WO2024071099A1 - ５'－シクロプロピレン修飾を含む核酸分子 - Google Patents

５'－シクロプロピレン修飾を含む核酸分子 Download PDF

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WO2024071099A1
WO2024071099A1 PCT/JP2023/034892 JP2023034892W WO2024071099A1 WO 2024071099 A1 WO2024071099 A1 WO 2024071099A1 JP 2023034892 W JP2023034892 W JP 2023034892W WO 2024071099 A1 WO2024071099 A1 WO 2024071099A1
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nucleic acid
modified
nucleoside
aso
group
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French (fr)
Japanese (ja)
Inventor
隆徳 横田
耕太郎 吉岡
隆之 黒田
聡 小比賀
卓男 山口
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Tokyo Medical and Dental University NUC
University of Osaka NUC
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Osaka University NUC
Tokyo Medical and Dental University NUC
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Priority to JP2024549412A priority Critical patent/JPWO2024071099A1/ja
Priority to EP23872322.5A priority patent/EP4596694A1/en
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    • 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
    • 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
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • 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
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • the present invention relates to nucleic acid molecules, double-stranded nucleic acid complexes, and compositions that contain 5'-cyclopropylene modifications.
  • oligonucleotides have attracted attention in the ongoing development of pharmaceuticals known as nucleic acid drugs, and in particular, the development of nucleic acid drugs using the antisense method is being actively pursued, given their high selectivity for target genes and low toxicity.
  • the antisense method involves selectively modifying or inhibiting the expression of proteins encoded by target genes or the activity of miRNA by introducing complementary oligonucleotides (antisense oligonucleotides: often referred to as "ASOs (Antisense Oligonucleotides)" in this specification) into cells, using a partial sequence of mRNA or miRNA transcribed from a target gene as the target sense strand.
  • ASOs Antisense Oligonucleotides
  • the present inventors have developed a double-stranded nucleic acid complex (heteroduplex oligonucleotide, HDO) in which an antisense oligonucleotide is annealed to its complementary strand (Patent Documents 1 and 2, Non-Patent Documents 1 and 2).
  • the double-stranded nucleic acid complex is a technological technology with a strong antisense effect.
  • nucleic acid drugs such as antisense drugs
  • antisense drugs progresses and the results of preclinical trials accumulate
  • avoiding toxicity is important.
  • gene suppression effect there is a trade-off between gene suppression effect and toxicity in nucleic acid drugs.
  • the stronger the gene suppression effect of a nucleic acid drug the higher the toxicity generally tends to be.
  • gapmer-type antisense nucleic acids that induce cleavage of the target transcript and antisense nucleic acids that contain cross-linked nucleic acids have a strong gene suppression effect, but are highly toxic and can be fatal.
  • the goal is to provide a new nucleic acid molecule that can simultaneously achieve both high gene control effects and reduced toxicity.
  • the present inventors have conducted intensive research to solve the above problems, and have introduced 5'-cyclopropylene modifications (often abbreviated as "5'-CP modifications" in this specification) into DNA nucleosides in the gap region of gapmer-type nucleic acid molecules.
  • 5'-CP modifications also abbreviated as "5'-CP modifications” in this specification
  • the toxicity of a nucleic acid molecule can be significantly reduced or eliminated by introducing 5'-CP modifications into specific positions of the nucleic acid molecule.
  • This toxicity suppression effect depends on the base position at which the 5'-CP modification is introduced, and it has been found that a significant toxicity reduction effect can be obtained by placing a 5'-CP modified nucleoside at the first, third, or ninth base position from the 5' side of the gap region.
  • the antisense effect of the nucleic acid molecule is not impaired, and both toxicity reduction and high efficacy are achieved.
  • the present invention is based on the above findings, and provides the following.
  • a nucleic acid molecule comprising a base sequence complementary to at least a portion of a target gene or its transcription product, and having an antisense effect on the target gene or its transcription product, [1] a central region containing at least three consecutive deoxyribonucleosides; [2] a 5' wing region comprising an unnatural nucleoside arranged on the 5' end of the central region; and [3] a 3' wing region comprising an unnatural nucleoside arranged on the 3' end of the central region, wherein the 1st, 3rd, and/or 9th base positions from the 5' end of the central region are represented by the following formula (I):
  • the nucleic acid molecule comprising a 5'-modified nucleoside represented by: (2) The nucleic acid molecule according to (1), wherein the internucleoside bond on the 5' side of the 5'-modified nucleoside is a modified internucleoside bond or a phosphodiester bond.
  • nucleic acid molecule according to (2) wherein the modified internucleoside bond is a phosphorothioate bond.
  • the nucleic acid molecule described in (1) comprising a 2'-modified nucleoside at the terminal base position adjacent to the central region in the 5' wing region, the second base position from the 5' side of the central region, and/or the eighth base position from the 5' side of the central region.
  • nucleic acid molecule described in (4) or (5) wherein the 2'-modified nucleoside is a 2'-O-methyl modified nucleoside, a 2'-O-methoxyethyl modified nucleoside, a 2'-O-[2-(N-methylcarbamoyl)ethyl] modified nucleoside, or a 2'-fluoro modified nucleoside.
  • the nucleic acid molecule described in (1), wherein the 5' wing region and the 3' wing region comprise bridged nucleosides and/or 2'-modified nucleosides.
  • a double-stranded nucleic acid complex comprising a first nucleic acid strand consisting of the nucleic acid molecule according to any one of (1) to (14) and a second nucleic acid strand comprising a base sequence complementary to the first nucleic acid strand.
  • the double-stranded nucleic acid complex according to (15) or (16), wherein the second nucleic acid strand comprises at least one selected from the group consisting of deoxyribonucleosides, 2'-modified nucleosides, 5'-modified nucleosides, and bridged nucleosides.
  • the alkylamino group is a hexylamino group.
  • (31) The double-stranded nucleic acid complex according to any one of (15) to (30), wherein all or a part of the internucleoside linkages of the second nucleic acid strand are modified internucleoside linkages.
  • (32) The double-stranded nucleic acid complex according to (31), wherein the modified internucleoside bond is a phosphorothioate bond.
  • a composition comprising the nucleic acid molecule according to any one of (1) to (14) or the double-stranded nucleic acid complex according to any one of (15) to (32).
  • the central nervous system is selected from the group consisting of the frontal lobe, temporal lobe, hippocampus, parahippocampal gyrus, parietal lobe, occipital lobe, striatum, globus pallidus, claustrum, thalamus, subthalamic nucleus, midbrain, substantia nigr
  • composition according to (33) which is administered intrathecally, intranasally, intravenously, subcutaneously, intraperitoneally, or intramuscularly.
  • composition according to (37), wherein the intrathecal administration is intraventricular administration, posterior fossa puncture, or lumbar puncture.
  • This specification includes the disclosure of Japanese Patent Application No. 2022-155777, which is the priority basis of this application.
  • the present invention provides a new nucleic acid drug that can simultaneously achieve both high gene control effects and reduced toxicity.
  • FIG. 1 shows the structures of various natural and non-natural nucleotides.
  • FIG. 2 shows the structures of various bridged nucleic acids.
  • FIG. 3 shows the structure of the nucleic acid used in Example 1. 4 shows the results of measuring the number of surviving cells after transfection with various nucleic acid agents. Error bars indicate standard error. 5 shows the LDH activity in the supernatant after transfection with various nucleic acid agents. Error bars indicate standard error.
  • Figure 6 shows the expression levels of Cdkn1a mRNA after transfection with various nucleic acid agents. Error bars indicate standard error.
  • Figure 7 shows the expression levels of Il-6 mRNA after transfection with various nucleic acid agents. Error bars indicate standard error.
  • FIG. 1 shows the structures of various natural and non-natural nucleotides.
  • FIG. 2 shows the structures of various bridged nucleic acids.
  • FIG. 3 shows the structure of the nucleic acid used in Example 1. 4 shows the results of measuring the number of surviving cells
  • FIG. 8 shows the structure of the nucleic acid used in Example 2.
  • 9 shows the results of measuring the number of surviving cells after transfection with various nucleic acid agents. Error bars indicate standard error.
  • 10 shows LDH activity in the supernatant after transfection with various nucleic acid agents. Error bars indicate standard error.
  • FIG. 11 shows the structure of the nucleic acid used in Example 3.
  • Figure 12 shows the body weight and food intake of mice 20 days after intracerebroventricular administration of various nucleic acid agents.
  • Figure 12A shows the body weight.
  • Figure 12B shows the food intake. Error bars show standard error.
  • 13 shows the results of evaluating motor function in mice intracerebroventricularly administered with various nucleic acid agents 10, 14, 17, and 20 days after administration.
  • FIG. 15 shows the structure of the nucleic acid used in Example 4.
  • 16 shows the results of measuring the number of surviving cells after transfection with various nucleic acid agents. Error bars indicate standard error.
  • 17 shows LDH activity in the supernatant after transfection with various nucleic acid agents. Error bars indicate standard error.
  • Figure 18 shows the expression levels of Cdkn1a mRNA after transfection with various nucleic acid agents. Error bars indicate standard error.
  • FIG. 19 shows the structure of the nucleic acid used in Example 5.
  • FIG. 20 shows the results of measuring the number of surviving cells after transfection with various nucleic acid agents. Error bars indicate standard error.
  • 21 shows LDH activity in the supernatant after transfection with various nucleic acid agents. Error bars indicate standard error.
  • FIG. 22 shows the structure of the nucleic acid used in Example 6. 23 shows the results of measuring the number of surviving cells after transfection with various nucleic acid agents. Error bars indicate standard error. 24 shows LDH activity in the supernatant after transfection with various nucleic acid agents. Error bars indicate standard error.
  • FIG. 25 shows the structure of the nucleic acid used in Example 7.
  • Figure 26 shows the expression levels of Hdac2 mRNA after transfection with various nucleic acid agents. Error bars indicate standard error.
  • FIG. 27 shows the structure of the nucleic acid used in Example 8.
  • FIG. 28 shows the scoring system used to assess the acute tolerability scores.
  • Figure 29 shows the body weight and food intake after intracerebroventricular administration of various nucleic acid agents in mice.
  • Figure 29A shows the body weight.
  • Figure 29B shows the food intake. Error bars show standard error.
  • Figure 30 shows the results of evaluating delayed central neurotoxicity 6 days after intracerebroventricular administration of various nucleic acid agents in mice.
  • Figure 30A shows motor function.
  • Figure 30B shows behavioral evaluation. Error bars show standard error.
  • FIG. 31 shows the structure of the nucleic acid used in Example 9.
  • 32 shows the body weight of mice after intracerebroventricular administration of various nucleic acid agents. Error bars indicate standard error.
  • FIG. 33 shows food intake in mice after intracerebroventricular administration of various nucleic acid agents. Error bars indicate standard error.
  • 34 shows the results of evaluating motor function after intracerebroventricular administration of various nucleic acid agents in mice. The vertical axis indicates the maximum movement speed in the open field test. Error bars indicate standard error.
  • Figure 35 shows behavioral evaluation by a scoring system for mice intracerebroventricularly administered with various nucleic acid agents. Error bars indicate standard error.
  • Figure 36 shows the expression levels of Tnf- ⁇ mRNA and Gfap mRNA in the hippocampus of mice administered various nucleic acid agents intracerebroventricularly.
  • Figure 36A shows the expression level of Tnf- ⁇ mRNA.
  • Figure 36B shows the expression level of Gfap mRNA.
  • Figure 37 shows the body weight and food intake after intracerebroventricular administration of various nucleic acid agents in mice.
  • Figure 37A shows the body weight.
  • Figure 37B shows the food intake.
  • Error bars show standard error.
  • 38 shows the results of evaluating motor function after intracerebroventricular administration of various nucleic acid agents in mice. The vertical axis indicates the maximum movement speed in the open field test. Error bars indicate standard error.
  • 39 shows the results of evaluating motor function after intracerebroventricular administration of various nucleic acid agents in mice. The vertical axis indicates the maximum movement speed in the open field test. Error bars indicate standard error.
  • Figure 40 shows the expression levels of Hdac2 mRNA in the left hippocampus 7 days after intracerebroventricular administration of various nucleic acid agents.
  • the first aspect of the present invention is a nucleic acid molecule.
  • the nucleic acid molecule of the present invention is a nucleic acid molecule that contains a base sequence complementary to at least a part of a target gene or its transcription product, has an antisense effect on the target gene or its transcription product, and contains a 5'-cyclopropylene-modified nucleoside at a specific base position.
  • the nucleic acid molecule of the present invention can simultaneously achieve a high gene regulation effect and low or no toxicity.
  • the "transcription product" of a target gene refers to any RNA synthesized by RNA polymerase. Specifically, it may include mRNA (including mature mRNA, pre-mRNA, and mRNA without base modification) transcribed from a target gene, non-coding RNA (ncRNA) such as miRNA, long non-coding RNA (lncRNA), and natural antisense RNA.
  • ncRNA non-coding RNA
  • miRNA miRNA
  • lncRNA long non-coding RNA
  • lncRNA long non-coding RNA
  • a "target gene” refers to a gene whose transcription or translation product expression level can be suppressed or enhanced, whose transcription or translation product function can be inhibited, or whose steric blocking, splicing control (e.g., splicing switch, exon skipping, exon inclusion), base editing, or RNA editing can be induced by the antisense effect of a nucleic acid molecule or double-stranded nucleic acid complex.
  • the type of target gene is not particularly limited.
  • Examples include genes derived from an organism into which a nucleic acid strand, nucleic acid molecule, or double-stranded nucleic acid complex is introduced, and include genes whose expression increases in various diseases (e.g., central nervous system diseases) and genes expressed in vivo, such as the central nervous system.
  • diseases e.g., central nervous system diseases
  • genes expressed in vivo such as the central nervous system.
  • SR-B1 scavenger receptor B1
  • Malat1 metastasis associated lung adenocarcinoma transcript 1
  • Mapt microtubule-associated protein tau
  • BACE1 beta-secretase 1
  • Hdac2 histone deacetylase 2
  • DMPK dystrophia myotonic-protein kinase
  • target transcript refers to a transcript that is the direct target of a nucleic acid molecule or a double-stranded nucleic acid complex
  • transcript of a target gene also falls under the category of target transcript.
  • Information on the base sequences of target transcripts and target genes can be obtained from publicly known databases, such as the NCBI (National Center for Biotechnology Information) database.
  • antisense nucleic acid refers to a single-stranded nucleic acid molecule that contains a base sequence capable of hybridizing (i.e., complementary) to at least a portion of a target transcript (mainly a transcript of a target gene) and can exert an antisense effect on the target transcript.
