.
  • the second antisense oligonucleotide is the base sequences of the gap region, the 3' wing region and the 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 the 5' wing region have the same structures as those of the first antisense oligonucleotide; comprising at least one 5'-CP nucleic acid in the gap region; Compared to the first antisense oligonucleotide, delayed central toxicity is reduced.
  • the gap region does not contain a 5'-CP nucleic acid;
  • the structure of the sugar moiety other than the 5'-CP nucleic acid in the gap region is preferably the same as that of the first antisense oligonucleotide.
  • the first antisense oligonucleotide and the second antisense oligonucleotide are The number of bases in the gap region is 5 to 20 mer, the 3' wing region is a 3-5 mer modified nucleic acid having a substituent at the 2' position, The 5' wing region is preferably a 3- to 5-mer modified nucleic acid having a substituent at the 2' position.
  • the base length is preferably 15 to 30 mer.
  • the cytosine placed in the gap region is preferably 5-methylcytosine.
  • the modified nucleic acid having a substituent at the 2'-position in the 3'-wing region comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA;
  • the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region preferably comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.
  • the GC ratio in the base sequence is preferably 0.2 to 0.6.
  • the first antisense oligonucleotide and the second antisense oligonucleotide are single-stranded antisense oligonucleotides.
  • the 5'-CP nucleic acid is preferably located at least at the second position counting from the 5' side of the gap region.
  • the 5'-CP nucleic acid is preferably arranged in succession in 2- to 4-mer sequences at least at one site in the gap region.
  • the 5'-CP nucleic acid is located at a position 1/9 or more relative to the gap region.
  • the 5'-CP nucleic acid is arranged in a position that is one-fifth or more of the gap region.
  • the 5'-CP nucleic acid is preferably located on the 5'-end side of the gap region.
  • the 5'-CP nucleic acid is preferably located on the 5'-terminal side and the 3'-terminal side of the gap region.
  • the percentage of phosphorothioate bonds is preferably 50% to 80%.
  • the percentage of phosphorothioate bonds is preferably 50% to 70%.
  • the bond between the 5'-CP nucleic acid and the nucleoside adjacent to the 3' side of the 5'-CP nucleic acid is preferably a phosphorothioate bond (except when the 5'-CP nucleic acid is positioned at the 3' end of the gap region).
  • the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is preferably a phosphorothioate bond (excluding the cases where the 5'-CP nucleic acid is positioned at the 3' end of the gap region and where the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is a 5'-CP nucleic acid).
  • the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 5' side of the 5'-CP nucleic acid is preferably a phosphodiester bond.
  • the number of bases composed of deoxyribose (but excluding 5'-CP nucleic acid) which may contain nucleic acid with a modified sugar moiety is preferably 5 or more.
  • the number of bases composed of deoxyribose (but excluding 5'-CP nucleic acid) which may contain nucleic acid with a modified sugar moiety is preferably 5 to 10.
  • a method for producing the antisense oligonucleotide or a pharma- ceutical acceptable salt thereof of the present invention comprises the steps of: 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 [1] to [24] above; and synthesizing the second antisense oligonucleotide.
  • the antisense oligonucleotide of the present invention comprises: An antisense oligonucleotide or a pharma- ceutical acceptable salt thereof, which has reduced delayed central toxicity,
  • the antisense oligonucleotide has each nucleoside linked by a phosphate group and/or a modified phosphate group;
  • the antisense oligonucleotide comprises a gap region, a 3' wing region attached to the 3' end of the gap region, and a 5' wing region attached to the 5' end of the gap region;
  • the gap region is a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the gap region comprises at least one 5'-CP nucleic acid;
  • the 3' wing region and the 5' wing region are modified nucleic acids having a substitution at the 2'position;
  • the base length of the antisense oligonucleotide is 12 to 30
  • the antisense oligonucleotide complex of the present invention or a pharma- ceutical acceptable salt thereof comprises: The antisense oligonucleotide according to [26] or a pharma- ceutical acceptable salt thereof; and an additional substance bound to the antisense oligonucleotide, or a pharma- ceutically acceptable salt thereof,
  • the additional substance is selected from the group consisting of polyethylene glycol, peptides, alkyl chains, nucleic acids, ligand compounds, antibodies, proteins, and sugar chains.
  • the therapeutic agent for a disease comprises A therapeutic agent for a disease, comprising the antisense oligonucleotide of [26] above or a pharma- ceutical acceptable salt thereof, or the antisense oligonucleotide complex of [27] above or a pharma-ceutical acceptable salt thereof,
  • the therapeutic agent is adapted to be administered so as to be exposed to the central nervous system.
  • the therapeutic agent for a disease comprises A therapeutic agent for a disease, comprising the antisense oligonucleotide of [26] above or a pharma- ceutical acceptable salt thereof, or the antisense oligonucleotide complex of [27] above or a pharma-ceutical acceptable salt thereof,
  • the therapeutic agent is administered to a subject susceptible to delayed central toxicity.
  • the therapeutic agent for a disease comprises A therapeutic agent for a disease, comprising the antisense oligonucleotide of [26] above or a pharma- ceutical acceptable salt thereof, or the antisense oligonucleotide complex of [27] above or a pharma-ceutical acceptable salt thereof,
  • the therapeutic agent for the above disease is a disease of the central nervous system.
  • the present invention makes it possible to provide a method for designing and producing an antisense oligonucleotide that has reduced delayed central nervous system toxicity while retaining antisense activity.
  • FIG. 1 is a schematic diagram showing an example of the configuration of an antisense oligonucleotide according to this embodiment.
  • FIG. 2 is a schematic diagram illustrating the mechanism by which the expression of a target RNA is suppressed when the antisense oligonucleotide according to this embodiment is used.
  • this embodiment is not limited to this.
  • the notation in the form "I to J” means the upper and lower limits of a range (i.e., I or more and J or less).
  • the method for designing an antisense oligonucleotide having reduced delayed central toxicity includes the steps of: Designing a second antisense oligonucleotide based on the first antisense oligonucleotide; A method for designing an antisense oligonucleotide having reduced delayed central toxicity, comprising the steps of:
  • the first antisense oligonucleotide is each nucleoside is linked by a phosphate group and/or a modified phosphate group; a gap region, a 3' wing region attached to the 3' end of the gap region, and a 5' wing region attached to the 5' end of the gap region;
  • the gap region is a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the 3' wing region and the 5' wing region are modified nucle
  • the second antisense oligonucleotide is the base sequences of the gap region, the 3' wing region and the 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 the 5' wing region have the same structures as those of the first antisense oligonucleotide; comprising at least one 5'-CP nucleic acid in the gap region; Compared to the first antisense oligonucleotide, delayed central toxicity is reduced.
  • single-stranded antisense oligonucleotide refers to an oligonucleotide or a pharmacologically acceptable salt thereof that is complementary to the mRNA, pre-mRNA, or ncRNA (non-coding RNA) of a target gene (hereinafter, these three may be collectively referred to as "target RNA”).
  • target RNA refers to an oligonucleotide or a pharmacologically acceptable salt thereof that is complementary to the mRNA, pre-mRNA, or ncRNA (non-coding RNA) of a target gene (hereinafter, these three may be collectively referred to as "target RNA”).
  • Antisense oligonucleotides are composed of DNA, RNA, and/or analogs thereof.
  • Antisense oligonucleotides form a double strand with the target mRNA, pre-mRNA, or ncRNA to suppress the action of the target mRNA, pre-mRNA, or ncRNA.
  • Antisense oligonucleotides include those that have a completely complementary base sequence to the base sequence of the target mRNA, pre-mRNA, or ncRNA, those that have a base sequence in which one or several bases are deleted, substituted, inserted, or added in the complementary base sequence, and those that contain a base that forms a wobble base pair in their base sequence.
  • the antisense oligonucleotide of the present invention may further contain modified nucleotides known in the art other than the "modified nucleic acid whose sugar moiety is a modified sugar" (sugar-modified modified nucleotides) described below.
