WO2021230286A1 - Ａｔａｘｉｎ ３発現を調節するための化合物、方法及び医薬組成物 - Google Patents

Ａｔａｘｉｎ ３発現を調節するための化合物、方法及び医薬組成物 Download PDF

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WO2021230286A1
WO2021230286A1 PCT/JP2021/018040 JP2021018040W WO2021230286A1 WO 2021230286 A1 WO2021230286 A1 WO 2021230286A1 JP 2021018040 W JP2021018040 W JP 2021018040W WO 2021230286 A1 WO2021230286 A1 WO 2021230286A1
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seq
sequence
modified
modified oligonucleotide
sequence listing
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French (fr)
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浩昭 澤本
拓也 肥後
俊平 村田
友 荒木
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田辺三菱製薬株式会社
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Priority to CN202180034948.7A priority Critical patent/CN115667513A/zh
Priority to JP2022522177A priority patent/JPWO2021230286A1/ja
Priority to US17/998,463 priority patent/US20230227824A1/en
Priority to EP21805226.4A priority patent/EP4151237A4/en
Publication of WO2021230286A1 publication Critical patent/WO2021230286A1/ja

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    • 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
    • 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
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • 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
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • 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
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===

Definitions

  • the present invention relates to a compound for reducing at least one of the pre-mRNA level, mRNA level and protein level of Ataxin 3 (ATXN3) in an animal, a method using the compound, and a pharmaceutical composition containing the compound. ..
  • the methods of the invention are useful for treating, preventing, or delaying progression of ATXN3-related diseases, such as spinocerebellar degeneration type 3 (SCA3, Machado-Joseph disease, also referred to as MJD).
  • SCA3 spinocerebellar degeneration type 3
  • MJD Machado-Joseph disease
  • Spinocerebellar degeneration type 3 is one of the most common autosomal dominant hereditary spinocerebellar degeneration types. This disease develops at an average age of 36 years, and initially, nystagmus, increased deep tendon reflex, articulation disorder, etc. appear. After that, the condition gradually worsens, and symptoms such as difficulty walking, dysphagia, eye paralysis, and compound eyes appear, and it is said that he will die in about 20 years due to aspiration pneumonia, a fall accident, and the like. There is no curative treatment to control the progression of the condition, only symptomatic treatment for motor dysfunction, extrapyramidal tract disorders, painful muscle cramps and depression, and fatigue, and rehabilitation treatment to control the progression of the condition. be.
  • Non-Patent Document 1 Takayama et al., Nature Genetics, 1993, 4,300-304.
  • the novel gene ATXN3 or MJD1 was found to have CAG repeat elongation and was identified as the responsible gene (Non-Patent Document 2 Kawaguchi et al., Nature Genetics, 1994, 8, 221-228.).
  • CAG repeat elongation is 14 to 37 in normal alleles, whereas it is 61 to 84 in extended alleles (Non-Patent Document 3 Takayama et al., Neurology, 1997, 49, 604-606), and CAG repeat elongation and onset age. Has been reported to have a negative correlation (Non-Patent Document 2, Kawaguchi et al., Nature Genetics, 1994, 8, 221-228).
  • CAG repeat elongation is the result of various pathological hypotheses such as RNA toxicity of itself or mitochondrial damage due to its translation product polyglutamine, transcriptional abnormalities, calcium homeostasis abnormalities, autophagy abnormalities, axonal transport abnormalities, etc. (non-patented) Document 4 M. M. Evers et al., Molecular Neurobiology, 2014, 49, 1513-1531), which is thought to cause motor dysfunction following cerebral purkine cell dysfunction and shedding.
  • Antisense oligonucleotides are being clinically applied one after another as an effective means for regulating the expression of certain gene products. In recent years, it has been clarified that it shows a very excellent effect on central genetic diseases such as spinraza (Non-Patent Document 5 Artsma-Rus, A. Nucleic Acid Ther. 2017, 27, 67). ing. Antisense oligonucleotides may therefore prove to be useful in several therapeutic, diagnostic, and research applications for regulating ATXN3 mRNA.
  • Patent Document 7 WO2020 / 172559
  • a genetically modified animal model spikenocerebellar degeneration type 3 animal model
  • Non-Patent Document 6 Hayley S. McLoughlin et al., Anals of Neurology). , 2018, 84, 64-77
  • the medicinal dose at this time was 700 ⁇ g per mouse, which could not be said to have sufficient medicinal potential for clinical application.