  • antisense oligonucleotide refers to an antisense nucleic acid composed of an oligonucleotide.
  • antisense nucleic acid or “antisense oligonucleotide” is often referred to as "ASO.”
  • the nucleic acid molecule of the present invention or the first nucleic acid strand of the double-stranded nucleic acid complex functions as an ASO, and the target region may include the 3'UTR, 5'UTR, exon, intron, coding region, translation initiation region, translation termination region, or any other nucleic acid region.
  • the target region of the target transcript can be at least 8 bases long, for example, 10-35 bases long, 12-25 bases long, 13-20 bases long, 14-19 bases long, or 15-18 bases long, or 13-22 bases long, 16-22 bases long, or 16-20 bases long.
  • the term "antisense effect” refers to any effect that occurs when an ASO hybridizes to a target transcript (e.g., an RNA sense strand), such as the effect of regulating the expression or editing of a target transcript.
  • a target transcript e.g., an RNA sense strand
  • Regulatory the expression or editing of a target transcript refers to suppression or reduction of the expression of a target gene or the expression level of a target transcript (herein, "expression level of a target transcript” is often referred to as “level of a target transcript”), inhibition of translation, RNA editing, base editing, splicing control or splicing function modification effects (e.g., splicing switch, exon inclusion, exon skipping, etc.), steric blocking, or degradation of a transcript.
  • RNA oligonucleotide when introduced into a cell as an ASO, the ASO forms a partial duplex with the mRNA, which is the transcription product of the target gene, by annealing.
  • This partial duplex acts as a cover to prevent translation by ribosomes, thereby inhibiting the expression of a target protein encoded by the target gene at the translation level (steric blocking).
  • an oligonucleotide containing DNA is introduced into a cell as an ASO, a partial DNA-RNA heteroduplex is formed.
  • This heteroduplex structure is recognized by RNase H, resulting in degradation of the mRNA of the target gene and inhibition of expression of the protein encoded by the target gene at the expression level.
  • an antisense effect can also be achieved by targeting an intron in a pre-mRNA.
  • an antisense effect can also be achieved by targeting an miRNA.
  • inhibition of the function of the miRNA can increase the expression of a gene whose expression is normally controlled by the miRNA.
  • the modulation of expression of the target transcript can be a reduction in the amount of the target transcript.
  • the antisense effect can be measured, for example, by administering a test nucleic acid compound to a subject (e.g., a mouse) and measuring, for example, several days later (e.g., 2 to 7 days later), the expression level of a target gene or the level (amount) of a target transcript (e.g., the amount of mRNA or RNA such as microRNA, the amount of cDNA, the amount of protein, etc.) whose expression is regulated by the antisense effect provided by the test nucleic acid compound.
  • a test nucleic acid compound e.g., a mouse
  • the expression level of a target gene or the level (amount) of a target transcript e.g., the amount of mRNA or RNA such as microRNA, the amount of cDNA, the amount of protein, etc.
  • a decrease in the measured expression level of the target gene or the level of the target transcript by at least 10%, at least 20%, at least 25%, at least 30%, or at least 40% compared to a negative control (e.g., vehicle administration) indicates that the test nucleic acid compound can produce an antisense effect (e.g., a reduction in the amount of the target transcript).
  • nucleic acid strand may affect the antisense effect provided by the nucleic acid strand, nucleic acid molecule or nucleic acid complex.
  • the choice of modification may vary depending on the sequence of the target gene, etc., but a person skilled in the art can determine a suitable embodiment by referring to the descriptions in the literature related to antisense methods (e.g., WO 2007/143315, WO 2008/043753, and WO 2008/049085).
  • the relevant modification can be evaluated if the measured value thus obtained is not significantly lower than that of the nucleic acid complex before modification (e.g., if the measured value obtained after modification is 70% or more, 80% or more, or 90% or more of the measured value of the nucleic acid complex before modification).
  • translation product of a target gene refers to any polypeptide or protein synthesized by translation of the target transcript or the transcription product of a target gene that is the direct target of a nucleic acid molecule or a double-stranded nucleic acid complex.
  • decoy refers to a nucleic acid that has a sequence of the binding site of a transcription factor (e.g., NF-kB) or a similar sequence, and is introduced into a cell as a “decoy” to suppress the action of the transcription factor (if it is a transcription activator, it suppresses transcription, and if it is a transcription repressor, it promotes transcription).
  • a transcription factor e.g., NF-kB
  • Decoy nucleic acids can be easily designed based on information on the binding sequence of the target transcription factor.
  • bait refers to a nucleic acid molecule that specifically binds to a specific target molecule within a cell and modifies the function of the target molecule.
  • a target that interacts with a bait is also called a "prey.”
  • nucleic acid or “nucleic acid molecule” as used herein may refer to a monomeric nucleotide or nucleoside, an oligonucleotide composed of multiple monomers, or multiple nucleosides linked by internucleoside bonds, and also includes polynucleotides if they are polymers.
  • Natural nucleic acid refers to a nucleic acid that exists in nature. Natural nucleic acids include natural nucleosides and natural nucleotides, etc., described below.
  • Non-natural nucleic acid or “artificial nucleic acid” refers to any nucleic acid other than natural nucleic acid. Non-natural nucleic acid or artificial nucleic acid includes non-natural nucleosides and non-natural nucleotides, etc., described below.
  • nucleic acid strand refers to two or more nucleosides linked by internucleoside bonds, and may be, for example, an oligonucleotide or a polynucleotide.
  • a nucleic acid strand may be made full length or partial by chemical synthesis, for example, using an automated synthesizer, or by enzymatic processes using polymerases, ligases, or restriction reactions.
  • a nucleic acid strand may contain natural and/or non-natural nucleotides.
  • Nucleoside generally refers to a molecule that is composed of a combination of a base and a sugar.
  • the sugar portion of a nucleoside is typically, but not limited to, a pentofuranosyl sugar, examples of which include ribose and deoxyribose.
  • the base portion of a nucleoside is typically a heterocyclic base moiety, including, but not limited to, adenine, cytosine, guanine, thymine, or uracil, as well as other modified nucleobases (modified bases).
  • Nucleotide refers to a molecule in which a phosphate group is covalently linked to the sugar portion of the nucleoside.
  • the phosphate group is usually linked to the hydroxyl group at the 2', 3', or 5' position of the sugar.
  • Oligonucleotide refers to a linear oligomer formed by covalently linking several to several dozen hydroxyl groups and phosphate groups in the sugar moieties between adjacent nucleotides.
  • Polynucleotide refers to a linear polymer formed by linking several dozen or more, preferably several hundred or more, nucleotides that are more numerous than an oligonucleotide, by the covalent bonds.
  • the phosphate groups are generally considered to form internucleoside bonds.
  • natural nucleosides refer to nucleosides that exist in nature. Examples include ribonucleosides consisting of ribose and bases such as adenine, cytosine, guanine, or uracil, and deoxyribonucleosides consisting of deoxyribose and bases such as adenine, cytosine, guanine, or thymine.
  • ribonucleosides found in RNA and deoxyribonucleosides found in DNA are often referred to as “RNA nucleosides” and “DNA nucleosides,” respectively.
  • natural nucleotide refers to a nucleotide that exists in nature and is a molecule in which a phosphate group is covalently bonded to the sugar portion of the natural nucleoside.
  • examples include ribonucleotides, which are known as the building blocks of RNA and in which a phosphate group is bonded to a ribonucleoside, and deoxyribonucleotides, which are known as the building blocks of DNA and in which a phosphate group is bonded to a deoxyribonucleoside.
  • non-natural nucleotide refers to any nucleotide other than a natural nucleotide, and includes modified nucleotides and nucleotide mimetics.
  • modified nucleotide refers to a nucleotide having one or more of a modified sugar moiety, a modified internucleoside linkage, and a modified nucleobase.
  • nucleotide mimetics includes structures used to replace nucleosides and linkages at one or more positions of an oligomeric compound.
  • Peptide nucleic acids PNA are nucleotide mimetics with a backbone in which N-(2-aminoethyl)glycine is linked by amide bonds in place of sugars.
  • nucleic acid strands including non-natural oligonucleotides often have desirable properties, such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity. Thus, they are preferred over natural nucleotides.
  • unnatural nucleoside refers to any nucleoside other than a natural nucleoside. For example, it includes modified nucleosides and nucleoside mimetics.
  • modified nucleoside refers to a nucleoside having a modified sugar moiety and/or a modified nucleobase.
  • mimetic refers to functional groups that replace the sugar, nucleobase, and/or internucleoside linkage. Generally, a mimetic is used in place of a sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.
  • nucleoside mimic includes structures that are used to replace the sugar, or the sugar and base, at one or more positions of an oligomeric compound, or to replace the linkage between the monomeric subunits that make up the oligomeric compound, etc.
  • oligomeric compound is meant a polymer of linked monomeric subunits that is at least capable of hybridizing to a region of a nucleic acid molecule.
  • Nucleoside mimetics include, for example, morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclic or tricyclic sugar mimetics, e.g., nucleoside mimetics having non-furanose sugar units.
  • Modified sugar refers to a sugar having a substitution and/or any change from a naturally occurring sugar moiety (i.e., a sugar moiety found in DNA (2'-H) or RNA (2'-OH)), and "sugar modification” refers to a substitution and/or any change from a naturally occurring sugar moiety.
  • a nucleic acid strand may optionally include one or more modified nucleosides, including modified sugars.
  • “Sugar-modified nucleoside” refers to a nucleoside having a modified sugar moiety. Such sugar-modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to a nucleic acid strand.
  • the nucleoside includes a chemically modified ribofuranose ring moiety.
  • chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (including 5' and 2' substituents), bridging of non-geminal ring atoms to form bicyclic nucleic acids (bridged nucleic acids, BNAs), replacement of ribosyl ring oxygen atoms with S, N(R), or C(R1)(R2) (wherein R, R1, and R2 each independently represent H, C1 - C12 alkyl, or a protecting group), and combinations thereof.
  • sugar modified nucleosides include, but are not limited to, nucleosides containing 5'-cyclopropylene, 5'-vinyl, 5'-alkyl (e.g., 5'-methyl (R or S), 5'-ethyl (R or S)), 5'-allyl (R or S), 4'-S, 2'-F (2'-fluoro group), 2'- OCH3 (2'-O-Me group or 2'-O-methyl group), 2'-O-[2-(N-methylcarbamoyl)ethyl] (2'-O-MCE group), and 2'-O-methoxyethyl (2'-O-MOE or 2- O ( CH2 ) 2OCH3 ) substituents.
  • “2'-modified sugar” refers to a furanosyl sugar modified at the 2'-position.
  • a nucleoside containing a 2'-modified sugar may also be referred to as a "2'-modified nucleoside” or a "2'-sugar modified nucleoside.”
  • “5'-modified sugar” refers to a furanosyl sugar modified at the 5'-position.
  • Nucleosides containing a 5'-modified sugar are referred to as “5'-modified nucleosides" or “5'-sugar-modified nucleosides,” and are specifically distinguished as “5'-modified deoxyribonucleosides” and "5'-modified ribonucleosides” and “5'-modified ribonucleosides,” respectively.
  • BNA nucleoside refers to a modified nucleoside that contains a bicyclic sugar moiety. Nucleics that contain a bicyclic sugar moiety are commonly referred to as bridged nucleic acids (BNAs). Nucleosides that contain a bicyclic sugar moiety are also sometimes referred to as “bridged nucleosides,” “bridged non-natural nucleosides,” or “BNA nucleosides.” Some examples of bridged nucleic acids are shown in Figure 2.
  • a bicyclic sugar may be a sugar in which the 2' and 4' carbon atoms are bridged by two or more atoms.
  • bicyclic sugars are known to those of skill in the art.
  • One subgroup of bicyclic sugar-containing nucleic acids (BNAs) or BNA nucleosides can be described as having the 2' and 4 ' carbon atoms bridged by 4'-( CH2 ) p -O-2', 4'-( CH2 ) p - CH2-2 ', 4'-( CH2 ) p -S-2', 4'-( CH2 )p-OCO-2', 4'-(CH2) n -N( R3 )-O-( CH2 ) m -2', where p, m and n represent integers from 1 to 4, 0 to 2 and 1 to 3, respectively; and R3 represents a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an ary
  • R 1 and R 2 are typically hydrogen atoms, but may be the same as or different from each other, and may also be a protecting group for a hydroxyl group for nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, a phosphate group, a phosphate group protected by a protecting group for nucleic acid synthesis, or P(R 4 )R 5 (wherein R 4 and R 5 may be the same as or different from each other and respectively represent a hydroxyl group, a hydroxyl group protected by a protecting group for nucleic acid synthesis, a mercapto group, a mercapto
  • amine BNAs also known as 2'-Amino-LNAs
  • 2'-Amino-LNAs e.g., 3-(Bis(3-aminopropyl)amino)propanoyl substitutions
  • 2'-O,4'-C-spirocyclopropylene bridged nucleic acids also known as scpBNAs
  • BNA nucleosides include methyleneoxy (4'-CH 2 -O-2') BNA nucleosides (also known as LNA nucleosides, 2',4'-BNA nucleosides) (e.g., ⁇ -L-methyleneoxy (4'-CH 2 -O-2') BNA nucleosides, ⁇ -D-methyleneoxy (4'-CH 2 -O-2') BNA nucleosides), ethyleneoxy (4'-(CH 2 ) 2 -O-2') BNA nucleosides (also known as ENA nucleosides), ⁇ -D-thio (4'-CH 2 -S-2') BNA nucleosides, aminooxy (4'-CH 2 -ON(R 3 )-2') BNA nucleosides, oxyamino (4'-CH 2 -N(R 3 )-O-2') BNA nucleosides (2',4'-BNA Also known as NC nucle
  • a "cationic nucleoside” is a modified nucleoside that exists in a cationic form relative to a neutral form (such as the neutral form of a ribonucleoside) at a certain pH (e.g., human physiological pH (about 7.4), the pH of a delivery site (e.g., an organelle, cell, tissue, organ, organism, etc.) etc.).
  • a cationic nucleoside may contain one or more cationic modifying groups at any position of the nucleoside.
  • Bicyclic nucleosides with a methyleneoxy (4'- CH2 -O-2') bridge are sometimes referred to as LNA nucleosides.
  • modified internucleoside linkage refers to an internucleoside linkage having a substitution or any change from a naturally occurring internucleoside linkage (i.e., a phosphodiester linkage).
  • Modified internucleoside linkages include internucleoside linkages that contain a phosphorus atom and internucleoside linkages that do not contain a phosphorus atom.