  • modified nucleotides known in the art include phosphate-modified modified nucleotides and nucleic acid base-modified modified nucleotides described below.
  • both ends of the antisense oligonucleotide in this embodiment is not particularly limited, and may be, for example, -OH or -OR (wherein R represents an alkyl chain, a phosphate ester, or an additional substance described later).
  • the single-stranded antisense oligonucleotide in this embodiment may be in the form of a single strand, or may hybridize with a second strand oligonucleotide described below to form a double strand.
  • the double-stranded oligonucleotide consisting of the single-stranded antisense oligonucleotide and the second strand oligonucleotide hybridized to the single-stranded antisense oligonucleotide may be referred to as a "double-stranded antisense oligonucleotide".
  • oligonucleotide refers to a polymer of nucleotides in which 2 to 30 identical or different nucleosides are linked together by phosphodiester bonds or other bonds.
  • the oligonucleotide can also be understood to be composed of a nucleic acid base portion, a phosphate portion, and a sugar portion, as shown in the following structural formula:
  • oligonucleotides are broadly classified into natural oligonucleotides and non-natural oligonucleotides.
  • Natural oligonucleotides refers to oligonucleotides made of naturally occurring nucleotides.
  • Non-natural oligonucleotides refers to oligonucleotides containing at least one modified nucleotide as a constituent unit, as described below.
  • Non-natural oligonucleotides preferably include modified sugar derivatives in which the sugar portion is modified; phosphorothioate derivatives in which one non-bridging oxygen atom of the phosphodiester bond is replaced with a sulfur atom; phosphorodithioate derivatives in which two non-bridging oxygen atoms of the phosphodiester bond are replaced with sulfur atoms; ester derivatives in which the phosphodiester bond is tri-esterified; phosphoamide derivatives in which the phosphodiester bond is amidated; boranophosphate derivatives in which the phosphodiester bond is boronated; alkylphosphonate (e.g., methylphosphonate, methoxypropylphosphonate, etc.) derivatives in which the non-bridging oxygen atom of the phosphodiester bond is replaced with an alkyl group; amide derivatives in which the phosphodiester bond is replaced with an amide bond; and modified base derivatives in which the nucleic acid base is modified.
  • the above-mentioned non-natural oligonucleotide includes a cross-linked modified sugar derivative in which the sugar portion is modified; a phosphorothioate derivative in which one non-bridging oxygen atom of the phosphodiester bond is replaced with a sulfur atom; an ester derivative in which the phosphodiester bond is esterified; and an alkylphosphonate derivative in which the sugar portion is modified with a modified sugar (e.g., a cross-linked sugar) described below and one non-bridging oxygen atom of the phosphodiester bond is replaced with a sulfur atom or the non-bridging oxygen atom of the phosphodiester bond is replaced with an alkyl group.
  • a modified sugar e.g., a cross-linked sugar
  • nucleoside refers to a compound in which a purine base or a pyrimidine base is bound to a sugar.
  • a naturally occurring nucleoside may be referred to as a "natural nucleoside”.
  • a modified nucleoside that does not exist in nature may be referred to as a "modified nucleoside”.
  • a modified nucleoside in which the sugar moiety is modified may be referred to as a "modified sugar nucleoside”.
  • nucleotide refers to a compound in which a phosphate group is bound to the sugar of the nucleoside.
  • a naturally occurring nucleotide may be referred to as a "natural nucleotide”.
  • a modified nucleotide that does not exist in nature may be referred to as a "modified nucleotide” or "modified nucleic acid”.
  • modified nucleotide or “modified nucleic acid” include a compound in which a phosphate group is bound to the sugar moiety of the modified nucleoside, a compound in which a modified phosphate group described below is bound to the sugar moiety of the modified nucleoside, and a compound in which a modified phosphate group described below is bound to the sugar moiety of a natural nucleoside.
  • sugar modification means that the sugar moiety of the nucleotide is modified.
  • the modified sugar moiety may be specifically referred to as a "modified sugar”.
  • Modified nucleotides with sugar modifications can be used as modified nucleic acids, and examples of such modified nucleic acids include 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).
  • nucleic acids examples include 2'-O,4'-C-Ethylene-Bridged Nucleic Acid (ENA), 2',4'-constrained Ethyl Nucleic Acid (S-cEt), and 5'-CP nucleic acid (5'-cyclopropyl Nucleic Acid).
  • LNA examples include those containing structures represented by the symbols “A(L)”, “5(L)”, “G(L)”, and “T(L)” as described below.
  • AmNA examples include those containing structures represented by the symbols "A(Y)”, “5(Y)", “G(Y)", and "T(Y)” as described below.
  • Examples of GuNA include those containing structures represented by the symbols “A(Gx)”, “5(Gx)”, “G(Gx)”, and “T(Gx)” as described below.
  • Examples of scpBNA include those containing structures represented by the symbols “A(S)”, “5(S)”, “G(S)”, and “T(S)” as described below.
  • Examples of 2'-MOE nucleic acids include those containing structures represented by the symbols “A(m)”, “5(m)”, “G(m)”, and “T(m)” as described below.
  • Examples of 5'-CP nucleic acids include those containing structures represented by the symbols “A(5'-CP)”, “5(5'-CP)”, “G(5'-CP)”, and “T(5'-CP)” as described below.
  • Examples of 2'-OMe nucleic acids include those containing structures represented by the symbols “A(M)”, “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.
  • modified nucleic acid having a substituent at the 2'-position refers to a modified nucleic acid having a substituent at the 2'-position of the sugar moiety of the above nucleotide.
  • non-bridged modified nucleic acids include 2'-O-alkyl (e.g., 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, MCE nucleic acid, etc.) and 2'-F, while bridged modified nucleic acids include LNA, AmNA, GuNA, scpBNA, ENA, S-cEt, etc.
  • Nucleotide modifications known in the art other than sugar modifications can be used as modified nucleic acids for producing the antisense oligonucleotides of the present invention.
  • nucleotide modifications phosphate group modifications and nucleic acid base modifications described below are known. Examples of such nucleotide modifications include nucleotide modifications described in W. Brad Wan et. Al. J. Med. Chem. (2016) 59: 9645-9667. (Non-Patent Document 3) and the like. These nucleotide modifications can be carried out based on methods known in the art described in the documents cited in the above documents.
  • phosphate group refers to a nucleotide in which the bond at the phosphate moiety is a naturally occurring phosphodiester bond (a bond indicated by the symbol "-" as described below).
  • phosphate group modification means that the phosphate moiety of the nucleotide is modified.
  • the modified phosphate moiety may be specifically referred to as a "modified phosphate group.”
  • nucleobase modification means that the nucleobase portion of the nucleotide is modified.
  • the modified nucleobase portion may be specifically referred to as a "modified nucleobase.”
  • modified nucleobases include 5-methylcytosine, 5-hydroxymethylcytosine, and 5-propynylcytosine.
  • DNA or RNA analogues means a molecule having a structure similar to that of DNA or RNA, such as peptide nucleic acid (pNA) and morpholino nucleic acid.
  • pNA peptide nucleic acid
  • ncRNA refers to a general term for RNA that is not involved in protein translation.
  • examples of the ncRNA include ribosomal RNA, transfer RNA, miRNA, and Natural Antisense Transcript (NAT).
  • nucleic acid base moiety of the oligonucleotide examples include thyminyl, cytosinyl, adeninyl, guaninyl, 5-methylcytosinyl, uracilyl, 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl, 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl, and 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl groups.
  • examples of the nucleic acid base moiety include thyminyl, cytosinyl, adeninyl, guaninyl, 5-methylcytosinyl, and uracilyl groups.
  • uracil (U) and thymine (T) are interchangeable, and both can form a base pair with adenine (A) of a complementary strand.