  • antisense oligonucleotides have been vigorously developed for the radical treatment of spinocerebellar degeneration type 3, they have not been sufficiently effective.
  • An object of the present invention is to provide compounds, methods, and pharmaceutical compositions for inhibiting ATXN3-expression and / or for treating, preventing, delaying, or ameliorating ATXN3-related diseases. be.
  • the present inventors have conducted diligent studies and found a modified oligonucleotide that strongly inhibits ATXN3 expression, and have completed the present invention.
  • a modified oligonucleotide having an activity of inhibiting the expression of Ataxin 3 is: 1) TCGGGTAAGTAGATTTTC (complementary sequence of 2159 to 2176 of SEQ ID NO: 1) (SEQ ID NO: 239 of the sequence listing) 2) GAAGTATCTGTAGGCCTA (complementary sequence of 2513 to 2530 of SEQ ID NO: 1) (SEQ ID NO: 240 of the sequence listing) 3) GGACTGTATAGGAGATTA (complementary sequence of 2646 to 2663 of SEQ ID NO: 1) (SEQ ID NO: 241 of the sequence listing) 4) GGTTATAGGATGCAGGTA (complementary sequence of 5844 to 5861 of SEQ ID NO: 1) (SEQ ID NO: 242 of the sequence listing) 5) AGGTTATAGGATGCAGGT (complementary sequence of 5844 to 5861 of SEQ ID NO: 1) (SEQ ID NO: 242 of the sequence listing) 5) AGGTTATAGGATGCAGGT (complementary
  • the modified sugar is selected from the group consisting of bicyclic sugars, sugars modified with 2'-MOE (2'-O-methoxyethyl), and sugars modified with 2'-OMe, [3]. Modified oligonucleotides.
  • Bicyclic sugars are selected from the sugar moieties of LNA, GuNA, ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Oxz], or ALNA [Trz], [4].
  • Modified oligonucleotide [6] The modified oligonucleotide according to any one of [1] to [5], wherein at least one nucleoside constituting the modified oligonucleotide contains a modified nucleobase.
  • the modified oligonucleotide of [6] wherein the modified nucleobase is 5-methylcytosine.
  • the modified oligonucleotide according to any one of [1] to [7], wherein at least one nucleoside-linked bond constituting the modified oligonucleotide is a modified nucleoside-modified bond.
  • the modified oligonucleotide is 1) Gap segment, 2) Includes 5'wing segment and 3) 3'wing segment, The gap segment is positioned between the 5'wing segment and the 3'wing segment.
  • modified oligonucleotide according to any one of [1] to [9], wherein the nucleoside constituting the 5'wing segment and the 3'wing segment contains a modified sugar.
  • a modified oligonucleotide consisting of 12 to 24 residues having an activity of inhibiting ATXN3 expression, wherein the nucleic acid base sequence of the modified oligonucleotide is at least in the equal length portion of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing.
  • At least one of the nucleic acids constituting the oligonucleotide having 85% complementarity is selected from the sugar moiety of ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Oxz], or ALNA [Trz].
  • a pharmaceutical composition comprising the modified oligonucleotide of any one of [1] to [11], or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of [13], wherein the ATXN3-related disease is a neurodegenerative disease.
  • Treatment, prevention or delay of progression of ATXN3-related disease in a subject characterized in that an effective amount of any of the modified oligonucleotides of [1] to [13] is administered to the subject in need thereof. Method for conversion.
  • a drug effective for preventing or treating an ATXN3-related disease for example, spinocerebellar degeneration type 3 can be improved and the disease is provided. Since the modified oligonucleotide of the present invention has excellent activity of inhibiting ATXN3 expression and has few side effects such as toxicity, it is excellent as an active ingredient of a prophylactic or therapeutic agent for ATXN3-related diseases.
  • Nucleobase means a heterocyclic moiety that can be paired with the base of another nucleic acid.
  • Nucleobase sequence means a continuous sequence of nucleobases constituting the oligonucleotide of the present invention.
  • Nucleoside means a molecule in which a sugar and a nucleobase are linked. In certain embodiments, the nucleoside is linked to a phosphate group.
  • Nucleotide means a molecule in which a phosphate group is bonded to the sugar portion of a nucleoside.
  • the naturally occurring nucleotide has a sugar moiety of ribose or deoxyribose, which is covalently bonded by a phosphodiester bond via a phosphate group.
  • Oligomer compound or "oligomer” means a polymer of linked monomer subunits that can hybridize to at least one region of a nucleic acid molecule.