  • Representative phosphorus-containing internucleoside bonds include phosphodiester bonds, phosphorothioate bonds, phosphorodithioate bonds, phosphotriester bonds (e.g., methyl phosphotriester bonds and ethyl phosphotriester bonds as described in U.S. Patent Registration No.
  • alkyl phosphonate bonds e.g., methyl phosphonate bonds as described in U.S. Patent Registration Nos. 5,264,423 and 5,286,717, and methoxypropyl phosphonate bonds as described in WO 2015/168172
  • alkylthiophosphonate bonds e.g., methylthiophosphonate bonds
  • boranophosphate bonds e.g., a cyclic guanidine moiety
  • internucleoside linkage containing a guanidine moiety e.g., a tetramethylguanidine (TMG) moiety
  • TMG tetramethylguanidine
  • Phosphorothioate linkages refer to internucleoside linkages in which the non-bridging oxygen atom of the phosphodiester bond is replaced with a sulfur atom. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known.
  • the modified internucleoside linkage is preferably one that is more resistant to nucleases than naturally occurring internucleoside linkages.
  • internucleoside linkage When an internucleoside linkage has a chiral center, the internucleoside linkage may be chiral controlled.
  • chiral controlled it is intended that the internucleoside linkage exists as a single diastereomer about a chiral center, e.g., a chiral phosphorus linkage.
  • the internucleoside linkage may be a phosphorothioate linkage chirally controlled in the Rp or Sp configuration, an internucleoside linkage containing a guanidine moiety substituted with one to four C1-6 alkyl groups (e.g., a tetramethylguanidine (TMG) moiety; see, for example, Alexander A. Lomzov et al., Biochem Biophys Res Commun., 2019, 513(4), 807-811), and/or an internucleoside linkage containing a cyclic guanidine moiety.
  • TMG tetramethylguanidine
  • Chirally controlled phosphorothioate linkages in the Rp or Sp configuration are also known, and phosphorothioate linkages chirally controlled in the Sp configuration are known to be more stable than those in the Rp configuration, and ASOs chirally controlled in the Sp configuration are also known to promote target RNA cleavage by RNase H1 and result in a more sustained response in vivo.
  • nucleobase refers to the base component (heterocyclic moiety) that constitutes a nucleic acid, and the main known bases are adenine, guanine, cytosine, thymine, and uracil.
  • nucleobase or “base” includes both modified and unmodified nucleic acid bases (bases), unless otherwise specified.
  • a purine base may be either a modified or unmodified purine base.
  • a pyrimidine base may be either a modified or unmodified pyrimidine base.
  • Modified nucleobase or “modified base” means any nucleobase other than adenine, cytosine, guanine, thymine, or uracil.
  • Unmodified nucleobase or “unmodified base” (natural nucleobase) means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).
  • modified nucleobases include, but are not limited to, hypoxanthine, 5-methylcytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, or N4-methylcytosine; N6-methyladenine, or 8-bromoadenine; 2-thio-thymine; and N2-methylguanine, or 8-bromoguanine.
  • the term "gapmer” generally refers to a single-stranded nucleic acid that includes or consists of a "central region” (a DNA gap region) and wing regions (referred to as the "5' wing region” and the “3' wing region”, respectively) located directly at the 5' and 3' ends of the central region.
  • the central region in a gapmer includes at least three or at least four consecutive deoxyribonucleosides. Additionally, each of the 5' wing region and the 3' wing region includes at least one non-natural nucleoside.
  • the boundary positions between the central region (DNA gap region) and the 5' and 3' wing regions can be easily determined by a person skilled in the art from the nucleoside sequence.
  • the central region can be functionally defined as a region that can be recognized by RNase H (e.g., RNase H1).
  • RNase H e.g., RNase H1
  • "recognizable by RNase H” means that when the gapmer binds to a target RNA, the paired sequence in the target RNA can be cleaved by RNase H.
  • the boundary positions can be determined by defining the region in the gapmer that can be recognized by RNase H as the central region, and the regions that are not recognized by RNase H (more specifically, regions in which cleavage activity by RNase H is not substantially detectable under physiological conditions) as the wing regions (5' wing region and 3' wing region).
  • the terminal nucleosides adjacent to the central region in the 5' wing region and the 3' wing region are non-natural nucleosides (e.g., 2'-modified nucleosides or bridged nucleosides), and the nucleosides adjacent to the 5' wing region or the 3' wing region in the central region are deoxyribonucleosides or sugar-modified versions thereof.
  • both nucleosides adjacent to the 5' wing region and the 3' wing region in the central region are deoxyribonucleosides.
  • the central region may contain modified nucleobases that are recognized by RNase H, for example, 5-methylcytosine.
  • the central region may also contain non-natural nucleosides, such as 2'-modified nucleosides and 5'-modified nucleosides, except for the two terminal nucleosides adjacent to the 5' and 3' wing regions.
  • the central region may be configured such that all internucleoside bonds in the region that it pairs with in its target RNA are susceptible to cleavage by RNase H.
  • the non-natural nucleosides contained in the wing region usually have a stronger binding strength with RNA than natural nucleosides and have a higher resistance to nucleic acid degrading enzymes (nucleases, etc.).
  • the non-natural nucleosides constituting the 5' wing region and the 3' wing region may be, for example, bridged nucleosides and/or 2'-modified nucleosides.
  • the gapmer is specifically referred to as a "BNA/DNA gapmer".
  • the number of bridged nucleosides contained in the 5' wing region and the 3' wing region is at least one, and may be, for example, two or three.
  • the bridged nucleosides contained in the 5' wing region and the 3' wing region may be present contiguously or non-contiguously in the 5' wing region and the 3' wing region.
  • the bridged nucleoside may further include a modified nucleobase (e.g., 5-methylcytosine).
  • the bridged nucleoside may be an LNA nucleoside or an ENA nucleoside.
  • the gapmer When the bridged nucleoside is an LNA nucleoside, the gapmer is referred to as an "LNA/DNA gapmer.” When the bridged nucleoside is an ENA nucleoside, the gapmer is referred to as an "ENA/DNA gapmer.” When the non-natural nucleosides that make up the 5' and 3' wing regions comprise or consist of peptide nucleic acids, the gapmer is specifically referred to as a "peptide nucleic acid gapmer.” When the non-natural nucleosides that make up the 5' and 3' wing regions comprise or consist of morpholino nucleic acids, the gapmer is specifically referred to as a "morpholino nucleic acid gapmer.” Furthermore, when the non-natural nucleosides constituting the 5' and 3' wing regions comprise or consist of 2'-modified nucleosides, the 2'-modified group of the 2'-modified nucleoside may be a 2'-O-methyl group or a 2
  • the number of 2'-modified nucleosides contained in the 5' and 3' wing regions is at least one, and may be, for example, two or three.
  • the 2'-modified nucleosides contained in the 5' and 3' wing regions may be contiguous or non-contiguous within the 5' and 3' wing regions.
  • the 2'-modified nucleoside may further comprise a modified nucleobase (e.g., 5-methylcytosine).
  • the nucleosides constituting the 5' wing region and the 3' wing region include or consist of a bridged nucleoside or a 2'-modified nucleoside
  • the nucleosides may be composed of a combination of two or more types of bridged nucleosides and/or 2'-modified nucleosides.
  • a nucleic acid strand having a wing region only on either the 5' end or the 3' end is called a "hemigapmer" in the art, and in this specification, hemi-gapmers are also included in the term "gapmer.”
  • nucleobases can form so-called Watson-Crick base pairs (natural base pairs) or Wobble base pairs (guanine-thymine or guanine-uracil) through hydrogen bonds, and a relationship in which similar base pairs can be formed between natural nucleobases and modified nucleobases or between modified nucleobases themselves.
  • the antisense region of the nucleic acid molecule does not necessarily have to be completely complementary to at least a portion of the target transcript (e.g., the transcript of the target gene), but is acceptable if the base sequence has at least 70%, preferably at least 80%, and even more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity.
  • the antisense region of the nucleic acid molecule can hybridize to the target transcript when the base sequence is complementary (typically when the base sequence is complementary to at least a portion of the base sequence of the target transcript).
  • the complementary region in the second nucleic acid strand does not necessarily have to be completely complementary to at least a portion of the nucleic acid molecule that is the first nucleic acid strand, but is acceptable if the base sequence has at least 70%, preferably at least 80%, and even more preferably at least 90% (e.g., 95%, 96%, 97%, 98%, or 99% or more) complementarity.
  • a complementary region in the second nucleic acid strand can anneal when the base sequence is complementary to at least a portion of the first nucleic acid strand.
  • the complementarity of the base sequence can be determined by using a BLAST program or the like.
  • Those skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which the two strands can anneal or hybridize, taking into account the degree of complementarity between the strands. Furthermore, those skilled in the art can easily design an antisense nucleic acid complementary to a target transcription product, for example, based on information on the base sequence of the target gene.
  • Hybridization conditions may be various stringent conditions, such as low stringency conditions and high stringency conditions.
  • Low stringency conditions may be conditions of relatively low temperature and high salt concentration, for example, 30°C, 2xSSC, 0.1% SDS.
  • High stringency conditions may be conditions of relatively high temperature and low salt concentration, for example, 65°C, 0.1xSSC, 0.1% SDS.
  • Hybridization stringency can be adjusted by changing conditions such as temperature and salt concentration.
  • 1xSSC contains 150 mM sodium chloride and 15 mM sodium citrate.
  • toxicity refers to an effect that causes undesirable objective or subjective symptoms or functional abnormalities in a subject. Toxicity may be toxicity in any organ, for example, neurotoxicity, hepatotoxicity, or nephrotoxicity.
  • Neurotoxicity refers to an effect that causes damage to nervous tissue, including central nervous tissue and peripheral nervous tissue, and interferes with normal activity of the nervous system. In particular, toxicity to the central nervous system is called central neurotoxicity.
  • Neurotoxicity may cause symptoms selected from death, respiratory abnormalities, cardiovascular abnormalities, headache, nausea or vomiting, unresponsiveness or hyporesponsiveness, impaired consciousness, mental disorders, personality changes, hallucinations, delusions, cognitive dysfunction, abnormal posture, involuntary movements, tremors, convulsions, hyperactive motor dysfunction, paralysis, sensory disorders, or autonomic dysfunction.
  • Neurotoxicity may be either acute neurotoxicity or delayed neurotoxicity.
  • Acute neurotoxicity is neurotoxicity that occurs within several hours to tens of hours, for example, within 1, 3, 6, 9, 12, 24, or 48 hours, after administration, and can be distinguished from delayed neurotoxicity that occurs thereafter.
  • Nephrotoxicity refers to the property of causing abnormal and/or impaired function in the kidney.
  • Hepatotoxicity and renal function can be evaluated by any method known to those skilled in the art, such as serum biochemical tests. Toxicity can be evaluated, for example, by acute tolerability scores, adverse event rates, or mortality rates, as described in the Examples below. Hepatotoxicity refers to the property of causing abnormal and/or impaired function in the liver.
  • the term "subject” refers to an object to which the nucleic acid molecule, double-stranded nucleic acid complex, or pharmaceutical composition of the present invention is applied.
  • Subjects include individuals, as well as organs, tissues, and cells. When the subject is an individual, it may be any animal, including humans. Examples of subjects other than humans include various livestock, poultry, pets, and laboratory animals. Without being limited thereto, the subject may be an individual in need of reducing the expression level of a target transcript, or an individual in need of treatment or prevention of a disease, such as a central nervous system disease.
  • multiple refers to, for example, 2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-10, 2-12, 2-14, 2-16, 2-18, 2-20, 2-25, 2-30, 2-35, 2-40, or more.
  • the nucleic acid molecule of this embodiment contains a base sequence complementary to at least a part of a target gene or its transcription product, and has an antisense effect on the target gene or its transcription product.
  • the nucleic acid molecule of this embodiment is characterized by a gapmer-type configuration and includes a central region containing at least three consecutive deoxyribonucleosides, a 5' wing region containing unnatural nucleosides located on the 5' end of the central region, and a 3' wing region containing unnatural nucleosides located on the 3' end of the central region.
  • the nucleic acid molecule of this embodiment has the following formula (I) at a specific base position in the central region:
  • the 5'-modified nucleoside represented by the formula (I) above includes a 5'-modified nucleoside represented by the formula (I) above. Since the modifying group at the 5' position in the formula (I) above is a cyclopropane group, the 5'-modified nucleoside represented by the formula (I) above is referred to herein as a "5'-cyclopropane modified nucleoside” or a "5'-cyclopropylene modified nucleoside", and is often abbreviated as a "5'-CP modified nucleoside".
  • the 5'-modified nucleoside represented by the formula (I) above can also be referred to as a 5'-cyclopropane modified deoxyribonucleoside or a 5'-cyclopropylene modified deoxyribonucleoside.
  • the nucleic acid molecule of this embodiment contains the 5'-modified nucleoside represented by formula (I) at the first, third, and/or ninth base position from the 5' side of the central region.
  • the Nth base position from the 5' side of the central region means the Nth base in the 5' to 3' direction starting from the terminal base adjacent to the 5' wing region in the central region. Therefore, the first base position from the 5' side of the central region is the terminal base adjacent to the 5' wing region in the central region, and the third base position from the 5' side of the central region means the third base in the 3' direction starting from the terminal base adjacent to the 5' wing region in the central region.
  • the nucleic acid molecule of this embodiment may contain the 5'-modified nucleoside represented by formula (I) at any of the first, third, and ninth base positions from the 5' side of the central region, but preferably contains the 5'-modified nucleoside represented by formula (I) at the third or ninth base position.
  • the number of 5'-modified nucleosides represented by the above formula (I) contained in the nucleic acid molecule of this embodiment is at least 1, and may be 2 or more or 3, but is preferably 2 or less, for example 1.
  • the type of nucleoside other than the 5'-CP nucleoside is not limited, so long as the central region can be recognized by RNase H.
  • all nucleosides other than the 5'-CP nucleoside in the central region may be deoxyribonucleosides, or the central region may contain one or more non-natural nucleosides in addition to deoxyribonucleosides.
  • non-natural nucleosides that can be introduced into the central region without impairing the antisense effect include non-natural deoxyribonucleosides such as 5'-modified deoxyribonucleosides.
  • the 5'-modified deoxyribonucleoside is not particularly limited, and examples include 5'-alkyl modified deoxyribonucleosides (e.g., 5'-methyl modified deoxyribonucleosides and 5'-ethyl modified deoxyribonucleosides) and 5'-allyl modified deoxyribonucleosides.
  • 5'-alkyl modified deoxyribonucleosides e.g., 5'-methyl modified deoxyribonucleosides and 5'-ethyl modified deoxyribonucleosides
  • 5'-allyl modified deoxyribonucleosides There is no restriction on the chirality of the 5'-modification in the deoxyribonucleoside, and it may be either R-type or S-type. It is preferable that there are no consecutive unnatural nucleosides in the central region.