  • Cytosine (C) and 5-methylcytosine (5(x)) are interchangeable and can form base pairs with guanine (G) in a complementary strand.
  • the same is true for the nucleic acid base portion of an antisense oligonucleotide.
  • Target RNA refers to an RNA whose function is controlled by binding to the antisense oligonucleotide.
  • binding to a target RNA means that the nucleic acid base of the antisense oligonucleotide forms a double-stranded nucleic acid together with the nucleic acid base of the target RNA due to complementarity with the target RNA.
  • the double-stranded nucleic acid may be formed in at least a part of the target RNA.
  • the strength of the binding to the target RNA can be measured, for example, by an index of thermal stability.
  • An example of the index of thermal stability is the melting temperature (Tm value) of the double-stranded nucleic acid.
  • the Tm value is preferably 40 to 90°C, more preferably 50 to 70°C.
  • the target region refers to a region in the target RNA that binds to the antisense oligonucleotide.
  • the binding with the above-mentioned target region means that the antisense oligonucleotide of the present invention forms a double strand with the target region.
  • the antisense oligonucleotide of the present invention does not necessarily need to form a double strand with the whole target region, and can be formed double strand with a part of the target region.
  • the antisense oligonucleotide of the present invention is preferably one that has complete complementarity with the target region, but can be complementary with at least a part of the target region as long as it binds with the target RNA.
  • the part of the target region means a region of the target region having a base length of 10 to 15 mer.
  • “Complementary to at least a portion of the target region” means complementary to the bases of at least a portion of the target region on the target RNA, including complementary to the bases of a region on an mRNA or pre-mRNA corresponding to the at least a portion of the region.
  • design of antisense oligonucleotides does not require the antisense oligonucleotide to be actually synthesized, but it is sufficient to imagine it in one's mind.
  • the imaged antisense oligonucleotide is embodied on a program (e.g., oligonucleotide design software, graphic design tool, office software, etc.) that operates on a computer or on paper.
  • a program e.g., oligonucleotide design software, graphic design tool, office software, etc.
  • designing an antisense oligonucleotide that is expected to reduce delayed central toxicity and summarizing the antisense oligonucleotide in the form of a table also corresponds to the implementation of the design method of the present invention.
  • central toxicity is also called “central neurotoxicity” and refers to a toxicological finding derived from the central nervous system identified by general condition changes and brain histopathological changes in rodents, non-human primates, and humans. The central toxicity is classified into acute central toxicity and delayed central toxicity. Toxicological findings derived from the central nervous system include, for example, in rodents, general condition changes including reversible and minor symptoms such as irritability, decreased spontaneous movement, ataxic gait, and ptosis, to severe symptoms such as convulsions and death. Specifically, "central toxicity” can be evaluated by a behavioral score performed by the modified Irwin method (Irwin S., Psychopharmacologia. 1968; 13(3): 222-257).
  • delayed central nervous system toxicity refers to central toxicity that appears after recovery from the period during which acute central toxicity may appear. Examples of symptoms observed due to delayed central toxicity include decreased spontaneous movement, abnormal gait and hind limb function, tremors, weakness of the hind limbs or tail, loss of hind limb reflexes, weight loss, etc.
  • delayed central toxicity is reduced means that the delayed central toxicity of the second antisense oligonucleotide is low or is expected to be low compared to the delayed central toxicity of the first antisense oligonucleotide described later.
  • This toxicity is calculated by scoring the general condition findings observed on the observation date according to the following criteria, and adding up the scores throughout the observation period to calculate a clinical sign score, but the criteria for scoring are not limited to these. Since the score is added depending on the intensity of the toxic findings, an antisense oligonucleotide with a low clinical sign score can be judged to be highly safe.
  • the pathological score is calculated by scoring the cases where abnormal findings were observed and those where they were not observed in the pathological examination of the brain, but the criteria for scoring are not limited to these.
  • subjects (individuals) sensitive to delayed central toxicity refers to subjects selected by biomarkers, etc.
  • Clinical sign score 0 points: no abnormality 1 point: abnormal hind leg function, tremor, decreased spontaneous movement 2 points: dragging of hind legs, weakness of tail or hind legs 3 points: complete hind leg dysfunction, paralysis of hind legs, recumbency, prone position 4 points: euthanasia Pathological score; 0 points: no abnormality 1 point: abnormality (single cell necrosis, vacuolation, etc.)
  • the first antisense oligonucleotide is each nucleoside is linked by a phosphate group and/or a modified phosphate group; a gap region, a 3' wing region attached to the 3' end of the gap region, and a 5' wing region attached to the 5' end of the gap region; the gap region is a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the 3' wing region and the 5' wing region are modified nucleic acids having a substitution at the 2'position;
  • the base length is 12 to 30 mer, and the antisense oligonucleotide is an antisense oligonucleotide.
  • the first antisense oligonucleotide can also be understood as the "base antisense oligonucleotide” or the "antisense oligonucleotide before improvement" in the design method
  • the second antisense oligonucleotide is the base sequences of the gap region, the 3' wing region and the 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 the 5' wing region have the same structures as those of the first antisense oligonucleotide; comprising at least one 5'-CP nucleic acid in the gap region; Compared to the first antisense oligonucleotide, delayed central toxicity is reduced.
  • the second antisense oligonucleotide can also be understood as the "improved antisense oligonucleotide" in the design method according to 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 includes cases in which the corresponding nucleic acid bases are replaced with modified versions of the nucleic acid bases (e.g., modified versions with the same base structure but different substituents). For example, cytosine and 5-methylcytosine can be considered to be identical.
  • the sugar moieties of the nucleic acids have the same structure
  • “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.
  • the base sequence of the antisense oligonucleotide of this embodiment is not particularly limited as long as it is identical between the first antisense oligonucleotide and the second antisense oligonucleotide, and examples thereof include the base sequences described in the examples below.
  • sequence identity refers to the percentage of identical bases in the total overlapping base sequence in the optimal alignment when two base sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm can take into account the introduction of gaps into one or both of the sequences for optimal alignment).
  • sequence identity of base sequences can be easily confirmed by those skilled in the art. For example, NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) can be used.
  • the base sequence of the single-stranded antisense oligonucleotide according to this embodiment preferably has 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.
  • a base sequence in which one or several bases have been deleted, substituted, inserted or added can be, for example, a base sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity due to the deletion, substitution, insertion or addition to the base sequence before the deletion, substitution, insertion or addition.
  • the specific number of "one or several bases” may be one, two, three, four or five of the above-mentioned deletions, substitutions, insertions or additions, each independently, or a combination of multiple bases.
  • stringent conditions refers to conditions such as, for example, incubation for 12 hours at room temperature in a solution containing 6xSSC (1xSSC composition: 0.15M NaCl, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5x Denhardt's, 100 ⁇ g/mL denatured salmon sperm DNA, and 50% (v/v) formamide, followed by washing with 0.5xSSC at a temperature of 50°C or higher.
  • more stringent conditions such as incubation for 12 hours at 45°C or 60°C, washing with 0.2xSSC or 0.1xSSC, and washing at temperatures of 60°C or 65°C or higher are also included.
  • the antisense oligonucleotide according to this embodiment may be in the form of a pharmacologically acceptable salt.
  • pharmaceutically acceptable salt refers to the salt of the single-stranded antisense oligonucleotide of the present invention, which is the physiologically acceptable salt of the antisense oligonucleotide of the present invention, that is, the salt that retains the desired biological activity of the antisense oligonucleotide and does not retain undesired toxicological effects.
  • the antisense oligonucleotide may be in the form of a pharma- ceutically acceptable salt.
  • pharmaceutically acceptable salt refers to a salt that is the pharmacologically acceptable salt described above and is an acid addition salt or a base addition salt.
  • acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate, and organic acid salts such as citrate, oxalate, phthalate, fumarate, maleate, succinate, malate, acetate, formate, propionate, benzoate, trifluoroacetate, methanesulfonate, benzenesulfonate, para-toluenesulfonate, and camphorsulfonate.
  • inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate
  • organic acid salts such as citrate, oxalate, phthalate, fumarate, maleate, succinate, malate, acetate, formate, propionate, benzoate, trifluoroacetate, methanesulfonate, benzenesulf
  • base addition salts include inorganic base salts such as sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, and aluminum salts, as well as organic base salts such as trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, dicyclohexylamine, and N,N-dibenzylethylamine.
  • inorganic base salts such as sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, and aluminum salts
  • organic base salts such as trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexy
  • salts with basic or acidic amino acids such as arginine, lysine, ornithine, aspartic acid, or glutamic acid (amino acid salts).
  • basic or acidic amino acids such as arginine, lysine, ornithine, aspartic acid, or glutamic acid (amino acid salts).
  • the antisense oligonucleotide includes a gap region, a 3' wing region bound to the 3' end of the gap region, and a 5' wing region bound to the 5' end of the gap region (see, for example, FIG. 1).
  • the antisense oligonucleotide is preferably in a single-stranded form (single-stranded antisense oligonucleotide).
  • the single-stranded antisense oligonucleotide may hybridize with a second-stranded oligonucleotide described below to form a double-stranded form (double-stranded antisense oligonucleotide).
  • the base sequence of the second-stranded oligonucleotide is preferably a base sequence having a sequence identity of 90% or more and 100% or less based on a base sequence complementary to the base sequence of the single-stranded antisense oligonucleotide.
  • the single-stranded antisense oligonucleotide is a so-called gapmer type single-stranded antisense oligonucleotide.
  • the gapmer type single-stranded antisense oligonucleotide inhibits the function of the target RNA by the following mechanism. First, the single-stranded antisense oligonucleotide binds to the target region of the target RNA (top to center of FIG. 2). Next, RNase H, an RNA degrading enzyme, recognizes and binds to the complex of the single-stranded antisense oligonucleotide and the target RNA (center of FIG. 2).
  • the target RNA is then cleaved and degraded by an enzymatic degradation reaction by RNase H.
  • the single-stranded antisense oligonucleotide is not affected by the enzymatic degradation by RNase H (lower part of FIG. 2). Therefore, the single-stranded antisense oligonucleotide can bind to another target RNA and cleave and degrade the RNA.
  • the gapmer type single-stranded antisense oligonucleotide functions as a catalyst in the enzymatic degradation reaction by RNase H described above, and is therefore considered to have a predetermined effect even when administered in small amounts.
  • the gap region comprises at least one 5'-CP nucleic acid.
  • the gap region is preferably a 5-20 mer nucleic acid composed of deoxyribose, which may contain a nucleic acid whose sugar moiety is modified.
  • the gap region can also be understood as a 5-20 mer nucleic acid containing deoxyribose, which may contain a nucleic acid whose sugar moiety is modified.
  • the number of bases in the gap region is preferably 5-20 mer, more preferably 6-17 mer, even more preferably 7-13 mer, and even more preferably 9-13 mer.
  • Examples of natural nucleotides in which the sugar moiety is deoxyribose include deoxyadenosine monophosphate, deoxyguanosine monophosphate, thymidine monophosphate, deoxycytidine monophosphate, and deoxy-5-methylcytidine monophosphate (also called 5-methyldeoxycytidine).
  • examples of natural nucleotides that make up the gap region include those that contain structural formulas corresponding to the symbols a, g, t, and c, which will be described later.
  • non-natural nucleotides in which the sugar moiety is deoxyribose or modified deoxyribose include 5'-CP nucleic acid, 2-thio-thymidine monophosphate, 2-aminoadenosine monophosphate, and 7-deazaguanosine monophosphate.
  • the above gap region may be a nucleic acid in which some of the sugar moieties of a natural nucleotide, the sugar moiety of which is deoxyribose, are modified sugars, as long as the effects of the present invention are achieved. That is, in one aspect of this embodiment, the above gap region may be a nucleic acid in which some of the sugar moieties are deoxyribose and other sugar moieties are modified sugars (e.g., modified deoxyribose).
  • the 5'-CP nucleic acid is located at least at the second position counting from the 5' side of the gap region.
  • the 5'-CP nucleic acid is arranged in a continuous 2-4 mer sequence at least at one location in the gap region.
  • the 5'-CP nucleic acid is preferably located on the 5'-end side of the gap region. Also, the 5'-CP nucleic acid is preferably located on the 5'-end side and the 3'-end side of the gap region.
  • “located on the 5'-end side of the gap region” means located on the 5'-end side of the center of the gap region.
  • “located on the 3'-end side of the gap region” means located on the 3'-end side of the center of the gap region.
  • the 5'-CP nucleic acid is preferably arranged in a ninth or more portion of the gap region, and more preferably in a fifth or more portion of the gap region.
  • the upper limit may be, for example, half or less.
  • the 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 two or more times. 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 at least one position in a continuous 2-4 mer arrangement. 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 at least one position in a continuous 2-4 mer arrangement. In the second antisense oligonucleotide, the 5'-CP nucleic acid may be located at the 5' end side and 3' end side of the gap region, and may be located at least one position in a continuous 2-4 mer arrangement.
  • the bond between the 5'-CP nucleic acid and the nucleoside adjacent to the 3' side of the 5'-CP nucleic acid is preferably a phosphorothioate bond (excluding the case where the 5'-CP nucleic acid is located at the 3' end of the gap region).
  • the 5'-CP nucleic acid is located at the 3' end of the gap region
  • the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is preferably a phosphorothioate bond (excluding the cases where the 5'-CP nucleic acid is positioned at the 3' end of the gap region and where the adjacent nucleoside on the 3' side of the 5'-CP nucleic acid is a 5'-CP nucleic acid).
  • the bond between the 5'-CP nucleic acid and the adjacent nucleoside on the 5' side of the 5'-CP nucleic acid is preferably a phosphodiester bond.
  • the 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.
  • the gap region constituting the second antisense oligonucleotide two or more of the 5'-CP nucleic acids may be arranged, and the number of bases composed of deoxyribose (but not including 5'-CP nucleic acid) which may contain a nucleic acid whose sugar portion is modified may be five or more.
  • 2 to 5 of the 5'-CP nucleic acid may be arranged, and the number of bases composed of deoxyribose (but not including 5'-CP nucleic acid) which may contain a nucleic acid whose sugar portion is modified may be five to ten.
  • the gap region does not include a 5'-CP nucleic acid
  • the structure of the sugar moiety other than the 5'-CP nucleic acid in the gap region is preferably the same as that of the first antisense oligonucleotide.
  • the cytosine located in the gap region in the first antisense oligonucleotide and the second antisense oligonucleotide may be 5-methylcytosine.
  • the 3' wing region is a modified nucleic acid having a substituent at the 2' position.
  • the 3' wing region can be understood to be composed of modified nucleotides having a substituent at the 2' position.
  • the modified nucleic acid having a substituent at the 2' position in the 3' wing region preferably includes at least one selected from the group consisting of 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, and MCE nucleic acid as non-bridged 2'-position modified nucleic acids, and LNA, AmNA, GuNA, and scpBNA as bridged modified nucleic acids.
  • the 3' wing region may be a modified nucleic acid in which the sugar moiety is a modified sugar.
  • modified nucleic acids in which the sugar moiety is a modified sugar include those listed above (sugar modification, modified sugar).
  • the modified nucleic acid having a substituent at the 2'-position in the 3'-wing region may be composed of only 2'-MOE nucleic acid. Note that, the modified nucleic acid in the 3'-wing region may be of multiple types contained in one single-stranded antisense oligonucleotide.
  • the modified nucleic acid having a substituent at the 2' position in the 3' wing region preferably includes at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA, and more preferably is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.
  • the number of bases in the 3' wing region is preferably 3 to 5 mer.
  • the 5' wing region is a modified nucleic acid having a substituent at the 2' position.