  • Oligonucleotide means a polymer of nucleosides in which each nucleoside and the bonds between each nucleoside are linked independently of each other.
  • Nucleoside bond refers to a chemical bond between nucleosides.
  • Naturally occurring nucleoside bond means a 3'-5'phosphodiester bond.
  • Modified nucleoside bond refers to a substitution or arbitrary change from a naturally occurring nucleoside bond (ie, a phosphodiester nucleoside bond). For example, there are, but are not limited to, phosphorothioate nucleoside linkages.
  • Phosphodiester bond between nucleosides means a bond between nucleosides in which the phosphodiester bond is modified by replacing one of the non-crosslinked oxygen atoms with a sulfur atom.
  • the phosphorothioate bond is an example of the modified nucleoside-linked bond.
  • Modified base refers to any nucleobase other than adenine, cytosine, guanine, thymidine or uracil. For example, there is, but is not limited to, 5-methylcytosine.
  • Unmodified nucleobase means the purine bases adenine (A) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified oligonucleotide means an oligonucleotide containing at least one of the modified nucleosides and / or the linkage between the modified nucleosides.
  • Salt is a general term for compounds in which one or more dissociable hydrogen ions contained in an acid are replaced with cations such as metal ions and ammonium ions, and the salt of the modified oligonucleotide is phosphorothioate.
  • a salt eg, sodium salt, magnesium salt
  • an inorganic ion eg, sodium ion, magnesium ion
  • a functional group eg, amino group
  • “Sugar” or “sugar moiety” means a natural sugar moiety or a modified sugar moiety.
  • Modified sugar refers to a substitution or change from a natural sugar.
  • Examples of the modified sugar include a substituted sugar moiety and a bicyclic sugar.
  • Substituted sugar moiety means furanosyl other than natural sugar in RNA or DNA.
  • Bicyclic sugar means a furanosyl ring modified by cross-linking two different carbon atoms present on the same ring.
  • Bicyclic nucleic acid refers to a nucleoside or nucleotide in which the furanose portion of the nucleoside or nucleotide comprises a "bicyclic sugar”.
  • Single-stranded oligonucleotide means an oligonucleotide that does not hybridize with the complementary strand.
  • ATXN3 means a nucleic acid or protein also called Ataxin 3.
  • ATXN3 may include, for example, various splicing variants transcribed from the ATXN3 gene, single nucleotide polymorphisms (SNPs) or CAG repeat extensions.
  • “Complementary” means the ability of the first nucleic acid to form a pair between the nucleobases of the second nucleic acid.
  • adenine is complementary to thymidine or uracil.
  • cytosine is complementary to guanine.
  • 5-methylcytosine is complementary to guanine.
  • “Completely complementary (also referred to as complementarity)” or “100% complementary (also referred to as complementary)” means that all of each nucleobase in the nucleic acid base sequence of the first nucleic acid is the second of the second nucleic acid. Means having a complementary nucleobase in the nucleobase sequence of.
  • the first nucleic acid is a modified oligonucleotide and the target nucleic acid is a second nucleic acid.
  • mismatch or “non-complementary nucleobase” refers to the case where the nucleobase of the first nucleic acid cannot be paired with the corresponding nucleobase of the second or target nucleobase.
  • Target nucleic acid refers to nucleic acids that can be targeted by modified oligonucleotides.
  • the target nucleic acid comprises a region of ATXN3 mRNA or ATXN3 pre-mRNA.
  • Microtif means a combination of chemically heterogeneous regions in a modified oligonucleotide.
  • Directly adjacent means that there is no intervening element between directly adjacent elements.
  • a “pharmaceutically acceptable salt” is a physiologically and pharmaceutically acceptable salt of a modified oligonucleotide of the invention, i.e., which retains the desired biological activity of the modified oligonucleotide and imparts an undesired toxic effect to it. Means no salt.
  • administering means giving the drug to an animal, including, but not limited to, administration by a medical professional or family member and self-administration.
  • “Improvement” refers to reducing at least one indicator, sign or symptom of a related disease, disorder or condition.
  • the severity of the indicator can be determined by subjective or objective measures known to those of skill in the art.
  • Animal refers to humans, or non-human animals including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and, but not limited to, non-human primates including monkeys and chimpanzees. Point to.
  • Effective amount means the amount of the modified oligonucleotide of the invention that is sufficient to achieve the desired physiological outcome in an individual in need of the drug. Effective amounts vary among individuals depending on the health and physical condition of the individual being treated, the taxon of the individual being treated, the formulation of the composition, the assessment of the individual's medical condition and other relevant factors. obtain.