  • the internucleoside bond at the 5'-side of the 5'-modified nucleoside represented by the above formula (I) is a modified internucleoside bond or a phosphodiester bond.
  • the modified internucleoside bond may be, for example, a phosphorothioate bond, without being limited thereto.
  • the term "internucleoside bond at the 5'-side of the 5'-modified nucleoside” refers to the internucleoside bond bonded to the 5' position of the nucleoside.
  • the structure in which the 5'-CP modified nucleoside is bonded to the phosphorothioate bond at the 5'-side is represented by the following formula (II):
  • the structure in which the 5'-CP modified nucleoside is bound to the 5' phosphodiester bond is represented by the following formula (III):
  • the internucleoside bond on the 5' side of the 5'-modified nucleoside represented by the above formula (I) is preferably a phosphorothioate bond.
  • the nucleic acid molecule of this aspect contains a 5'-modified nucleoside represented by the above formula (I) at the first base position from the 5' side of the central region, and the internucleoside bond on the 5' side of the 5'-modified nucleoside is, for example, a modified internucleoside bond or a phosphodiester bond, preferably a phosphorothioate bond.
  • the nucleic acid molecule of this aspect contains a 5'-modified nucleoside represented by the above formula (I) at the third base position from the 5' side of the central region, and the internucleoside bond on the 5' side of the 5'-modified nucleoside is, for example, a modified internucleoside bond or a phosphodiester bond, preferably a phosphorothioate bond.
  • the nucleic acid molecule of this aspect contains a 5'-modified nucleoside represented by the above formula (I) at the 9th base position from the 5' side of the central region, and the internucleoside bond on the 5' side of the 5'-modified nucleoside is, for example, a modified internucleoside bond or a phosphodiester bond, preferably a phosphorothioate bond.
  • the type of internucleoside bond other than the internucleoside bond on the 5' side of the 5'-CP nucleoside in the nucleic acid molecule of this aspect is not limited.
  • all internucleoside bonds other than the internucleoside bond on the 5' side of the 5'-CP nucleoside may be modified internucleoside bonds such as phosphorothioate bonds.
  • the nucleic acid molecule of this aspect comprises a 2'-modified nucleoside at the terminal base position adjacent to the central region in the 5' wing region, the second base position from the 5' side of the central region, and/or the eighth base position from the 5' side of the central region.
  • the terminal base position adjacent to the central region in the 5' wing region refers to the base position located at the 3' end in the 5' wing region.
  • the 2'-modified nucleoside may be located on either the 5' or 3' side of the 5'-modified nucleoside represented by formula (I) above, and may be located either adjacent to the 5'-modified nucleoside or not adjacent to it, but is preferably located adjacent to the 5' side of the 5'-modified nucleoside.
  • the 2'-modification in the 2'-modified nucleoside may be, for example, a 2'-O-methyl group, a 2'-O-[2-(N-methylcarbamoyl)ethyl] group, a 2'-O-methoxyethyl group, or a 2'-fluoro group, but a 2'-O-methyl group is preferred.
  • the nucleic acid molecule of this aspect contains a 5'-modified nucleoside at the fourth base position from the 5' side of the central region.
  • 5'-modified nucleosides include 5'-modified deoxyribonucleosides, and may be, for example, 5'-alkyl-modified deoxyribonucleosides (e.g., 5'-methyl-modified deoxyribonucleosides or 5'-ethyl-modified deoxyribonucleosides) or 5'-allyl-modified deoxyribonucleosides.
  • the chirality of the 5'-modification in the deoxyribonucleoside there is no restriction on the chirality of the 5'-modification in the deoxyribonucleoside, and it may be either the R-type or the S-type, but the R-type, such as (R-)5'-methyl-modified deoxyribonucleosides, is particularly preferred.
  • the central region may be, for example, 3 to 12 bases long, 4 to 11 bases long, 5 to 10 bases long, 6 to 9 bases long, or 7 to 8 bases long.
  • the base length of the 5' wing region and the 3' wing region may each independently be at least 2 bases long, for example, 2 to 10 bases long, 2 to 7 bases long, 3 to 5 bases long, 3 to 4 bases long, or 3 bases long.
  • the nucleic acid molecules of the present invention can include 2'-modified nucleosides and/or bridged nucleosides in the 5' and 3' wing regions.
  • the 2'-modified nucleosides can be, for example, 2'-O-methyl or 2'-O-methoxyethyl modified nucleosides.
  • the bridged nucleosides can be, for example, LNA nucleosides, 2',4'-BNA NC nucleosides, cEt BNA nucleosides, ENA nucleosides, AmNA nucleosides, GuNA nucleosides, scpBNA nucleosides, scpBNA2 nucleosides, or BANA3 nucleosides.
  • 2'-O-methyl modified nucleosides, 2'-O-methoxyethyl modified nucleosides, 2'-LNA or ENA may be combined, and the types of modifications may include 1 to 4 types, 2 to 3 types, for example 2 types, which may be the same or different in the 5' wing region and the 3' wing region.
  • one or two to three consecutive nucleosides at the terminal end adjacent to the central region in the 5' wing region and/or the 3' wing region are comprised of a 2'-modified nucleoside and/or a bridged nucleoside.
  • the 5' wing region and/or the 3' wing region are comprised of two or more 2'-modified nucleosides and/or bridged nucleosides linked by internucleoside linkages.
  • examples of base lengths of the 5' wing region, central region, and 3' wing region include 2-12-3, 3-12-2, 3-12-3, 4-12-3, 2-11-3, 3-11-2, 3-11-3, 4-11-3, 2-10-3, 3-10-2, 3-10-3, 4-10-3, 2-9-3, 3-9-2, 3-9-3, 4-9-3, 2-8-3, 3-8-2, 3-7-3, 4-6-3, 3-6-4, 4-5-4, 4-7-3, 3-7-4, 4-6-4, 5-6-3, 3-6-5, 3-7-5, 5-7-3, 4-7-4, 4-6-5, 5-6-4, 5-5-5, 5-6-5, etc.
  • A-B-C indicates the base length of the 5' wing region
  • B indicates the base length of the central region
  • C indicates the base length of the 3' wing region.
  • the base length of the nucleic acid molecule of this embodiment is not particularly limited, but may be at least 8 bases, at least 9 bases, at least 10 bases, at least 11 bases, at least 12 bases, at least 13 bases, at least 14 bases, or at least 15 bases.
  • the base length of the nucleic acid molecule may be 40 bases or less, 35 bases or less, 30 bases or less, 25 bases or less, 24 bases or less, 23 bases or less, 22 bases or less, 21 bases or less, 20 bases or less, 19 bases or less, 18 bases or less, 17 bases or less, or 16 bases or less.
  • the base length of the nucleic acid molecule may be, for example, 10 to 40 bases, 12 to 30 bases, or 15 to 25 bases.
  • the length can be selected by balancing the strength of the antisense effect and the specificity of the nucleic acid strand for the target, among other factors such as cost and synthesis yield.
  • the base length of the nucleic acid molecule as a whole may be the above-mentioned base length plus the base length of the bound nucleic acid.
  • the internucleoside bond in the nucleic acid molecule of this embodiment may be a naturally occurring internucleoside bond and/or a modified internucleoside bond. Although not limited thereto, it is preferable that at least one, at least two, or at least three internucleoside bonds from the end (5' end, 3' end, or both ends) of the nucleic acid molecule of this embodiment are modified internucleoside bond.
  • two internucleoside bonds from the end of the nucleic acid chain means the internucleoside bond closest to the end of the nucleic acid chain and the internucleoside bond adjacent to it and located on the opposite side to the end.
  • Modified internucleoside bonds in the terminal region of the nucleic acid chain are preferable because they can suppress or inhibit undesired degradation of the nucleic acid chain.
  • all or a portion of the internucleoside linkages of the nucleic acid molecule may be modified internucleoside linkages.
  • the modified internucleoside linkages may be phosphorothioate linkages.
  • the nucleic acid molecules of the present invention may comprise, in whole or in part, a nucleoside mimic or a nucleotide mimic.
  • the nucleotide mimic may be a peptide nucleic acid and/or a morpholino nucleic acid.
  • the antisense effect of the nucleic acid molecule of this embodiment on the target transcript can be measured by a method known in the art. For example, after introducing the nucleic acid molecule into cells, etc., the effect can be measured using known techniques such as Northern blotting, quantitative PCR, or Western blotting. By measuring the expression level of the target gene or the level of the target transcript (e.g., RNA amount such as mRNA amount, cDNA amount, etc.) in a specific tissue, it can be determined whether the target gene expression is suppressed by the nucleic acid molecule at those sites.
  • RNA amount such as mRNA amount, cDNA amount, etc.
  • test nucleic acid compound can have an antisense effect.
  • the nucleic acid molecule of the present invention can reduce or eliminate the toxicity of a nucleic acid drug (e.g., neurotoxicity such as central nervous toxicity) without compromising its high efficacy (e.g., steric blocking, exon skipping, exon inclusion, splicing control, decreased expression, increased expression, and/or base editing). In addition, it can reduce cell death, inflammation or gliosis, abnormal increase in cytokines or chemokines, motor function or behavior abnormality associated with administration of an antisense nucleic acid.
  • a nucleic acid drug e.g., neurotoxicity such as central nervous toxicity
  • high efficacy e.g., steric blocking, exon skipping, exon inclusion, splicing control, decreased expression, increased expression, and/or base editing.
  • it can reduce cell death, inflammation or gliosis, abnormal increase in cytokines or chemokines, motor function or behavior abnormality associated with administration of an antisense nucleic acid.
  • Gapmer-type antisense nucleic acids capable of cleaving targets, particularly gapmers containing bridged nucleosides, are highly effective but have increased toxicity, which has been a major obstacle to their practical application.
  • the nucleic acid molecule of the present invention has the surprising effect of maintaining efficacy while significantly reducing toxicity.
  • nucleic acid molecules of the present invention are less toxic than gapmer-type nucleic acid molecules that do not contain 5'-CP modified nucleosides (e.g., nucleic acid molecules that do not contain 5'-CP modified nucleosides at the first, third, and/or ninth base positions from the 5' side of the central region).
  • 5'-CP modified nucleosides e.g., nucleic acid molecules that do not contain 5'-CP modified nucleosides at the first, third, and/or ninth base positions from the 5' side of the central region.
  • the 5'-CP modification is a nucleic acid modification that can enhance nuclease resistance when using more biocompatible phosphodiester bonds to avoid phosphorothioate bonds, which are a concern due to their accumulation in certain organs (Patent Document 3, International Publication No. 2020/158910, Abstract).
  • Patent Document 3 International Publication No. 2020/158910, Abstract.
  • the effect of the 5'-CP modification itself in reducing toxicity was unexpected.
  • it was unexpected that a toxicity-reducing effect could be obtained regardless of whether the internucleoside bond bonded to the 5' side of the 5'-CP-modified nucleoside was a PS bond or a PO bond.
  • the present invention also provides a toxicity reducer for gapmer-type nucleic acid molecules, which comprises a 5'-CP modified nucleoside.
  • the toxicity reducer can reduce the toxicity of the nucleic acid molecule by being placed at a specific position in the gap region (central region) of a gapmer-type nucleic acid molecule.
  • the present invention also provides the use of a 5'-CP modified nucleoside to reduce the toxicity of a gapmer-type nucleic acid molecule.
  • Double-stranded nucleic acid complex 2-1 Overview
  • the second aspect of the present invention is a double-stranded nucleic acid complex.
  • the double-stranded nucleic acid complex of this aspect comprises a first nucleic acid strand capable of functioning as an antisense nucleic acid, and a second nucleic acid strand comprising a base sequence complementary to the first nucleic acid strand.
  • the double-stranded nucleic acid complex of this aspect can simultaneously achieve a high gene regulation effect and low or no toxicity.
  • the double-stranded nucleic acid complex of this embodiment includes a first nucleic acid strand and a second nucleic acid strand.
  • the first nucleic acid strand is the nucleic acid molecule described in the first embodiment, and the structure thereof is as described above, so that the description thereof will be omitted here.
  • the second nucleic acid strand is a nucleic acid molecule that contains a base sequence complementary to the first nucleic acid strand.
  • the second nucleic acid strand is annealed to the first nucleic acid strand via hydrogen bonds of complementary base pairs.
  • the second nucleic acid strand may contain deoxyribonucleosides, 2'-modified nucleosides, 5'-modified nucleosides, and/or bridged nucleosides.
  • nucleosides in the region of the second nucleic acid strand that is complementary to the central region of the first nucleic acid strand may be (a) deoxyribonucleosides; (b) deoxyribonucleosides and ribonucleosides; (c) deoxyribonucleosides and 2'-modified nucleosides; (d) ribonucleosides and 2'-modified nucleosides; or (e) deoxyribonucleosides, ribonucleosides, and 2'-modified nucleosides.
  • the second nucleic acid strand includes a region containing at least three or at least four consecutive ribonucleosides and/or deoxyribonucleosides that are complementary to at least three or at least four consecutive deoxyribonucleosides in a central region of the first nucleic acid strand.
  • the second nucleic acid strand may include a region consisting of a base sequence complementary to the 5' wing region and/or the 3' wing region of the first nucleic acid strand.
  • the region consisting of a base sequence complementary to the 5' wing region and/or the 3' wing region of the first nucleic acid strand may include at least one non-natural nucleoside, which may be a bridged nucleoside and/or a 2'-modified nucleoside.
  • the 2'-modified group of the 2'-modified nucleoside in the second nucleic acid strand may be a 2'-O-methyl group or a 2'-O-methoxyethyl group.
  • the bridged nucleoside and/or the 2'-modified nucleoside in the first nucleic acid strand and the second nucleic acid strand may be the same or different.
  • the internucleoside linkages in the second nucleic acid strand may be naturally occurring internucleoside linkages and/or modified internucleoside linkages. It is preferred, but not limited to, that at least one, at least two, or at least three internucleoside linkages from the ends (5' end, 3' end, or both ends) of the second nucleic acid strand are modified internucleoside linkages. In one embodiment, all or a portion of the internucleoside linkages in the second nucleic acid strand may be modified internucleoside linkages.
  • the second nucleic acid strand may include modified internucleoside linkages in a region consisting of a base sequence complementary to the 5' wing region and/or the 3' wing region of the first nucleic acid strand.
  • the modified internucleoside linkages may be phosphorothioate linkages.
  • the second nucleic acid strand can include 2'-modified nucleosides (e.g., 2'-O-methoxyethyl modified nucleosides).