  • the 5' wing region can be understood to be composed of modified nucleotides having a substituent at the 2' position.
  • the modified nucleic acid in the 5' wing region preferably includes at least one selected from the group consisting of 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, and MCE nucleic acid as non-bridged 2'-position modified nucleic acids, and LNA, AmNA, GuNA, and scpBNA as bridged modified nucleic acids.
  • the 5' wing region may be a modified nucleic acid in which the sugar moiety is a modified sugar.
  • modified nucleic acids in which the sugar moiety is a modified sugar include those listed above (sugar modification, modified sugar).
  • the modified nucleic acid in the 5' wing region having a substituent at the 2' position may be composed only of 2'-MOE nucleic acid. Note that multiple types of modified nucleic acids in the 5' wing region may be included in one single-stranded antisense oligonucleotide.
  • the modified nucleic acids in the 3' and 5' wing regions may consist solely of 2'-MOE nucleic acids.
  • the modified nucleic acid having a substituent at the 2' position in the 5' wing region preferably includes at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA, and more preferably is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.
  • the modified nucleic acid having a substituent at the 2'-position in the 3'-wing region comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA;
  • the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region preferably comprises at least one selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.
  • the modified nucleic acid having a substituent at the 2'-position in the 3'-wing region is composed of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA, More preferably, the modified nucleic acid having a substituent at the 2'-position in the 5'-wing region is comprised of a modified nucleic acid selected from the group consisting of 2'-MOE nucleic acid, LNA, AmNA, GuNA, and scpBNA.
  • the number of bases in the 5' wing region is preferably 3 to 5 mer.
  • the number of bases in the gap region is 5-20 mer
  • the number of bases in the 3' wing region is 3-5 mer
  • the number of bases in the 5' wing region is 3-5 mer.
  • the number of bases in the gap region is 7-13 mer
  • the number of bases in the 3' wing region is 3-5 mer
  • the number of bases in the 5' wing region is 3-5 mer.
  • the number of bases in the gap region is 9-13 mer
  • the number of bases in the 3' wing region is 3-5 mer
  • the number of bases in the 5' wing region is 3-5 mer.
  • the structure of the sugar moiety of the nucleic acid is not particularly limited as long as it is one of 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.
  • Table 1 structural examples 1, 5, 9, 12, 15, 20, 22, and 25 correspond to the structures of the sugar moiety in the first antisense oligonucleotide, and the rest correspond to the structures of the sugar moiety in the second antisense oligonucleotide.
  • the single-stranded antisense oligonucleotide may further comprise a natural nucleotide bound to the 3' end of the 3' wing region.
  • the number of bases of the natural nucleotide bound to the 3' end of the 3' wing region may be one or several, or may be one.
  • the single-stranded antisense oligonucleotide of the present invention is a gapmer type.
  • the notation method of "X-Y-Z” may be used. In the above notation method, "X” indicates the number of bases in the 5' wing region, “Y” indicates the number of bases in the gap region, and “Z” indicates the number of bases in the 3' wing region.
  • X-Y-Z is 2-8-4, 2-8-3, 2-8-5, 2-9-2, 2-9-3, 2-9-4, 2-9-5, 2-10-3, 2-10-4, 2-10-5, 2-11-3, 2-11-4, 2-11-5, 2-12-3, 2-12-4, 2-12-5, 3-8-2, 3-8-3, 3-8-4, 3-8-5, 3-9-3, 3-9-4, 3-9-5, 3-10-3, 3-10-4, 3-10-5, 3-11-3, 3-11-4, 3-11-5, 3-12-3, 3-12-4, 3-12-5, 3-1 Examples include 3-3, 3-13-4, 4-8-2, 4-8-3, 4-8-4, 4-8-5, 4-9-3, 4-9-4, 4-9-5, 4-10-3, 4-10-4, 4-10-5, 4-11-2, 4-11-3, 4-11-4, 4-11-5, 4-12-4, 4-13-3, 5-8-2, 5-8-3, 5-8-4, 5-8-5, 5-9-2, 5-9-3, 5-9-4, 5-9-5, 5-10-2, 5-10-3, 5-10-4,
  • “2-8-4" means that the 5' wing region is a 2-mer oligonucleotide, the gap region is an 8-mer oligonucleotide, and the 3' wing region is a 4-mer oligonucleotide.
  • the base length of the antisense oligonucleotide is 15-30 mer, preferably 15-20 mer, and more preferably 18-20 mer.
  • the base length of the single-stranded antisense oligonucleotide of the present invention is 15-20 mer or 18-20 mer, it has particularly strong binding to the target RNA and can regulate the expression of the target RNA more effectively.
  • the single-stranded antisense oligonucleotide has each nucleoside linked via a phosphate group and/or a modified phosphate group, and is preferably linked via a phosphodiester bond or a phosphorothioate bond.
  • At least one internucleoside bond in the single-stranded antisense oligonucleotide is a phosphorothioate bond. In another aspect of this embodiment, it is preferred that at least one internucleoside bond in the single-stranded antisense oligonucleotide is a phosphodiester bond.
  • the proportion of phosphorothioate bonds is preferably 50% to 80%, and more preferably 50% to 70%.
  • the GC ratio in the base sequence is preferably 0.2 to 0.6.
  • the "GC ratio" refers to the ratio of the numbers of guanine and cytosine (including 5-methylcytosine) to the entire base sequence of the antisense oligonucleotide.
  • the single-stranded antisense oligonucleotide of the present invention is a gapmer type single-stranded antisense oligonucleotide having a gap region of 5 to 20 mer, a 5' wing region of 3 to 5 mer, and a 3' wing region of 3 to 5 mer.
  • the gap region is positioned between the 5' wing region and the 3' wing region.
  • the gap region contains at least one 5'-CP nucleic acid. It is preferable that the 5' wing region and the 3' wing region each contain at least one 2'-MOE nucleic acid, LNA, AmNA, GuNA, or scpBNA.
  • the 5' wing region and the 3' wing region may contain 2'-O-alkylated or 2'-F-substituted nucleotides.
  • a 2'-O-alkylated nucleotide e.g., 2'-O-methylated, etc.
  • the above-mentioned gapmer-type single-stranded antisense oligonucleotide may form a double strand by hybridizing with a second strand oligonucleotide.
  • the antisense oligonucleotide comprises: An antisense oligonucleotide or a pharma- ceutical acceptable salt thereof, which has reduced delayed central toxicity, the antisense oligonucleotide, each nucleotide of which is linked via a phosphate group and/or a modified phosphate group; the antisense oligonucleotide comprises a gap region, a 3' wing region attached to the 3' end of the gap region, and a 5' wing region attached to the 5' end of the gap region; the gap region is a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the gap region comprises at least one 5'-CP nucleic acid; the 3' wing region and the 5' wing region are modified nucleic acids having a substitution at the 2'position; The base length of the antisense oligonucleotide is 12 to 30 mer.
  • 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.
  • “single-stranded antisense oligonucleotide of SEQ ID NO: 2 in Table 2-1” means a single-stranded antisense oligonucleotide whose sequence name in Table 2-1 is "h451-465-N". The same applies to other cases.
  • the double-stranded antisense oligonucleotide comprises the single-stranded antisense oligonucleotide (the second antisense oligonucleotide) and and a second strand oligonucleotide hybridized to the single-stranded antisense oligonucleotide, or a pharma- ceutically acceptable salt thereof.
  • the base sequence of the second strand oligonucleotide is preferably a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to the base sequence of the single-stranded antisense oligonucleotide.
  • the double-stranded antisense oligonucleotide can be dissociated in solution and separated into the single-stranded antisense oligonucleotide and the second-stranded oligonucleotide.
  • the separated single-stranded antisense oligonucleotide can bind to the target RNA.
  • the single-stranded antisense oligonucleotide can also be understood as a "first-stranded oligonucleotide" in relation to the second-stranded oligonucleotide.