  • “Individual” means a human or non-human animal selected for treatment or therapy.
  • Treatment reduces, eliminates, or slows the progression of a disease, disorder or unfavorable health condition, or one or more symptoms associated with the disease, disorder or unfavorable health condition. Or, it means partially eliminating or eradicating one or more causes of the disease, disorder, or unfavorable health condition itself.
  • Certain specific embodiments shown below are not limited to these, but provide a compound for inhibiting the expression of ATXN3, a method using the compound, and a pharmaceutical composition containing the compound.
  • Modified oligonucleotide The modified oligonucleotide of the present invention (hereinafter, may be referred to as "the compound of the present invention” or “the modified oligonucleotide of the present invention”) is an antisense oligonucleotide of ATXN3 having an activity of inhibiting ATXN3 expression.
  • Inhibition of ATXN3 expression (level) in the present invention means at least one of the pre-mRNA level transcribed from the ATXN3 gene, the pre-mRNA spliced mRNA level, and the ATXN3 protein level translated from the mRNA. It means to suppress.
  • the ATXN3 pre-mRNA may contain CAG repeat elongation found in patients with SNP and / or spinocerebellar degeneration type 3, eg, NC_0014.9: c92106621-92058552 Homo sapiens chromosome 14, GRCh38.
  • the one shown by the sequence (SEQ ID NO: 1 in the sequence listing) described in p13 Primary Assembury can be mentioned.
  • the ATXN3 mRNA may contain at least one of the CAG repeat elongations found in patients with variants, SNPs and spinocerebellar degeneration type 3, eg, the sequence described in GenBank Accession No. NM_004993.5.
  • the one shown by (SEQ ID NO: 2 in the sequence listing) can be mentioned.
  • the base sequence of DNA is shown in SEQ ID NOs: 1 and 2
  • T is read as U in the case of an RNA sequence.
  • the degree of inhibition of ATXN3 expression possessed by the compound of the present invention is reduced as compared with the case where at least one of the pre-mRNA level, the mRNA level and the protein level of ATXN3 is not administered with the compound, resulting in ATXN3-related diseases. Any degree may be used as long as the prevention and / or improvement of the accompanying symptoms is observed. Specifically, for example, in the method for measuring in vitro ATXN3 expression described later, after contacting the compound of the present invention with cells, the compound is contacted with the cells.
  • the ATXN3 expression level is at least 70% or less, preferably 50% or less, more preferably 40% or less, still more preferably 30% or less, and particularly preferably 20% or less, as compared with the case of non-contact or contact with a negative control substance. Is used.
  • the compound of the present invention is a modified oligonucleotide consisting of 12 to 24 residues, preferably 16 to 18 residues, more preferably 18 residues having an activity of inhibiting ATXN3 expression, and is the same as SEQ ID NO: 1 in the sequence listing. From the 5'end of the nucleic acid base sequence, positions 256 to 273, 404 to 427, 541 to 558, 647 to 665, 697 to 716, 767 to 784, 2043 to 2066, 2092 to 2109, 2159 to 2159.
  • ATXN3 complementary nucleic acid base sequence a nucleic acid base sequence complementary to the equal length portion of the above. It may be that one.
  • the ATXN3 complementary nucleobase sequence is preferably 8 to 18 consecutive nucleobase sequences, and more preferably 16, 17 or 18 consecutive nucleobase sequences.
  • the ATXN3 complementary nucleobase sequence includes positions 1512 to 1536, positions 1568 to 1583, positions 3931-1946, positions 5668 to 5683, and positions 7817 to 7832 from the 5'end of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing. , 9925 to 9940, 10409 to 10424, 10556 to 10571, 10584 to 10599, 11002 to 11017, 11617 to 11632, 12076 to 12091, 14074 to 14089, 14213 to 14228, 1621-1226.
  • the modified oligonucleotide of the present invention has a total length of 12 to 24 residues, preferably 16 to 18 residues, more preferably 18 residues in addition to the above ATXN3 complementary nucleobase sequence, and the same thereof. If the full-length nucleobase sequence has at least 85% complementarity to the equal-length portion of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing, it has an additional sequence on its 5'end and / or 3'end. You may. Furthermore, the addition sequence may be any as long as the modified oligonucleotide of the present invention has an activity of inhibiting ATXN3 expression.