  • the number of 2'-modified nucleosides (e.g., 2'-O-methoxyethyl modified nucleosides) in the second nucleic acid strand is not limited.
  • At least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or 100% of the total number of nucleosides in the second nucleic acid strand may be 2'-modified nucleosides (e.g., 2'-O-methoxyethyl modified nucleosides).
  • all of the nucleosides in the second nucleic acid strand are 2'-modified nucleosides (e.g., 2'-O-methoxyethyl modified nucleosides).
  • the second nucleic acid strand can include one or more consecutive 2'-modified nucleosides (e.g., 2'-O-methoxyethyl modified nucleosides) located at the 5' end and/or one or more consecutive 2'-modified nucleosides (e.g., 2'-O-methoxyethyl modified nucleosides) located at the 3' end.
  • the number of 2'-modified nucleosides (e.g., 2'-O-methoxyethyl modified nucleosides) located at the 5' end and/or 3' end is not limited.
  • the second nucleic acid strand may include one or two, three, four, five, six, or seven consecutive 2'-modified nucleosides (e.g., 2'-O-methoxyethyl modified nucleosides) located at the 5' end, and/or one or two, three, four, five, six, or seven consecutive 2'-modified nucleosides (e.g., 2'-O-methoxyethyl modified nucleosides) located at the 3' end.
  • 2'-modified nucleosides e.g., 2'-O-methoxyethyl modified nucleosides
  • the second nucleic acid strand may include modified nucleobases.
  • the number of modified nucleobases is not limited and may be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6.
  • the second nucleic acid strand may include non-complementary bases and/or an insertion sequence and/or deletion of one or more bases relative to the first nucleic acid strand.
  • the number of non-complementary bases in the second nucleic acid strand is not limited, but may be, for example, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 or 2.
  • the number of bases of the insertion sequence in the second nucleic acid strand is not limited, but may be, for example, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 or 2.
  • the length of the deleted contiguous bases in the second nucleic acid strand is not limited, but may be, for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 or 2.
  • the region composed of non-complementary bases or an inserted sequence may form a bulge.
  • the second nucleic acid strand may further include at least one overhang region located at one or both of the 5'-end and 3'-end of the complementary region.
  • An "overhang region” refers to a region adjacent to a complementary region, in which, when the first and second nucleic acid strands anneal to form a double-stranded structure, the 5'-end of the second nucleic acid strand extends beyond the 3'-end of the first nucleic acid strand and/or the 3'-end of the second nucleic acid strand extends beyond the 5'-end of the first nucleic acid strand, i.e., a nucleotide region in the second nucleic acid strand that protrudes from the double-stranded structure.
  • the overhang region in the second nucleic acid strand may be located at the 5'-end or 3'-end of the complementary region.
  • the overhang region in the second nucleic acid strand may be located at the 5'-end and 3'-end of the complementary region.
  • a functional moiety may be bound to the first nucleic acid strand and/or the second nucleic acid strand, for example, the second nucleic acid strand.
  • the bond between the first nucleic acid strand and/or the second nucleic acid strand and the functional moiety may be a direct bond or an indirect bond via another substance, but in one embodiment, it is preferable that the first nucleic acid strand and/or the second nucleic acid strand and the functional moiety are directly bound to each other via a covalent bond, an ionic bond, a hydrogen bond, or the like, and a covalent bond is more preferable from the viewpoint of obtaining a more stable bond.
  • the structure of the "functional portion” is not particularly limited, and it confers a desired function to the double-stranded nucleic acid complex to which it is bound.
  • the desired functions include a labeling function, a purification function, and a target delivery function.
  • Examples of the portion that confers a labeling function include compounds such as fluorescent proteins and luciferase.
  • Examples of the portion that confers a purification function include compounds such as biotin, avidin, His tag peptide, GST tag peptide, and FLAG tag peptide.
  • a molecule having an activity of delivering the double-stranded nucleic acid complex in a certain embodiment to a target site is bound as a functional portion to the first nucleic acid strand and/or the second nucleic acid strand.
  • the portion that confers a target delivery function include lipids, antibodies, aptamers, and ligands for a specific receptor.
  • the first nucleic acid strand and/or the second nucleic acid strand, e.g., the second nucleic acid strand is bound to a lipid.
  • the lipid may include, but is not limited to, tocopherol, cholesterol, fatty acids, phospholipids and their analogs; folic acid, vitamin C, vitamin B1, vitamin B2; estradiol, androstane and their analogs; steroids and their analogs; LDLR, SRBI or LRP1/2 ligands; FK-506, and cyclosporine; lipids described in WO2019/182109 and WO2019/177061, etc.
  • the lipid may be tocopherol or an analog thereof and/or cholesterol or an analog thereof, a substituted or unsubstituted C 1-30 alkyl group, a substituted or unsubstituted C 2-30 alkenyl group, or a substituted or unsubstituted C 1-30 alkoxy group.
  • the second nucleic acid strand may be bound to tocopherol or cholesterol or an analog thereof.
  • tocopherol is a methylated derivative of tocorol, a fat-soluble vitamin (vitamin E) with a ring structure called chroman.
  • Tocorol has a strong antioxidant effect, and therefore, as an antioxidant in the body, it has the function of eliminating free radicals generated by metabolism and protecting cells from damage.
  • Tocopherol is known in several different forms, consisting of ⁇ -tocopherol, ⁇ -tocopherol, ⁇ -tocopherol, and ⁇ -tocopherol, based on the position of the methyl group bound to the chroman.
  • tocopherol may be any tocopherol.
  • examples of tocopherol analogs include various unsaturated analogs of tocopherol, such as ⁇ -tocotrienol, ⁇ -tocotrienol, ⁇ -tocotrienol, and ⁇ -tocotrienol.
  • the tocopherol is ⁇ -tocopherol.
  • cholesterol refers to a type of sterol, also known as a steroid alcohol, and is found in large amounts in animals. Cholesterol plays an important role in metabolic processes in the body, and in animal cells, it is also a major component of the cell membrane system along with phospholipids. Furthermore, cholesterol analogs refer to various cholesterol metabolites and analogs, which are alcohols with a sterol skeleton, and include, but are not limited to, cholestanol, lanosterol, cerebrosterol, dehydrocholesterol, and coprostanol.
  • analog refers to a compound that has a similar structure and properties with the same or a similar basic skeleton.
  • Analogs include, for example, biosynthetic intermediates, metabolic products, compounds with substituents, etc. Whether or not a compound is an analog of another compound can be determined by a person skilled in the art based on common technical knowledge.
  • the functional moiety may be linked to the 5' end, or the 3' end, or both ends of the first and/or second nucleic acid strand.
  • the functional moiety may be linked to an internal nucleotide of the first and/or second nucleic acid strand.
  • the first and/or second nucleic acid strand may contain two or more functional moieties, such as lipids, which may be linked to multiple positions on the first and/or second nucleic acid strand and/or may be linked as a group to one position on the first and/or second nucleic acid strand.
  • the functional moieties may be linked to the 5' end and the 3' end of the first and/or second nucleic acid strand, one each.
  • the bond between the first and/or second nucleic acid strand and the functional moiety may be a direct bond or an indirect bond mediated by another substance. However, in certain embodiments, it is preferred that the functional moiety is directly bonded to the first and/or second nucleic acid strand via a covalent bond, ionic bond, hydrogen bond, etc., and a covalent bond is more preferred in terms of obtaining a more stable bond.
  • the functional moiety may also be linked to the first and/or second nucleic acid strands via a cleavable or uncleavable linker.
  • the first and second nucleic acid strands may be linked via a linker to form a single strand.
  • the functional region has the same structure as in the double-stranded nucleic acid complex, and therefore, in this specification, such a single-stranded nucleic acid is also included as an embodiment of the double-stranded nucleic acid complex of the present invention.
  • the linker may be any polymer. Examples include polynucleotides, polypeptides, and alkylenes.
  • the linker may be composed of natural nucleotides such as DNA and RNA, or non-natural nucleotides such as peptide nucleic acids and morpholino nucleic acids.
  • the linker is composed of a nucleic acid
  • the chain length of the linker may be at least 1 base, for example, 3 to 10 bases or 4 to 6 bases. The chain length is preferably 4 bases.
  • the linker may be in the form of a hinge (hairpin loop).
  • the linker can be located on either the 5' or 3' side of the first nucleic acid strand, but for example, in the case of a configuration in which the second nucleic acid strand is bound to the 5' side of the first nucleic acid strand, the 5' end of the first nucleic acid strand and the 3' end of the second nucleic acid strand are linked via a linker.
  • “Cleavable linker” refers to a linking group that is cleaved under physiological conditions, e.g., within a cell or within an animal (e.g., within the human body). In certain embodiments, the cleavable linker is selectively cleaved by an endogenous enzyme, such as a nuclease. Cleavable linkers include amides, esters, phosphodiesters or both esters, phosphate esters, carbamates, and disulfide bonds, as well as natural DNA linkers.
  • Non-cleavable linker means a linker that is not cleaved under physiological conditions, for example, within a cell or an animal body (for example, within the human body).
  • Non-cleavable linkers include, but are not limited to, linkers consisting of phosphorothioate bonds, and modified or unmodified deoxyribonucleosides or modified or unmodified ribonucleosides linked by phosphorothioate bonds.
  • the linker is a nucleic acid such as DNA or an oligonucleotide
  • the chain length is not particularly limited, but may be usually 2 to 20 bases, 3 to 10 bases, or 4 to 6 bases.
  • linker represented by the following formula (IV):
  • L 2 represents a substituted or unsubstituted C 1 -C 12 alkylene group (e.g., propylene, hexylene, dodecylene), a substituted or unsubstituted C 3 -C 8 cycloalkylene group (e.g., cyclohexylene), -(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 -, -(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 -, or CH(CH 2 -OH)-CH 2 -O- (CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 -;
  • L 3 represents -NH- or a bond;
  • L 4 represents a substituted or
  • L5 represents an alkylene group having 1 to 12 carbon atoms (e.g., ethylene, pentylene, heptylene, undecylene), a substituted or unsubstituted cycloalkylene group having 3 to 8 carbon atoms (e.g., cyclohexylene), -( CH2
  • the linker represented by formula (IV) is one in which L 2 is an unsubstituted C 3 to C 6 alkylene group (e.g., propylene, hexylene), -(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 -, or -(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 -, L 3 is -NH-, and L 4 and L 5 are bonds.
  • L 2 is an unsubstituted C 3 to C 6 alkylene group (e.g., propylene, hexylene), -(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 2 -O-(CH 2 ) 3 -, or -(CH 2 ) 2 -O-(CH 2 )
  • the linker includes a nucleic acid, a polyether group, and/or an alkylamino group.
  • the nucleic acid may be, for example, one or two to ten nucleosides and/or non-natural nucleosides linked by internucleoside bonds.
  • Examples of polyether groups include polyethylene glycol groups or triethylene glycol groups.
  • Examples of alkylamino groups include hexylamino groups.
  • the base length of the first nucleic acid strand and the second nucleic acid strand is not particularly limited, but may be at least 8 bases, at least 9 bases, at least 10 bases, at least 11 bases, at least 12 bases, at least 13 bases, at least 14 bases, or at least 15 bases.
  • the base length of the first nucleic acid strand and the second nucleic acid strand may be 40 bases or less, 35 bases or less, 30 bases or less, 25 bases or less, 24 bases or less, 23 bases or less, 22 bases or less, 21 bases or less, 20 bases or less, 19 bases or less, 18 bases or less, 17 bases or less, or 16 bases or less.
  • the first nucleic acid strand and the second nucleic acid strand may be the same length or different lengths (for example, one of them may be 1 to 3 bases shorter or longer).
  • the double-stranded structure formed by the first nucleic acid strand and the second nucleic acid strand may include a bulge.
  • the length can be selected based on the balance between the strength of the antisense effect and the specificity of the nucleic acid strand for the target, among other factors such as cost and synthesis yield.
  • the overall base length of the first nucleic acid strand and the second nucleic acid strand may be the above-mentioned base length plus the base length of the bound nucleic acid.
  • the base length of the bound nucleic acid is not limited, but may be, for example, at least 10 bases, at least 15 bases, or at least 20 bases, or may be 100 bases or less, 80 bases or less, 60 bases or less, 40 bases or less, or 30 bases or less.
  • the double-stranded nucleic acid complex of the present invention can achieve high efficacy (e.g., steric blocking, exon skipping, exon inclusion, splicing control, expression downregulation, expression upregulation, and/or base editing) while at the same time achieving low or no toxicity.
  • high efficacy e.g., steric blocking, exon skipping, exon inclusion, splicing control, expression downregulation, expression upregulation, and/or base editing
  • composition 3-1 Overview
  • the third aspect of the present invention is a composition.
  • the composition of the present invention contains at least one nucleic acid molecule of the first aspect or one double-stranded nucleic acid complex of the second aspect, and can be used to treat diseases.
  • the pharmaceutical composition of the present invention can simultaneously achieve a high gene regulation effect and low or no toxicity.
  • the composition of this aspect can exhibit an excellent antisense effect in the central nervous system while reducing neurotoxicity.
  • composition 3-2-1 Active ingredient
  • the composition of the present invention contains, as an active ingredient, at least the nucleic acid molecule according to the first aspect or the double-stranded nucleic acid complex according to the second aspect.
  • the composition of the present invention may contain one or more nucleic acid molecules and/or double-stranded nucleic acid complexes.
  • the amount (content) of the nucleic acid molecule or double-stranded nucleic acid complex contained in the composition of the present invention varies depending on the type of nucleic acid molecule or double-stranded nucleic acid complex, the delivery site, the formulation of the composition, the dosage of the composition, and the type of carrier described below. Therefore, it may be appropriately determined taking into account each condition.
  • the composition is adjusted so that an effective amount of the nucleic acid molecule or double-stranded nucleic acid complex is contained in a single dose.
  • the "effective amount” refers to the amount necessary for the nucleic acid molecule or double-stranded nucleic acid complex to function as an active ingredient, and to an amount that gives little or no harmful side effects to the living body to which it is applied.
  • Subject information refers to various individual information of the living body to which the composition is applied. For example, if the subject is a human, it includes age, weight, sex, diet, health condition, progression and severity of the disease, drug sensitivity, and the presence or absence of concomitant drugs.
  • composition of the present invention may contain a pharma- ceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to an additive commonly used in the field of formulation technology. Examples include solvents, vegetable oils, bases, emulsifiers, suspending agents, surfactants, pH adjusters, stabilizers, excipients, vehicles, preservatives, binders, diluents, isotonicity agents, sedatives, bulking agents, disintegrants, buffers, coating agents, lubricants, thickeners, dissolution aids, and other additives.