  • the first-stranded oligonucleotide has an antisense strand to the target RNA, but for convenience, the double-stranded oligonucleotide consisting of the first-stranded oligonucleotide and the second-stranded oligonucleotide will be referred to as a "double-stranded antisense oligonucleotide.”
  • the method for producing the antisense oligonucleotide or a pharma- ceutical acceptable salt thereof includes the steps of: 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; and synthesizing the second antisense oligonucleotide.
  • 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-phase support using a commercially available automatic nucleic acid synthesizer. Next, the synthesized single-stranded oligonucleotide is cut out from the solid-phase support using a basic substance or the like, and deprotected to obtain a crude single-stranded oligonucleotide.
  • the crude single-stranded oligonucleotide obtained is then purified using HPLC or the like.
  • the single-stranded antisense oligonucleotide of the present invention can be produced by appropriately changing the base sequence, modification site, etc. of the nucleic acid according to a method known to those skilled in the art, without being limited to the above-mentioned production method.
  • 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 by using amidites that are commercially available as reagents.
  • 5'-CP nucleic acids can be produced by the method described in WO 2020/158910 (Patent Document 3).
  • LNAs can be produced by the method described in WO 99/14226 (Patent Document 8).
  • the double-stranded antisense oligonucleotide of the present invention can be produced by first producing an oligonucleotide (second strand oligonucleotide) having a predetermined sequence identity based on the base sequence complementary to the single-stranded antisense oligonucleotide using the same production method as the single-stranded antisense oligonucleotide, and then hybridizing the single-stranded antisense oligonucleotide and the second strand oligonucleotide.
  • the antisense oligonucleotide complex comprises: the single-stranded antisense oligonucleotide (second antisense oligonucleotide) or a pharma- ceutically acceptable salt thereof, or the double-stranded antisense oligonucleotide or a pharma- ceutically acceptable salt thereof; an additional substance bound to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide;
  • the additional substance is selected from the group consisting of polyethylene glycol, peptides, alkyl chains (e.g., saturated aliphatic hydrocarbons, etc.), nucleic acids, ligand compounds, antibodies, proteins, and sugar chains (e.g., carbohydrates, polysaccharides, etc.).
  • the antisense oligonucleotide conjugate comprises: The single-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof; and an additional substance bound to the single-stranded antisense oligonucleotide, the additional substance being selected from the group consisting of polyethylene glycol, peptides, alkyl chains (e.g., saturated aliphatic hydrocarbons, etc.), nucleic acids, ligand compounds, antibodies, proteins, and sugar chains (e.g., carbohydrates, polysaccharides, etc.).
  • the antisense oligonucleotide conjugate comprises: The double-stranded antisense oligonucleotide or a pharma- ceutical acceptable salt thereof; and an additional substance bound to the single-stranded antisense oligonucleotide or the second strand oligonucleotide, the additional substance being selected from the group consisting of polyethylene glycol, peptides, alkyl chains (e.g., saturated aliphatic hydrocarbons, etc.), nucleic acids, ligand compounds, antibodies, proteins, and sugar chains (e.g., carbohydrates, polysaccharides, etc.).
  • additive substance refers to a substance bound to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide, and used to impart a predetermined action.
  • the additive substance may be bound to the 5' end, the 3' end, or both the 5' end and the 3' end of the single-stranded antisense oligonucleotide.
  • the additive substance may be bound to the 5' end, the 3' end, or both the 5' end and the 3' end of the second-stranded oligonucleotide.
  • the additive substance is preferably bound to either the 5' end or the 3' end of the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide.
  • the additive substance may be directly bound to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide by a covalent bond.
  • the additive substance may be bound to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide via a linker substance.
  • linker substance examples include linkers composed of alkyl, polyethylene glycol, peptide, disulfide, nucleic acid, etc., and/or combinations thereof.
  • the method for binding the additional substance to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide can be, for example, the method described in the Examples below.
  • Peptides used as the above-mentioned additional substances include, but are not limited to, the following: CPPs (Cell Penetrating Peptides), nuclear transport peptides, TAT (Trans-Activator of Transcription protein), polyarginine, glucagon-like peptide-1 analogue peptides, synthetic cyclic RGD peptides, and brain transport peptides.
  • ligand compounds used as the above-mentioned additional substances include, but are not limited to, the following: N-acetylgalactosamine (GalNAc), sugars (glucose, mannose, etc.), lipids (cholesterol, palmitic acid, docosahexaenoic acid, etc.), vitamins (folic acid, vitamin A, vitamin E (tocopherol), etc.), amino acids, monoamine receptor ligands (indatraline, etc.)
  • antibodies that can be used as the additional substance include, but are not limited to, the following: anti-insulin receptor antibody, anti-transferrin receptor antibody, anti-LDL receptor-related protein antibody, anti-CD22 antibody, anti-CD30 antibody, anti-HER2 antibody
  • Proteins that can be used as the above-mentioned additional substances include, but are not limited to, the following: Albumin
  • nucleic acid used as the additional substance examples include the following: natural nucleotides, aptamers
  • the nucleic acid used as the additional substance is not counted in the base length of the antisense oligonucleotide.
  • the pharmaceutical composition according to this embodiment contains the single-stranded antisense oligonucleotide (second antisense oligonucleotide) of the present invention or a pharma- ceutical acceptable salt thereof, the above-mentioned double-stranded antisense oligonucleotide or a pharma-ceutical acceptable salt thereof, or the above-mentioned antisense oligonucleotide complex or a pharma-ceutical acceptable salt thereof as an active ingredient.
  • the pharmaceutical composition include a disease treatment agent, a disease prevention agent, and the like.
  • the administration method and formulation of the pharmaceutical composition according to this embodiment can be any administration method and formulation known in the art.
  • the pharmaceutical composition may be referred to as a "pharmaceutical composition of antisense oligonucleotide, etc.”
  • the pharmaceutical compositions are used for treating or preventing diseases of the central nervous system.
  • diseases of the central nervous system include diseases related to the RPS25 gene, i.e., diseases that can be caused by dipeptide repeats produced by RNA translation (sometimes referred to as "repeat diseases").
  • repeat diseases include various psychiatric and neurological diseases and muscular diseases, including C9orf72 ALS, C9orf72 FTLD, Huntington's disease, spinocerebellar ataxia (types 1, 2, 3, 6, 7, 8, 12, and 17), dentatorubral-pallidoluysian atrophy, spinal-bulbar muscular atrophy, Friedreich ataxia, fragile X-associated tremor ataxia syndrome, and myotonic dystrophy.
  • C9orf72 ALS C9orf72 FTLD
  • Huntington's disease Huntington's disease
  • spinocerebellar ataxia types 1, 2, 3, 6, 7, 8, 12, and 17
  • dentatorubral-pallidoluysian atrophy spinal-bulbar muscular atrophy
  • Friedreich ataxia fragile X-associated tremor ataxia syndrome
  • myotonic dystrophy myotonic dystrophy.
  • the above-mentioned individual means a mammal.
  • the above-mentioned individual is preferably a human, a monkey, a marmoset, a dog, a pig, a rabbit, a guinea pig, a rat, or a mouse.
  • the above-mentioned individual is more preferably a human.
  • the single-stranded antisense oligonucleotide (second antisense oligonucleotide) or a pharmaceutical composition thereof can be administered by a suitable administration route to a subject (individual) sensitive to delayed central toxicity.
  • delayed central toxicity refers to central toxicity that appears after a period during which acute central toxicity may appear has passed and recovery has occurred. Symptoms observed due to delayed central toxicity include, for example, decreased spontaneous movement, abnormal gait and abnormal hind limb function, tremors, weakness of the hind limbs or tail, loss of hind limb reflexes, weight loss, etc.
  • the administration method and dosage form are not particularly limited.
  • any administration method and formulation known in the art can be used as the administration method and formulation of the antisense oligonucleotide of the present invention.