  • the full-length nucleobase sequence of the modified oligonucleotide has at least 85% complementarity to the equilength portion of SEQ ID NO: 1 in the sequence listing, with the complementarity preferably 90%, more preferably 95%. More preferably, it is 100%.
  • the equal-length portion is the nucleic acid base sequence of the modified oligonucleotide of the present invention and the nucleic acid base sequence of ATXN3 pre-mRNA or mRNA, and is aligned using software such as BLAST and Genetyx software (GENETYX CORPORATION). In particular, it means a portion detected as a portion having homology.
  • the nucleobase sequence of the oligonucleotide of the present invention is completely complementary to the equal length portion of the ATXN3 pre-mRNA or the nucleobase sequence of the mRNA, but it has one or more mismatched nucleobases. It may be used, and those having a complementarity of 85% or more, 90% or more, preferably 95% or more are used.
  • the mismatched nucleobase may be continuous or may be interspersed with an ATXN3 complementary nucleobase sequence.
  • the percent complementarity of the antisense oligonucleotide of the present invention with ATXN3 pre-mRNA or mRNA is described, for example, in BLAST programs (basic local alignment sequences) and PowerBLAST programs (Altschul et al., J. Mol.) Known in the art. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649, 656), can be determined conventionally by using Genetyx software (GENETYX CORPORATION).
  • Percentage homology, sequence identity or complementarity can be determined, for example, by the Gap Program (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group, UniversityResearchAman.Park, Madison). It can be determined using the default setting using the algorithm of 1981, 2, 482, 489), Genetyx software (GENETYX CORPORATION).
  • Gap Program WidesinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group, UniversityResearchAman.Park, Madison. It can be determined using the default setting using the algorithm of 1981, 2, 482, 489), Genetyx software (GENETYX CORPORATION).
  • the modified oligonucleotide has 90% complementarity when 18 of the 20 nucleobases of the modified oligonucleotide are complementary and hybridize to the equal length portion of the ATXN3 pre-mRNA.
  • the method for verifying the activity of the compound of the present invention may be any method as long as it can be verified that the compound of the present invention inhibits ATXN3 expression. Specifically, for example, an in vitro ATXN3 expression measuring method described later may be used. Used.
  • modified oligonucleotides of the present invention thus selected include positions 256 to 273, positions 404 to 421, positions 405 to 422, positions 407 to 424, and positions 408 to the 5'end of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing.
  • the full-length nucleobase sequence of the modified oligonucleotide has 100% complementarity to the equal-length portion of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing, the 5'end side and / or the 3'end thereof. It may have an additional sequence of 1 or 2 residues on the side.
  • the modified oligonucleotide was added to SH-SY5Y cells by the gymnosis method so as to have a final concentration of 3 ⁇ M, and the ATXN3 mRNA level was reduced to 70% or less as compared with the case where the modified oligonucleotide was not contacted.
  • the nucleic acid base sequence of the modified oligonucleotide that inhibits include positions 698 to 715, 699 to 716, 2043 to 2060, 2046 to 2063, and 2047 from the 5'end of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing.
  • TCGGGTAAGTAGATTTTC (complementary sequence of 2159-2176 of SEQ ID NO: 1) (SEQ ID NO: 239 of the sequence listing), GAAGTATCTGTAGGCCTA (complementary sequence of 2513 to 2530 of SEQ ID NO: 1) (SEQ ID NO: 240 of the sequence listing), GGACTGTATAGGAGATTA (complementary sequence of 2646 to 2663 of SEQ ID NO: 1) (SEQ ID NO: 241 of the sequence listing), GGTTATAGGATGCAGGTA (Complementary sequence of 5844 to 5861 of SEQ ID NO: 1) (SEQ ID NO: 242 of the sequence listing), AGGTTATAGGATGCAGGT (complementary sequence of 5845 to 5862 of SEQ ID NO: 1) (SEQ ID NO: 243 of the sequence listing), GAAGCTAAGTAGGTGACT (complementary sequence of 15115 to 15132
  • TCGGGTAAGTAGATTTTC (complementary sequence of 2159-2176 of SEQ ID NO: 1) (SEQ ID NO: 239 of the sequence listing), AGGTTATAGGATGCAGGT (complementary sequence of 5845 to 5862 of SEQ ID NO: 1) (SEQ ID NO: 243 of the sequence listing), GAAGCTAAGTAGGTGACT (complementary sequence of 15115 to 15132 of SEQ ID NO: 1) (SEQ ID NO: 244 of the sequence listing), GTCATCCCTATGTCTTAT (complementary sequence of 36607 to 36624 of SEQ ID NO: 1) (SEQ ID NO: 259 of the sequence listing), GTCATATGGTCAGGGTAT (complementary sequence of 40453 to 40470 of SEQ ID NO: 1) (SEQ ID NO: 260 in the sequence listing), TGTCATATGGTCAGGGTA (complementary sequence of 40454 to
  • GTCATATGGTCAGGGTAT Complementary sequence of 40453 to 40470 of SEQ ID NO: 1 (SEQ ID NO: 260 of the sequence listing)
  • SEQ ID NO: 260 of the sequence listing The nucleic acid base sequence described in the above, or a continuous 17-base nucleic acid base sequence contained in the nucleic acid base sequence can be mentioned.