  • the solvent may be, for example, water or any other pharma- ceutically acceptable aqueous solution, or a pharma-ceutically acceptable organic solvent.
  • aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffer, and sodium acetate buffer.
  • adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, and other low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc.
  • the above-mentioned carriers are used to avoid or inhibit the decomposition of the active ingredient, the double-stranded nucleic acid complex, by enzymes and other factors in the body, as well as to facilitate formulation and administration methods and maintain the dosage form and efficacy, and may be used appropriately as needed.
  • the dosage form of the composition of the present invention is not particularly limited as long as it is a form that can deliver the active ingredient, a nucleic acid molecule or a double-stranded nucleic acid complex, to a target site without inactivating the active ingredient through degradation or the like, and can exert the pharmacological effect of the active ingredient in the body (antisense effect on the expression of a target gene).
  • the specific dosage form may be a dosage form suitable for intrathecal administration.
  • the preferred dosage form is a liquid that can be administered intrathecally.
  • An example of a liquid is an injection.
  • An injection can be formulated by appropriately combining the above-mentioned excipients, elixirs, emulsifiers, suspending agents, surfactants, stabilizers, pH regulators, etc., and mixing them in a unit dose form required for generally accepted pharmaceutical practice.
  • composition of the present invention may be formulated according to the usual methods in the art.
  • the nucleic acid molecule or double-stranded nucleic acid complex of the present invention has excellent properties as a pharmaceutical, such as excellent solubility in water, Japanese Pharmacopoeia Dissolution Test Fluid 2, or Japanese Pharmacopoeia Disintegration Test Fluid 2, excellent pharmacokinetics (e.g., drug half-life in blood, brain transferability, metabolic stability, CYP inhibition), low toxicity (e.g., superior as a pharmaceutical in terms of acute toxicity, chronic toxicity, genotoxicity, neurotoxicity, reproductive toxicity, cardiotoxicity, drug interactions, carcinogenicity, cytotoxicity, etc.), and few side effects (e.g., suppression of sedation, avoidance of lamellar necrosis).
  • excellent pharmacokinetics e.g., drug half-life in blood, brain transferability, metabolic stability, CYP inhibition
  • low toxicity e.g., superior as a pharmaceutical in terms of acute toxicity, chronic toxicity, genotoxicity, neurotoxicity, reproductive toxicity, cardiotoxicity, drug interactions
  • parenteral administration includes intrathecal administration (intraventricular administration, posterior fossa puncture, or lumbar puncture), nasal administration, intravenous administration, intraarterial administration, administration by blood transfusion, intraperitoneal administration, intraocular administration, intramuscular administration, subcutaneous administration (including implantable continuous subcutaneous administration), intradermal administration, intravesical administration, intravaginal administration, rectal administration, inhalation or nasal administration, and tracheal/bronchial administration.
  • intrathecal administration or nasal administration which is advantageous for delivery to the target site, is preferable, but it can also be delivered to the central nervous system by passing through the blood-brain barrier by, for example, intravenous administration, subcutaneous administration, intraperitoneal administration, or intramuscular administration.
  • the compositions of the present invention can be administered intrathecally.
  • Intrathecal administration can be, for example, intraventricular administration, posterior fossa puncture, or lumbar puncture.
  • Intrathecal administration can also be administered using a shunt, an indwelling catheter, or a subcutaneous port.
  • the double-stranded nucleic acid complex may be administered in an amount of 0.01 mg or more, 0.1 mg or more, or 1 mg or more, for example, 2 mg or more, 3 mg or more, 4 mg or more, 5 mg or more, 10 mg or more, 20 mg or more, 30 mg or more, 40 mg or more, 50 mg or more, 75 mg or more, 100 mg or more, 200 mg or more, 300 mg or more, 400 mg or more, or 500 mg or more, or 0.01 mg to 1000 mg, 0.1 mg to 200 mg, or 1 mg to 20 mg, and in the case of mice, 1 ⁇ g or more may be administered.
  • the dosage of the composition may be, for example, 0.00001 mg/kg/day to 10,000 mg/kg/day, or 0.001 mg/kg/day to 100 mg/kg/day, of the nucleic acid molecule or double-stranded nucleic acid complex contained therein.
  • the composition may be administered in a single dose or multiple doses. In the case of multiple doses, the composition may be administered daily or at appropriate time intervals (e.g., at intervals of 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month), for example, 2 to 20 times.
  • the dosage of the nucleic acid molecule or double-stranded nucleic acid complex per administration can be, for example, 0.001 mg/kg or more, 0.005 mg/kg or more, 0.01 mg/kg or more, 0.25 mg/kg or more, 0.5 mg/kg or more, 1.0 mg/kg or more, 2.0 mg/kg or more, 3.0 mg/kg or more, 4.0 mg/kg or more, 5 mg/kg or more, 10 mg/kg or more, 20 mg/kg or more, 30 mg/kg or more, 40 mg/kg or more, 50 mg/kg or more, 75 mg/kg or more, 100 mg /kg or more, 150 mg/kg or more, 200 mg/kg or more, 300 mg/kg or more, 400 mg/kg or more, or 500 mg/kg or more, and can be appropriately selected from any amount within the range of, for example, 0.001 mg/kg to 500 mg/kg (e.g., 0.001 mg/kg, 0.01 mg/kg, 0.1 mg/kg, 1 mg/kg, 5 mg
  • the nucleic acid molecule or double-stranded nucleic acid complex may be administered at a dose of 0.01 to 10 mg/kg (e.g., about 6.25 mg/kg) twice a week for four doses.
  • the nucleic acid molecule or double-stranded nucleic acid complex may be administered at a dose of 0.05 to 30 mg/kg (e.g., about 25 mg/kg) once or twice a week for two to four doses, e.g., twice a week for two doses.
  • toxicity e.g., avoiding a decrease in platelets
  • the burden on the subject can be reduced compared to a single administration of a higher dose.
  • the pharmaceutical composition exerts an additive inhibitory effect within cells even when administered repeatedly. Furthermore, when administering repeatedly, the effectiveness can be improved by leaving a certain interval between administrations (for example, half a day or more).
  • the target diseases for the pharmaceutical composition include, for example, central nervous system diseases.
  • the target diseases may involve genes whose expression levels of transcription products or translation products may be suppressed or enhanced, whose functions of transcription products or translation products may be inhibited, or whose steric blocking, splicing switch, RNA editing, exon skipping, or exon inclusion may be induced by the antisense effect of the nucleic acid molecule or double-stranded nucleic acid complex of the present invention.
  • the composition may be used in animals, including humans, as subjects. However, there is no particular limitation on animals other than humans, and various livestock, poultry, pets, laboratory animals, etc. may be subjects in some embodiments.
  • the subject may be one in which it is necessary to reduce the expression of a target transcript in the central nervous system.
  • the subject may also be one in which it is necessary to treat a central nervous system disorder.
  • the disease to be treated may be a central nervous system disease associated with increased or decreased gene expression, particularly a disease (such as a tumor) associated with increased expression of a target transcript or target gene.
  • central nervous system diseases include, but are not limited to, brain tumors, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, etc.
  • the nervous system is divided into the central nervous system and the peripheral nervous system.
  • the central nervous system consists of the brain and spinal cord.
  • the brain includes the cerebrum (cerebral cortex, cerebral white matter, basal ganglia), diencephalon (thalamus, subthalamic nucleus), cerebellum (cerebellar cortex, cerebellar nuclei) and brainstem (midbrain, substantia nigra, pons, medulla oblongata).
  • the spinal cord includes the cervical, thoracic, lumbar, sacral and coccygeal spinal cord.
  • the central nervous system in this specification may be any of these regions, but in particular may be the cerebral cortex (frontal lobe, temporal lobe, parietal lobe, occipital lobe), cerebellum, striatum, globus pallidus, claustrum, hippocampus, parahippocampal gyrus, brainstem, cervical spinal cord, thoracic spinal cord or lumbar spinal cord.
  • the peripheral nerves consist of the cranial nerves and spinal nerves.
  • FTD frontotemporal dementia
  • SD semantic dementia
  • PNFA progressive non-fluent aphasia
  • Pick's disease drug delivery to the frontal lobe, temporal lobe and/or substantia nigra may be effective.
  • Parkinson's disease dementia drug delivery to the occipital lobe, substantia nigra and/or striatum may be effective.
  • drug delivery to the substantia nigra and/or striatum may be effective.
  • corticobasal degeneration In the treatment of corticobasal degeneration (CBD), drug delivery to the frontal lobe, parietal lobe, basal ganglia and/or substantia nigra may be effective. In the treatment of progressive supranuclear palsy (PSP), drug delivery to the frontal lobe, basal ganglia and/or substantia nigra may be effective. In the treatment of amyotrophic lateral sclerosis, drug delivery to the frontal lobe, parietal lobe, basal ganglia, and/or substantia nigra may be effective.
  • CBD corticobasal degeneration
  • PPSP progressive supranuclear palsy
  • amyotrophic lateral sclerosis drug delivery to the frontal lobe, parietal lobe, basal ganglia, and/or substantia nigra may be effective.
  • SCD spinocerebellar degeneration
  • DPLA dentatorubral-pallidoluysian degeneration
  • SBMA spinal-bulbar atrophy
  • FA Friedreich's ataxia
  • striatum In the treatment of Huntington's disease, drug delivery to the striatum, frontal lobe, parietal lobe, and/or basal ganglia may be effective.
  • prion diseases mad cow disease, GSS
  • drug delivery to the cerebral cortex, cerebral white matter, basal ganglia and/or substantia nigra In the treatment of cerebral leukoencephalopathy, drug delivery to the cerebral white matter may be effective.
  • encephalitis viral, bacterial, fungal, tuberculous
  • meningitis viral, bacterial, fungal, tuberculous
  • drug delivery to the entire brain may be effective.
  • drug delivery to the cerebral white matter may be effective.
  • cerebral infarction cerebral hemorrhage, subarachnoid hemorrhage, moyamoya disease, and anoxic encephalopathy
  • drug delivery to the entire brain may be effective.
  • drug delivery to the cerebral white matter may be effective.
  • drug delivery to the cerebral white matter may be effective.
  • drug delivery to the cerebral white matter may be effective.
  • drug delivery to the cerebral white matter may be effective.
  • drug delivery to the cerebral white matter may be effective.
  • head trauma drug delivery to the entire brain may be effective.
  • MM ⁇ sclerosis multiple sclerosis
  • NMO neuromyelitis optica
  • drug delivery to the cerebral white matter, cerebral cortex, optic nerve, and/or spinal cord may be effective.
  • myotonic dystrophy DM1, DM2
  • drug delivery to skeletal muscle, cardiac muscle, cerebral cortex, and/or cerebral white matter may be effective.
  • HSP familial spastic paraplegia
  • drug delivery to the parietal lobe and/or spinal cord may be effective.
  • Fukuyama muscular dystrophy drug delivery to skeletal muscle, cerebral cortex, and/or cerebral white matter may be effective.
  • ⁇ nigra In the treatment of dementia with Lewy bodies (DLB), drug delivery to the substantia nigra, striatum, occipital lobe, frontal lobe, and/or parietal lobe may be effective.
  • MSA multiple system atrophy
  • drug delivery to the striatum, basal ganglia, cerebellum, substantia nigra, frontal lobe, and/or temporal lobe In the treatment of Alexander disease, drug delivery to the cerebral white matter may be effective. In the treatment of CADASIL and CARASIL, drug delivery to the cerebral white matter may be effective.
  • composition of the present invention can achieve preventive or therapeutic effects on central nervous system diseases by intrathecal administration.
  • the present invention also provides a method for treating and/or preventing diseases, such as central nervous system diseases, which comprises administering the above-mentioned composition to a subject, such as a human.
  • Example 1 Toxicity evaluation of Mapt-targeted ASO with 5'-CP modification introduced into the gap region (the purpose)
  • a 5'-cyclopropylene modification (hereinafter abbreviated as "5'-CP modification”) is introduced into the gap region of a gapmer-type antisense nucleic acid (hereinafter abbreviated as "ASO") targeting the Mapt gene.
  • ASO gapmer-type antisense nucleic acid
  • the ASO (control) used in this example is an LNA/DNA gapmer ASO that targets mouse microtubule-associated protein tau (Mapt) mRNA, has a base sequence complementary to a portion of Mapt mRNA, and has a structure in which three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and ten DNA nucleosides between them are linked by phosphorothioate bonds.
  • the three LNA nucleosides at the 5' end correspond to the 5' wing region of the gapmer ASO
  • the three LNA nucleosides at the 3' end correspond to the 3' wing region
  • the ten DNA nucleosides between them correspond to the gap region (central region).
  • the ASO(5'-CP gap1) to ASO(5'-CP gap10) used in this example contained the following formula (I):
  • the 5'-cyclopropylene modification refers to a modification in which a cyclopropane group is attached to the 5' carbon of ribose.
  • the base position located on the 5'-most side in the gap region is referred to as "gap 1”
  • the Nth base position in the 3' direction is referred to as "gap N.” Therefore, in ASO(5'-CP gap1) to ASO(5'-CP gap10), the nucleosides located at gap 1 to gap 10, respectively, have a 5'-CP modification.
  • the ASOs used in the following examples were contract-synthesized by Gene Design Inc. (Osaka, Japan).
  • the ASO prepared in (1) was introduced into mouse neuroblastoma cells (Neuro-2a cell line) at 50 nM using lipofection (lipofectamine 2000). 72 hours after introduction, neurotoxicity was evaluated by measuring the number of viable cells, lactate dehydrogenase (LDH) activity in the cell supernatant, and the expression levels of Cdkn1a and Il-6 mRNA. The number of viable cells was determined by aspirating the medium, preparing a cell suspension, and measuring the number of viable cells using Scepter 3.0 (Millipore).
  • LDH lactate dehydrogenase
  • LDH activity was measured using the Cytotoxicity LDH Assay Kit-WST (Dojindo Laboratories) according to the attached protocol. The value normalized against the LDH activity in the PBS-administered group was used as the relative LDH release level.
  • Cdkn1a mRNA and Il-6 mRNA were measured as follows. After measuring the number of cells, RNA was extracted from the cell solution using the Isogen-LS kit (Gene Design Co., Ltd.). cDNA was synthesized using Transcriptor Universal cDNA Master, DNase (Roche Diagnostics) according to the protocol. Next, the expression levels of Cdkn1a mRNA, Il-6 mRNA, and Gapdh mRNA (internal standard gene) were measured by performing quantitative RT-PCR using the obtained cDNA as a template. Quantitative RT-PCR was performed using TaqMan (Roche Applied Science). Primers used in quantitative RT-PCR were products designed and manufactured by Thermo Fisher Scientific (formerly Life Technologies Corp).