  • the administration method include oral administration and parenteral administration.
  • parenteral administration include ophthalmic administration, intravaginal administration, intrarectal administration, intranasal administration, transdermal administration, intravenous injection, drip infusion, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration by aspiration or inhalation, intrathecal administration, and intraventricular administration.
  • the single-stranded antisense oligonucleotide or pharmaceutical composition thereof is preferably administered so as to be exposed to the central nervous system.
  • the administration method that exposes the central nervous system include intrathecal administration and intraventricular administration.
  • the antisense oligonucleotides and other formulations of the present invention may be mixed with various pharmaceutical additives, such as excipients, binders, wetting agents, disintegrants, lubricants, diluents, flavoring agents, fragrances, solubilizing agents, suspending agents, emulsifiers, stabilizers, preservatives, and isotonicity agents, as necessary.
  • various pharmaceutical additives such as excipients, binders, wetting agents, disintegrants, lubricants, diluents, flavoring agents, fragrances, solubilizing agents, suspending agents, emulsifiers, stabilizers, preservatives, and isotonicity agents, as necessary.
  • formulations such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, etc. can be used.
  • the pharmaceutical composition of the present invention such as the antisense oligonucleotide
  • it can be in the form of, for example, a powder, granules, a suspension or solution dissolved in water or a non-aqueous medium, capsules, powders, tablets, or other formulations.
  • compositions such as the antisense oligonucleotides of the present invention parenterally, intrathecally, or intraventricularly
  • formulations such as sterile aqueous solutions can be used.
  • the effective dosage of the single-stranded antisense oligonucleotide of the present invention can be determined arbitrarily depending on the sex, age, weight, symptoms, etc. of the individual to be administered. Furthermore, it can also be determined arbitrarily depending on the method, route, frequency, etc. of administration. For example, the dosage can be 0.01 to 100 mg/kg, etc. Preferably, it is 0.1 to 50 mg/kg, and more preferably, it is 0.1 to 10 mg/kg.
  • the above describes the design method and manufacturing method of the antisense oligonucleotide according to this embodiment.
  • the single-stranded antisense oligonucleotide obtained by the above-mentioned design method and manufacturing method contains at least one 5'-CP nucleic acid in the gap region, and therefore has reduced delayed central nervous toxicity.
  • R 1 and R 2 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 3 carbon atoms.
  • R 3 , R 4 , and R 5 each independently represent a hydrogen atom, a linear or branched alkyl group having 1 to 7 carbon atoms, or a cycloalkyl group having 3 to 7 carbon atoms.
  • R 3 and R 5 in the GuNA shown by “Gx” above are both hydrogen atoms and R 4 is a methyl group, it is represented as “Gm”
  • R 3 is a hydrogen atom and R 4 and R 5 are both methyl groups
  • Gdm when R 3 and R 5 are hydrogen atoms and R 4 is a tert-butyl group, it is represented as "GtB”.
  • single-stranded antisense oligonucleotides were prepared by the following procedure.
  • modified nucleic acids single-stranded antisense oligonucleotides containing 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, AmNA, scpBNA, 5'-CP nucleic acid, and/or GuNA, and/or a nucleic acid whose nucleic acid base is 5-methylcytosine, were synthesized on a 0.2 ⁇ mol scale using an automatic nucleic acid synthesizer (nS-8 type, manufactured by Gene Design Co., Ltd.).
  • Single-stranded antisense oligonucleotides containing 2'-MOE nucleic acid, AmNA and/or scpBNA were obtained in which 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.
  • the single-stranded antisense oligonucleotide was cut out from the solid phase support by alkali treatment and recovered in the form of a solution. Thereafter, the solvent was distilled off from the recovered solution to obtain a crude product.
  • the obtained crude product was purified by reverse phase HPLC to obtain a purified single-stranded antisense oligonucleotide.
  • the purity and structure of each of the obtained single-stranded antisense oligonucleotides were confirmed by LC-MS (Waters Corporation). The measurement results are shown in Tables 2-1 to 2-8.
  • Clinical sign score 0 points: no abnormality 1 point: abnormal hind leg function, tremor, decreased spontaneous movement 2 points: dragging of hind legs, weakness of tail or hind legs 3 points: complete hind leg dysfunction, paralysis of hind legs, recumbency, prone position 4 points: euthanasia Pathological score; 0 points: no abnormality 1 point: abnormality (single cell necrosis, vacuolation, etc.)
  • Tables 3-1 to 3-7 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.
  • Tables 3-1 to 3-8 the items marked with “-” indicate that no measurement evaluation was performed.
  • the "Observation date” and “Clinical sign score” columns in Table 3-7 are the evaluation results obtained from the experiment described in "Evaluation of central nervous system toxicity of antisense oligonucleotides (2)" described below.
  • Evaluation items were 1) posture, 2) external abnormality, 3) stereotypic behavior, 4) reactivity to stimuli, 5) grip strength, 6) respiration, and 7) tremors/convulsions, each of which was scored as 0 for normal, 1 for slightly abnormal, and 2 for extremely abnormal (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 findings, 2 representing moderate findings, and 3 representing severe findings (the column for histopathological findings in Table 3-8).
  • Tables 3-7 to 3-8 the evaluation results of delayed central 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 "Sample Date” and “Pathological score” columns in Table 3-7 are the evaluation results obtained from the experiment described in "Evaluation of Central Toxicity of Antisense Oligonucleotides (1)" above.
  • the expression evaluation of the RPS25 gene was performed by expression evaluation using human fetal kidney cells according to the single-stranded antisense oligonucleotide produced.
  • the expression evaluation of the RPS25 gene can also be performed using human iPS cell-derived nerve cells.
  • gene expression evaluation means evaluating the amount of mRNA by measuring the amount of complementary DNA (cDNA) obtained by reverse transcription reaction. The specific procedures for each expression evaluation are described below.
  • Human embryonic kidney cells HEK293T (ATCC® CRL-3216TM) were cultured in a culture medium at 37° C. and 5% CO 2.
  • the culture medium for HEK293T cells had the following composition:
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • S1820 100-fold diluted penicillin-streptomycin mixed solution: Nacalai Tesque Cat#09367-34 (penicillin 10,000 units/ml, streptomycin 10,000 ⁇ g/ml, stabilizer included)
  • HEK293T cells (12,000 cells/well) were seeded in a 96-well plate and cultured overnight at 37°C and 5% CO2 . Then, each single-stranded antisense oligonucleotide (final concentration 0.5 nM, 5 nM, 15 nM, or 50 nM) diluted with phosphate-buffered saline (PBS) was transfected into the above-mentioned cells by lipofection. As a negative control, cells transfected with PBS in which the single-stranded antisense oligonucleotide was not dissolved were used.
  • PBS phosphate-buffered saline
  • the transfected cells were cultured in growth medium at 37°C and 5% CO2 for 48 hours.
  • the growth medium was then removed, and the extracted total RNA was subjected to reverse transcription using the Taqman Fast Cells-to-CT Kit (Thermo Fisher Scientific, Cat#4399003).
  • the complementary DNA (cDNA) obtained from this reverse transcription reaction was used to perform real-time PCR using pre-designed gene-specific probes (see below) in Taqman gene expression assays (Applied Biosystems) (40 cycles of 95°C for 3 seconds and 60°C for 30 seconds).
  • the expression ratio of human RPS25 mRNA in each single-stranded antisense oligonucleotide relative to human RPS25 mRNA, determined by the above-mentioned method, is shown in Tables 4-1 to 4-7 (column "In vitro evaluation"). At this time, the expression ratio of human RPS25 mRNA determined in the negative control group was set to 1.00. Those with an expression ratio of 0.80 or less were determined to be single-stranded antisense oligonucleotides capable of suppressing the expression of human RPS25 mRNA. In the table, "-" indicates that no measurement was performed. In general, it is believed that when mRNA expression is suppressed, the subsequent translation into protein, etc. is also suppressed. Therefore, those with the above expression ratio of 0.80 or less can be determined to be single-stranded antisense oligonucleotides capable of regulating the function of the human RPS25 gene.