  • Amplification was performed by repeating 40 cycles of 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 1 second (1 cycle).
  • the ratios of the expression levels of Cdkn1a mRNA and Il-6 mRNA to the expression level of Gapdh mRNA (internal control gene) were calculated, and the values normalized to the values in the PBS-administered group were used as the expression levels of Cdkn1a mRNA and Il-6 mRNA.
  • Figure 4 shows the results of measuring the number of viable cells after transfection with PBS or various nucleic acid agents to determine cytotoxicity in the Neuro-2a cell line.
  • the number of viable cells in the ASO (control) group was lower than that in the PBS group, indicating cytotoxicity.
  • the ASO (5'-CP gap1), ASO (5'-CP gap3), and ASO (5'-CP gap9) groups showed higher numbers of viable cells.
  • Figure 5 shows the results of measuring LDH activity in the supernatant as an indicator of cytotoxicity when various nucleic acid agents were introduced into cells.
  • LDH activity was increased in the ASO (control) administration group, indicating cytotoxicity.
  • ASO (5'-CP gap1) to ASO (5'-CP gap10) administration groups ASO (5'-CP gap1), ASO (5'-CP gap3), and ASO (5'-CP gap9) showed low LDH activity, indicating an effect of reducing cytotoxicity.
  • Figures 6-7 show the results of evaluating the expression levels of Cdkn1a mRNA and Il-6 mRNA as a measure of cytotoxicity when various nucleic acid agents were introduced into cells.
  • ASO control
  • the expression levels of both genes were increased, indicating cytotoxicity.
  • the expression of both genes was reduced in ASO (5'-CP gap1), ASO (5'-CP gap3), and ASO (5'-CP gap9), indicating the effect of reducing cytotoxicity.
  • Example 2 Toxicity evaluation of Mapt-targeted ASOs with 5'-CP and 2'-O-methyl modifications introduced into the gap region (the purpose) 5'-CP modifications and 2'-O-methyl modifications (hereinafter referred to as "2'-OMe modifications") will be introduced into the gap region of a gapmer-type ASO targeting the Mapt gene, and the toxicity reduction effect will be verified through in vitro experiments.
  • the ASO (control) and ASO (5'-CP gap3) used in this example were the same as those used in Example 1.
  • the ASO (2'-OMe gap2) and ASO (2'-OMe gap2/5'-CP gap3) used in this example have a 2'-OMe modified nucleoside (a ribonucleoside in which the hydroxyl group at the 2' position of the ribose is replaced with a methoxy group) at the second base position (gap 2) from the 5' side of the gap region of the above ASO (control) and the above ASO (5'-CP gap3), respectively.
  • a 2'-OMe modified nucleoside a ribonucleoside in which the hydroxyl group at the 2' position of the ribose is replaced with a methoxy group
  • the ASO prepared in (1) was introduced into mouse neuroblastoma-derived cells (Neuro-2a cell line) at 50 nM using the lipofection method (lipofectamine 2000). 72 hours after introduction, the number of viable cells and lactate dehydrogenase (LDH) activity in the cell supernatant were measured in the same manner as in Example 1 to evaluate neurotoxicity.
  • LDH lactate dehydrogenase
  • Figure 9 shows the results of measuring the number of viable cells after transfection with PBS or various nucleic acid agents as cytotoxicity against the Neuro-2a cell line.
  • the number of viable cells decreased in the ASO (control) administration group compared to the PBS administration group, indicating cytotoxicity.
  • the number of viable cells increased in the ASO (2'-OMe gap2) administration group compared to the ASO (control) administration group, indicating reduced cytotoxicity.
  • the number of viable cells in the ASO (5'-CP gap3) administration group was higher than that in the ASO (2'-OMe gap2) administration group, indicating a further toxicity reduction effect.
  • the ASO (2'-OMe gap2/5'-CP gap3) administration group which combines the two modifications, showed an even higher number of viable cells than the ASO (5'-CP gap3) administration group, indicating a significant toxicity reduction effect.
  • Figure 10 shows the results of measuring LDH activity in the supernatant as an indicator of cytotoxicity when various nucleic acid agents were introduced into cells.
  • ASO control
  • LDH activity increased, indicating cytotoxicity.
  • ASO (2'-OMe gap2) administration group 5'-CP gap3) administration group
  • ASO (2'-OMe gap2/5'-CP gap3) administration group LDH activity decreased to the same level as the PBS administration group, indicating a significant reduction in cytotoxicity.
  • Example 3 In vivo toxicity evaluation of Mapt-targeted ASOs with 5'-CP and 2'-O-methyl modifications introduced into the gap region (the purpose) We will use in vivo experiments to evaluate the effect of introducing 5'-CP and 2'OMe modifications into the gap region to reduce central neurotoxicity observed when gapmer-type ASOs targeting the Mapt gene are administered intracerebroventricularly.
  • the ASO(2'-OMe gap PS-3(-)), ASO(2'-OMe gap3 PS-3(-)), and ASO(5'-CP gap3 PS-3(-)) used in this example have a 2'-OMe modified nucleoside, a 5'-CP modified nucleoside, and a 5'-CP modified nucleoside at the second (gap 2), third (gap 3), and third (gap 3) base positions, respectively, from the 5' side of the gap region of the above ASO (control), and the internucleoside bond between gap 2 and gap 3 in all cases is a phosphodiester bond (PO bond).
  • PO bond phosphodiester bond
  • a negative control group of mice was also prepared that received only PBS.
  • mice after administration of various nucleic acid agents delayed central neurotoxicity was evaluated by measuring body weight/food intake and evaluating motor function by an open field test.
  • Body weight/food intake was measured 20 days after administration of the nucleic acid agent.
  • Motor function was evaluated 10, 14, 17, and 20 days after administration of the nucleic acid agent.
  • the mouse was placed in the center of a cage (50 cm wide x 50 cm diameter x 40 cm high), and the trajectory of the mouse's movement was recorded for 5 minutes. The maximum movement speed (m/s) based on the recorded data was measured using video tracking software (ANY-maze).
  • Quantitative RT-PCR was performed using the obtained cDNA as a template to measure the expression levels of Mapt mRNA and Actb mRNA (internal control genes). Quantitative RT-PCR was performed using TaqMan (Roche Applied Science). Primers used in quantitative RT-PCR were products designed and manufactured by Thermo Fisher Scientific (formerly Life Technologies Corp). Amplification conditions (temperature and time) were as follows: 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 1 second (1 cycle), repeated 40 cycles.
  • the ratio of the expression level of Mapt mRNA to the expression level of Actb mRNA (internal control gene) was calculated, and the value normalized to the value of the PBS-administered group was used as the relative Mapt mRNA level.
  • Figure 12 shows the results of measuring body weight/food intake 20 days after intracerebroventricular administration of various nucleic acid agents in mice.
  • Body weight/food intake was lower in the ASO (control) group than in the PBS group, indicating central neurotoxicity.
  • Body weight was higher in the ASO (2'-OMegap2 PS-3(-)) and ASO (2'-OMe gap3 PS-3(-)) groups than in the ASO (control) group, but no difference was observed in food intake.
  • both body weight and food intake were higher in the ASO (5'-CP gap3 PS-3(-)) group than in the ASO (control) group.
  • Figure 13 shows the results of evaluating motor function in mice that had been intracerebroventricularly administered various nucleic acid agents 10, 14, 17, and 20 days after administration.
  • the ASO (5'-CP gap3 PS-3(-)) group had a higher maximum movement speed and reduced delayed neurotoxicity compared to the ASO (control) group.
  • Figure 14 shows the Mapt mRNA expression levels in the lumbar spinal cord 21 days after intracerebroventricular administration of various nucleic acid agents.
  • the gene silencing effects were reduced in the ASO (2'-OMegap2), ASO (2'-OMe gap2 PS-3(-)), and ASO (2'-OMegap3 PS-3(-)) administration groups compared to the ASO (control) administration group.
  • the ASO (5'-CP gap3 PS-3(-)) administration group showed a gene silencing effect equivalent to that of the ASO (control) administration group.
  • ASO(5'-CP gap3 PS-3(-)) reduced delayed toxicity to the same extent as ASO(2'-OMegap2) and showed lower delayed toxicity than ASO(2'-OMe gap2 PS-3(-)) and ASO(2'-OMegap3 PS-3(-)), while having a higher gene suppression effect than ASO(2'-OMe gap2), ASO(2'-OMegap2 PS-3(-)), and ASO(2'-OMe gap3 PS-3(-)).
  • Example 4 Toxicity evaluation of Snca-targeting ASO with 5'-CP modification introduced into the gap region (the purpose) A 5'-CP modification will be introduced into the gap region of a gapmer-type ASO targeting the Snca gene, and the toxicity reduction effect will be verified through in vitro experiments (human cells).
  • the ASO (control) used in this example is an LNA/DNA gapmer ASO that targets mouse alpha-synuclein (Snca) mRNA, has a base sequence complementary to a portion of Snca mRNA, and has a structure in which three LNA nucleosides at the 5' end, four LNA nucleosides at the 3' end, and 12 DNA nucleosides between them are linked by phosphorothioate bonds.
  • the three LNA nucleosides at the 5' end correspond to the 5' wing region of the gapmer ASO
  • the three LNA nucleosides at the 3' end correspond to the 3' wing region
  • the 12 DNA nucleosides between them correspond to the gap region (central region).
  • the ASO(5'-CP gap1) to ASO(5'-CP gap12) used in this example contain 5'-CP modified nucleosides at the 1st to 12th base positions (gap 1 to gap 12) from the 5' side of the gap region of the above ASO(control).
  • the ASO prepared in (1) was introduced into human neuroblastoma-derived cells (BE(2)-M17 cell line) at 50 nM using the lipofection method (lipofectamine2000). 72 hours after introduction, neurotoxicity was evaluated by measuring the number of viable cells, lactate dehydrogenase (LDH) activity in the cell supernatant, and Cdkn1a mRNA expression level. The number of viable cells, LDH activity, and the expression level of Cdkn1a mRNA were measured in the same manner as in Example 1.
  • LDH lactate dehydrogenase
  • Figure 16 shows the results of evaluating the number of viable cells as an indicator of cytotoxicity against the BE(2)-M17 cell line.
  • the number of viable cells was lower in the ASO (control)-treated group compared to the PBS-treated group, indicating cytotoxicity.
  • the number of viable cells was higher in the groups administered ASO with 5'-CP modifications introduced into gaps 1 to 3 and gaps 7 to 9, demonstrating the effect of reducing cytotoxicity.
  • Figure 17 shows the results of evaluating LDH activity in the supernatant as an indicator of cytotoxicity when various nucleic acid agents were introduced into cells.
  • LDH activity was increased in the ASO (control) administration group, indicating cytotoxicity.
  • ASO control
  • ASO ASO
  • ASO ASO
  • ASO ASO
  • 5'-CP gap 12 ASO
  • LDH activity was low, particularly in the ASO groups in which 5'-CP modifications were introduced into gaps 1 to 3 and gaps 7 to 9, indicating an effect of reducing cytotoxicity.
  • Figure 18 shows the results of evaluating the expression level of Cdkn1a mRNA as a measure of cytotoxicity when various nucleic acid agents were introduced into cells.
  • ASO control
  • the expression level of Cdkn1a mRNA was increased, indicating cytotoxicity.
  • the expression level of Cdkn1a mRNA was reduced in ASOs that introduced 5'-CP modifications to gaps 1 to 3 and gaps 7 to 9, indicating an effect of reducing cytotoxicity.
  • Example 5 Toxicity evaluation of Snca-targeting ASO with 5'-CP modification introduced into the gap region (the purpose) A 5'-CP modification will be introduced into the gap region of a gapmer-type ASO targeting the Snca gene, and its toxicity-reducing effect will be verified through in vitro experiments (mouse cells).
  • ASO (control), ASO (5'-CP gap1), ASO (5'-CP gap3), and ASO (5'-CP gap9) used in this example were the same as those used in Example 4.
  • the ASO prepared in (1) was introduced into mouse neuroblastoma-derived cells (Neuro-2a cell line) at 50 nM using the lipofection method (lipofectamine2000). 72 hours after introduction, neurotoxicity was evaluated by measuring the number of viable cells, lactate dehydrogenase (LDH) activity in the cell supernatant, and the expression level of Cdkn1a mRNA using the same methods as in Example 1.
  • LDH lactate dehydrogenase
  • Figure 20 shows the results of measuring the number of viable cells after transfection with PBS or various nucleic acid agents to measure cytotoxicity against the Neuro-2a cell line.
  • the number of viable cells was reduced in the ASO (control) administration group compared to the PBS administration group, indicating cytotoxicity.
  • the number of viable cells was high in the ASO (5'-CP gap1), ASO (5'-CP gap3), and ASO (5'-CP gap9) administration groups, indicating a toxicity-reducing effect.
  • Figure 21 shows the results of measuring LDH activity in the supernatant as an indicator of cytotoxicity.
  • LDH activity was increased in the ASO (control) administration group, indicating cytotoxicity.
  • LDH activity was low in the ASO (5'-CP gap1), ASO (5'-CP gap3), and ASO (5'-CP gap9) administration groups, indicating a toxicity-reducing effect.
  • Example 6 Toxicity evaluation of Hdac2-targeting ASO with 5'-CP and 2'-O-methyl modifications introduced into the gap region (the purpose) 5'-CP and 2'-O-methyl modifications will be introduced into the gap region of gapmer-type ASO targeting the Hdac2 gene, and the toxicity reduction effect will be verified through in vitro experiments.
  • the ASO (control) used in this example is an LNA/DNA gapmer-type ASO that targets mouse histone deacetylase 2 (Hdac2) mRNA, has a base sequence complementary to a portion of Hdac2 mRNA, and has a structure in which three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and 10 DNA nucleosides between them are linked by phosphorothioate bonds (PS bonds).
  • Hdac2 mouse histone deacetylase 2
  • the ASO (2'-OMe gap2) used in this example contains a 2'-OMe modified nucleoside at the second base position from the 5' side (gap 2) of the gap region of the above ASO (control).
  • the 2'-OMe modification is a modification in which the hydroxyl group at the 2' position of ribose is replaced with a methoxy group.
  • ASO (5'-CP gap3) contains a 5'-CP modified nucleoside at the third base position from the 5' side (gap 3) of the gap region of ASO (control).
  • ASO (5'-CP gap3 PS-3(-)) contains a 5'-CP modified nucleoside at the third base position from the 5' side (gap 3) of the gap region of ASO (control), and the internucleoside bond between gap 2 and gap 3 is a phosphodiester bond (PO bond).
  • ASO(5'-CP gap9 PS-9(-)) contains a 5'-CP modified nucleoside at the 9th base position (gap 9) from the 5' side of the gap region of ASO(control), and the internucleoside bond between gap 8 and gap 9 is a phosphodiester bond (PO bond).
  • the ASO prepared in (1) was introduced into human neuroblastoma-derived cells (BE(2)-M17 cell line) at 20 nM using the lipofection method (lipofectamine2000). 72 hours after introduction, cell counts and lactate dehydrogenase (LDH) activity in the cell supernatant were measured according to the method described in Example 1 to evaluate neurotoxicity.
  • LDH lactate dehydrogenase
  • FIG. 23 shows the results of measuring the number of viable cells after transfection with PBS or various nucleic acid agents as cytotoxicity against the BE(2)-M17 cell line.
  • the number of viable cells was reduced in the ASO(control)-administered group compared to the PBS-administered group, indicating cytotoxicity.
  • the number of viable cells was lower in the ASO(control)-administered group in which the 2'-OMe modification was introduced into gap 2 (2'-OMe gap2), indicating enhanced cytotoxicity.
  • the number of viable cells was higher in the ASO(control)-administered group in which the 5'-CP modification was introduced into gap 3 or gap 9, regardless of whether the internucleoside bond attached to the 5' side of the 5'-CP-modified nucleoside was a PS bond or a PO bond, indicating the effect of reducing cytotoxicity.
  • Figure 24 shows the results of measuring LDH activity in the supernatant as an indicator of cytotoxicity when various nucleic acid agents were introduced into cells.
  • LDH activity was increased in the ASO (control) administration group, indicating cytotoxicity.
  • ASOs in which 5'-CP modification was introduced into gap 3 or gap 9 showed lower LDH activity than the ASO (control) administration group, regardless of whether the internucleoside bond attached to the 5' side of the 5'-CP modified nucleoside was a PS bond or a PO bond, indicating the effect of reducing cytotoxicity.
  • the 5'-CP modification is a nucleic acid modification that can enhance nuclease resistance when using more biocompatible phosphodiester bonds to avoid phosphorothioate bonds, which are a concern due to their accumulation in certain organs.
  • the effect of the 5'-CP modification itself in reducing toxicity was unexpected.
  • it is surprising that a toxicity-reducing effect can be obtained regardless of whether the internucleoside bond attached to the 5' side of the 5'-CP modified nucleoside is a PS bond or a PO bond.
  • Example 7 Toxicity evaluation of Hdac2-targeting ASO with 5'-CP modification introduced into the gap region (the purpose) 5'-CP modifications will be introduced into the gap region of gapmer-type ASO targeting the Hdac2 gene, and the effect on gene suppression will be verified through in vitro experiments.
  • the ASO (control) used in this example is an LNA/DNA gapmer-type ASO that targets mouse histone deacetylase 2 (Hdac2) mRNA, has a base sequence complementary to a portion of Hdac2 mRNA, and has a structure in which three LNA nucleosides at the 5' end, three LNA nucleosides at the 3' end, and 10 DNA nucleosides between them are linked by phosphorothioate bonds (PS bonds).
  • Hdac2 mouse histone deacetylase 2
  • the ASO(5'-CP gap1) used in this example contains a 5'-CP modified nucleoside at the first base position from the 5' side (gap 1) of the gap region of ASO(control).
  • ASO(5'-CP gap1 PS-3(-)) contains a 5'-CP modified nucleoside at the first base position from the 5' side (gap 1) of the gap region of ASO(control), and the internucleoside bond on the 5' side of gap 1 is a phosphodiester bond (PO bond).
  • ASO(5'-CP gap3) contains a 5'-CP modified nucleoside at the third base position from the 5' side (gap 3) of the gap region of ASO(control).
  • ASO(5'-CP gap3 PS-3(-)) contains a 5'-CP modified nucleoside at the third base position from the 5' side of the gap region of ASO(control) (gap 3), and the internucleoside bond between gap 2 and gap 3 is a phosphodiester bond (PO bond).
  • ASO(5'-CP gap9) contains a 5'-CP modified nucleoside at the ninth base position from the 5' side of the gap region of ASO(control) (gap 9).
  • ASO(5'-CP gap9 PS-9(-)) contains a 5'-CP modified nucleoside at the ninth base position from the 5' side of the gap region of ASO(control) (gap 9), and the internucleoside bond between gap 8 and gap 9 is a phosphodiester bond (PO bond).
  • the ASO prepared in (1) was introduced into mouse neuroblastoma-derived cells (Neuro-2a cell line) using the lipofection method (lipofectamine2000). 72 hours after ASO introduction, RNA was extracted from the cells using an IsogenI kit (Gene Design Co., Ltd.), and cDNA synthesis and quantitative RT-PCR were performed according to the method described in Example 1. The ratio of Hdac2 mRNA expression level to Actb mRNA (internal control gene) expression level was calculated, and the value standardized against the value of the PBS-administered group was used as the relative Hdac2 mRNA level.
  • Figure 26 shows the results of measuring Hdac2 mRNA expression levels in Neuro-2a cell lines transfected with various nucleic acid agents.
  • gene suppression effects equivalent to those of the ASO (control) were achieved, regardless of whether the internucleoside bond attached to the 5' side of the 5'-CP-modified nucleoside was a PS bond or a PO bond.
  • Example 8 Toxicity evaluation of Hdac2-targeting ASO with 2'-O-methyl modification in the gap region (the purpose)
  • the ASO (control) and ASO (2'-OMe gap2) used in this example were the ASO (control) and ASO (2'-OMe gap2) described in Example 6.
  • nucleic acid agent was administered intracerebroventricularly in the same manner as in Example 3. However, the nucleic acid agent was administered at a dose of 18.96 nmol/mouse, and a negative control group of mice was also prepared to which only nuclease free water (NFW) was administered.
  • NFW nuclease free water
  • Body weight and food intake were measured 3 and 6 days after administration of the nucleic acid agent.
  • Behavioral evaluation was performed 6 days after administration of the nucleic acid agent using the scoring system shown in Figure 28.
  • behaviors belonging to five categories are evaluated ( Figure 28, categories 1 to 5).
  • Each category includes two behavioral evaluation items.
  • Each behavioral evaluation item is scored on a five-point scale from 0 to 4 points ( Figure 28, scores 0 to 4), with normal being scored as 0 and higher scores indicating higher toxicity.
  • scores 0 to 4 the higher score of the two behavioral evaluation items is adopted as the score for that category.
  • the sum of the scores for the five categories represents the acute tolerability score (0 to 20 points).
  • Figure 29 shows the results of measuring body weight/food intake 3 and 6 days after intracerebroventricular administration of various nucleic acid agents in mice.
  • Body weight and food intake were lower in the ASO (control) group than in the PBS group, indicating central neurotoxicity.
  • Body weight and food intake were further lower in the ASO (2'-OMe gap2) group than in the ASO (control) group, indicating enhanced delayed central neurotoxicity.
  • Figure 30A shows the results of evaluating motor function 6 days after intracerebroventricular administration of various nucleic acid agents in mice.
  • the ASO (2'-OMegap2)-administered group had a lower maximum movement speed than the ASO (control)-administered group, indicating enhanced delayed central neurotoxicity.
  • Figure 30B shows the results of behavioral assessment 6 days after intracerebroventricular administration of various nucleic acid agents in mice.
  • the ASO (2'-OMegap2) group had higher scores than the ASO (control) group, indicating enhanced delayed central neurotoxicity.
  • Example 9 Toxicity evaluation of Hdac2-targeting ASO with 5'-CP modification introduced into the gap region (the purpose)
  • the ASOs used in this Example and listed in Table 9 above are the same as the ASO (control), ASO (5'-CP gap3), ASO (5'-CP gap3 PS-3(-)), ASO (5'-CP gap9), and ASO (5'-CP gap9 PS-9(-)) used in Example 7.
  • Body weight/food intake was measured 3, 6, 10, and 13 days after administration of the nucleic acid agent.
  • Tnf- ⁇ mRNA and Gfap mRNA were measured as follows.
  • the left hippocampus was excised from the mice 14 days after administration of various nucleic acid agents.
  • RNA was extracted from the excised left hippocampus using an IsogenI kit (Gene Design Co., Ltd.).
  • cDNA was synthesized using Transcriptor Universal cDNA Master, DNase (Roche Diagnostics) according to the protocol.
  • the expression levels of Tnf- ⁇ mRNA, Gfap mRNA, and Actb mRNA were measured by quantitative RT-PCR using the obtained cDNA as a template in the same manner as above.
  • Tnf- ⁇ mRNA and Gfap mRNA were calculated, and the values normalized to the values of the NFW-administered group were used as the Tnf- ⁇ mRNA expression levels and Gfap mRNA expression levels.
  • Figures 32 and 33 show the results of body weight/food intake.
  • body weight/food intake was lower than in the PBS-administered group, indicating central neurotoxicity.
  • ASO(5'-CP gap3), ASO(5'-CP gap3 PS-3(-)), ASO(5'-CP gap9), and ASO(5'-CP gap9 PS-9(-))-administered groups body weight/food intake was improved compared to the ASO(control)-administered group and was equivalent to that of the PBS-administered group, indicating an effect of reducing delayed central neurotoxicity.
  • Figure 34 shows the results of evaluating motor function using an open field test. The maximum movement speed was increased at all time points in the ASO(5'-CP gap3), ASO(5'-CP gap3 PS-3(-)), ASO(5'-CP gap9), and ASO(5'-CP gap9 PS-9(-)) administration groups compared to the ASO(control) administration group, indicating a reduction in delayed central neurotoxicity.
  • Figure 35 shows the results of behavioral assessment using a scoring system.
  • the ASO(5'-CP gap3), ASO(5'-CP gap3 PS-3(-)), ASO(5'-CP gap9), and ASO(5'-CP gap9 PS-9(-)) groups had lower scores at all time points compared to the ASO(control) group, indicating reduced delayed central neurotoxicity.
  • Figure 36 shows the results of measuring the expression levels of Tnf- ⁇ mRNA and Gfap mRNA in the left hippocampus as an index of delayed neurotoxicity.
  • ASO control
  • the expression of both was increased, indicating cytotoxicity.
  • the expression of both was decreased in the ASO (5'-CP gap3), ASO (5'-CP gap3 PS-3(-)), ASO (5'-CP gap9), and ASO (5'-CP gap9 PS-9(-)) administration groups, indicating an effect of reducing delayed neurotoxicity.
  • Example 10 In vivo toxicity evaluation of Mapt-targeted ASO with 5'-CP modification introduced into the gap region (the purpose) We will use in vivo experiments to evaluate the effect of introducing 5'-CP modifications into the gap region to reduce central neurotoxicity observed when gapmer-type ASOs targeting the Mapt gene are administered intracerebroventricularly.
  • the ASOs used in this example and listed in Table 10 above have the same configuration as the ASO (control), ASO (5'-CP gap1), and ASO (5'-CP gap3) used in Example 1.
  • mice after administration of various nucleic acid agents delayed central neurotoxicity was evaluated by body weight/food amount measurement and motor function evaluation by open field test. Body weight/food amount was measured 21 days after administration of nucleic acid agents. Motor function evaluation was performed 3, 7, 10, 14, 17, and 21 days after administration of nucleic acid agents. Specifically, mice were placed in the center of a cage (width 50 cm ⁇ diameter 50 cm ⁇ height 40 cm), and the trajectory of mouse movement was recorded for 5 minutes. The maximum movement speed (m/s) based on the recorded data was measured using video tracking software (ANY-maze).
  • Figure 37 shows the results of measuring body weight (Figure 37A) and food intake (Figure 37B) 21 days after intracerebroventricular administration of various nucleic acid agents in mice.
  • Body weight and food intake were lower in the ASO(control) group than in the PBS group, indicating central neurotoxicity.
  • both body weight and food intake were higher in the ASO(5'-CP gap1) and ASO(5'-CP gap3) groups than in the ASO(control) group, indicating a toxicity-reducing effect.
  • Figure 38 shows the results of evaluating motor function in mice that had been intracerebroventricularly administered various nucleic acid agents 3, 7, 10, 14, 17, and 21 days after administration.
  • the ASO(5'-CP gap1) and ASO(5'-CP gap3) groups had higher maximum movement speeds than the ASO(control) group.
  • Example 11 Toxicity evaluation of Hdac2-targeting ASO in which one of the PS bonds in the gap region was changed to a PO bond (the purpose)
  • PS bond phosphorothioate bonds
  • PO bond phosphodiester bond
  • the ASO (control) used in this example is an LNA/DNA gapmer-type ASO that targets mouse Hdac2 mRNA, and has the same structure as the ASO (control) used in Example 6. In the ASO (control), all internucleoside bonds are PS bonds.
  • the internucleoside bond between the 5' wing region and gap region of ASO (control) (the internucleoside bond adjacent to the 5' side of the 5'-most base position in the gap region (gap 1)) has been changed from a PS bond to a PO bond.
  • ASO (PS-3(-)) the internucleoside bond between the second base position from the 5' side (gap 2) and gap 3 in the gap region of ASO (control) is a phosphodiester bond (PO bond).
  • ASO (PS-9(-) the internucleoside bond between gap 8 and gap 9 of ASO (control) is a phosphodiester bond (PO bond).
  • Quantitative RT-PCR was performed using the obtained cDNA as a template to measure the expression levels of Hdac2 mRNA and Actb mRNA (internal control gene). Quantitative RT-PCR was performed using TaqMan. The primers used in quantitative RT-PCR were products designed and manufactured by Thermo Fisher Scientific. The amplification conditions (temperature and time) were as follows: 95°C for 15 seconds, 60°C for 30 seconds, and 72°C for 1 second (1 cycle), repeated 40 times.
  • the ratio of the expression level of Hdac2 mRNA to the expression level of Actb mRNA (internal control gene) was calculated, and the value normalized to the value of the PBS-administered group was used as the relative Hdac2 mRNA level.
  • Figure 39 shows the results of evaluating motor function by an open field test. The maximum movement speed was equivalent in the ASO (PS-1(-)), ASO (PS-3(-)), and ASO (PS-9(-)) administration groups compared with the ASO (control) administration group, and delayed central neurotoxicity was not reduced.
  • Figure 40 shows the Hdac2 mRNA expression levels in the left hippocampus 7 days after intracerebroventricular administration of various nucleic acid agents.
  • the gene suppression effect was weakened in the ASO (PS-1(-)), ASO (PS-3(-)), and ASO (PS-9(-)) groups compared to the ASO (control) group.

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