  • the expression evaluation of the RPS25 gene was performed by administering the gene intracerebroventricularly to mice and measuring the amount of mRNA in each area of the prefrontal cortex.
  • the gene expression evaluation means evaluating the amount of mRNA by measuring the amount of complementary DNA (cDNA) obtained by reverse transcription reaction. The specific procedures for each expression evaluation are described below.
  • FVB mice (CLEA Japan) were anesthetized with isoflurane (Pfizer, Cat#114133403).
  • antisense oligonucleotides dissolved in artificial cerebrospinal fluid (Tocris Bioscience, Cat#3525/25mL) were administered to the anesthetized FVB mice at 10 ⁇ L/individual using a two-stage needle (Top, Medical Device Approval Number 15800BZZ01460000) attached to a 50 ⁇ L Hamilton syringe (Hamilton, Cat#705LT).
  • Mice in the negative control group were administered only artificial cerebrospinal fluid at 10 ⁇ L/individual.
  • RNA extraction from the stored tissue samples was performed using RNeasy Mini Kit (QIAGEN, Cat#74106).
  • Reverse transcription reaction from the extracted mRNA was performed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystem, Cat#4368814). For the reverse transcription reaction, 1 ⁇ g of mRNA was diluted to 20 ⁇ L and used.
  • cDNA complementary DNA obtained from this reverse transcription reaction
  • real-time PCR was performed using pre-designed gene-specific probes (see below) in Taqman expression assays (Applied Biosystems) (95°C; 3 seconds, 60°C; 30 seconds, 40 cycles).
  • Tables 4-1 to 4-7 show the expression ratios of mouse RPS25 mRNA obtained by the above-mentioned method for the single-stranded antisense oligonucleotides listed in Tables 2-1 to 2-7 (column "In vivo evaluation"). At this time, the expression ratio of mouse RPS25 RNA obtained in the negative control group was set to 1.00. "Sampling date" in Tables 4-1 to 4-7 indicates the number of days from administration of the single-stranded antisense oligonucleotide to collection of the specified tissue. Since it is generally believed that when mRNA expression is suppressed, subsequent translation into protein, etc.
  • 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.
  • a cell line not treated with antisense oligo was used as a control.
  • KELLY cells (ECACC, EC92110411-F0) were used as human neuroblastoma lines.
  • Each antisense oligonucleotide was introduced into KELLY cells using a commercially available transfection reagent (ThermoFisher Scientific, Lipofectamine 3000), and the expression level of mRNA was measured by qRT-PCR to examine knockdown activity (suppression of mRNA expression). The specific procedure is shown below.
  • KELLY cells were seeded at 2.0 x 104 cells/well in wells of a 96-well plate (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).
  • RNA extraction reagent MagMAX mirVana Total RNA Isolation Kit, ThermoFisher Scientific.
  • RNA extraction reagent MagMAX mirVana Total RNA Isolation Kit, ThermoFisher Scientific.
  • 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 with temperature cycling of 45°C, 10 minutes ⁇ 95°C, 5 minutes ⁇ [(95°C, 5 seconds ⁇ 60°C, 30 seconds) x 40 cycles].
  • the amount of mRNA of the housekeeping gene ⁇ -actin (ACTB) was also quantified at the same time, and the amount of UBE3A-ATS mRNA relative to the amount of ACTB mRNA was evaluated by the ⁇ Ct method.
  • the amount of mRNA produced 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.
  • the primer sets used are as follows: (Primer set for PTEN detection) TaqMan Gene Expression Assay Hs01372957_m1 (ThermoFisher Scientific) (Primer set for ACTB detection) TaqMan Gene Expression Assay Hs99999903_m1 (ThermoFisher Scientific)
  • caspase 3/7 activity by antisense nucleotides in human cervical cancer-derived HelaS3 cell line was evaluated by the following procedure.
  • HelaS3 cells ATCC, CCL-2.2
  • Each antisense oligonucleotide was introduced into HelaS3 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 shown below.
  • logarithmic growth phase HelaS3 cells were seeded at 2.0 x 104 cells/well in wells of a 96-well plate (Dulbecco's modified Eagle 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 incubated for 24 hours. After incubation, the cells were harvested and caspase 3/7 activity was examined using a kit. The activity value of each antisense oligonucleotide was shown as a relative value when the activity value of cells that did not incorporate the antisense oligonucleotide (untreated cells, "control”) was set at 100.
  • DMEM Dulbecco's modified Eagle medium
  • FBS fetal bovine serum
  • results are shown in Table 5.
  • the results in Table 5 confirm 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.

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Abstract

L'invention concerne un procédé de conception d'un oligonucléotide antisens ayant une toxicité centrale retardée réduite, le procédé comprenant une étape de conception d'un deuxième oligonucléotide antisens sur la base d'un premier oligonucléotide antisens. Le premier oligonucléotide antisens est conçu de telle sorte que les nucléosides sont liés entre eux par un groupe phosphate et/ou un groupe phosphate modifié. Le premier oligonucléotide antisens comporte une région lacunaire, une région d'aile 3' liée à l'extrémité 3' de la région lacunaire et une région d'aile 5' liée à l'extrémité 5' de la région lacunaire. La région lacunaire comporte des acides nucléiques constitués de désoxyribose pouvant inclure un acide nucléique dans lequel le sucre est modifié ; la région de l'aile 3' et la région de l'aile 5' sont des acides nucléiques modifiés présentant un substituant en position 2' ; et la longueur des bases est comprise entre 12 et 30 mer. Le deuxième oligonucléotide antisens présente les mêmes séquences de bases pour la région lacunaire, la région de l'aile 3' et la région de l'aile 5' que le premier oligonucléotide antisens, et présente les mêmes structures de sucre des acides nucléiques dans la région de l'aile 3' et la région de l'aile 5' que celles du premier oligonucléotide antisens. La région lacunaire contient au moins un acide nucléique 5'-CP, et la toxicité centrale retardée est réduite par comparaison avec le premier oligonucléotide antisens.
PCT/JP2024/021614 2023-06-16 2024-06-14 Procédé de conception d'oligonucléotide antisens ayant une toxicité centrale retardée réduite, et son procédé de production Ceased WO2024257848A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022147209A1 (fr) * 2020-12-31 2022-07-07 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et utilisations associées pour le traitement de la dystrophie myotonique
WO2022234855A1 (fr) * 2021-05-06 2022-11-10 ルクサナバイオテク株式会社 Procédé de conception d'un oligonucléotide ayant une toxicité centrale réduite
JP2023509793A (ja) * 2020-01-10 2023-03-09 ダイン セラピューティクス，インコーポレーテッド 筋緊張性ジストロフィーを処置するための筋標的化複合体およびその使用
WO2024071099A1 (fr) * 2022-09-29 2024-04-04 国立大学法人東京医科歯科大学 Molécule d'acide nucléique contenant une modification 5'-cyclopropylène

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2023509793A (ja) * 2020-01-10 2023-03-09 ダイン セラピューティクス，インコーポレーテッド 筋緊張性ジストロフィーを処置するための筋標的化複合体およびその使用
WO2022147209A1 (fr) * 2020-12-31 2022-07-07 Dyne Therapeutics, Inc. Complexes de ciblage musculaire et utilisations associées pour le traitement de la dystrophie myotonique
WO2022234855A1 (fr) * 2021-05-06 2022-11-10 ルクサナバイオテク株式会社 Procédé de conception d'un oligonucléotide ayant une toxicité centrale réduite
WO2024071099A1 (fr) * 2022-09-29 2024-04-04 国立大学法人東京医科歯科大学 Molécule d'acide nucléique contenant une modification 5'-cyclopropylène

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