  • one or more cytosins may be 5-methylcytosine, which is a modified nucleic acid base described later, and specifically, for example, SEQ ID NO: 23, SEQ ID NO: 38, SEQ ID NO: 63, SEQ ID NO: 131, SEQ ID NO: Examples thereof include the sequences shown in 148, SEQ ID NO: 149, SEQ ID NO: 150 or SEQ ID NO: 151, preferably the sequences shown in SEQ ID NO: 148.
  • the 16-base nucleic acid base sequence of the modified oligonucleotide of the present invention includes, for example, SEQ ID NO: 264, SEQ ID NO: 179, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 192, SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 204, SEQ ID NO: 269, SEQ ID NO: 214, SEQ ID NO: 220, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 228 or SEQ ID NO: 230, preferably SEQ ID NO: 265, SEQ ID NO: 268, SEQ ID NO: 204, Examples thereof include the sequences shown in SEQ ID NO: 269, SEQ ID NO: 214, SEQ ID NO: 220, SEQ ID NO: 270, SEQ ID NO: 271 or SEQ ID NO: 230.
  • one or more cytosins may be 5-methylcytosine, which is a modified nucleic acid base described later, and specifically, for example, SEQ ID NO: 168, SEQ ID NO: 184, SEQ ID NO: 187, SEQ ID NO: 194, SEQ ID NO: Examples thereof include the sequences shown in 203, SEQ ID NO: 208, SEQ ID NO: 224, or SEQ ID NO: 225, preferably the sequences shown in SEQ ID NO: 184, SEQ ID NO: 203, SEQ ID NO: 208, SEQ ID NO: 224 or SEQ ID NO: 225.
  • the modified oligonucleotide of the present invention may be a double-stranded modified oligonucleotide, but a single-stranded modified oligonucleotide is preferably used.
  • modified sugar As the modified oligonucleotide of the present invention, one in which at least one nucleoside constituting the oligonucleotide contains a modified sugar is preferably used.
  • the modified sugar means a modified sugar moiety, and a modified oligonucleotide containing one or more of the modified sugars has advantageous features such as enhanced nuclease stability and increased binding affinity.
  • At least one of the modified sugars is preferably selected from the group consisting of bicyclic sugars, 2'-MOE modified sugars, and 2'-OMe modified sugars.
  • bicyclic sugars include sugar moieties of LNA, GuNA, ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Oxz], or ALNA [Trz], as shown below.
  • ALNA [Ms] sugar moiety is preferably used.
  • substituted sugar moieties are, but are not limited to, 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH 3 (2'-OMe). , 2'-OCH 2 CH 3 , 2'-OCH 2 CH 2 F and 2'-O (CH 2 ) 2 OCH 3 (2'-MOE) nucleosides containing substituents.
  • Substituents at the 2'position are allyl, amino, azide, thio, O-allyl , OC 1 to C 10 alkyl, OCF 3 , OCH 2 F, O (CH 2 ) 2 SCH 3 , O (CH 2 ).
  • each R l , R m and R n are independently H or substituted or unsubstituted C 1 to C 10 alkyl). You can choose from.
  • nucleosides with bicyclic sugars include, but are not limited to, nucleosides that include crosslinks between the 4'and 2'ribosyl ring atoms.
  • the oligonucleotides provided herein comprise a nucleoside having one or more bicyclic sugars whose cross-linking comprises one of the following formulas: 4'-(CH 2). ) -O-2'(LNA);4'-(CH 2 ) -S-2';4'-(CH 2 ) 2- O-2'(ENA);4'-CH (CH 3 ) -O -2'and 4'-CH (CH 2 OCH 3 ) -O-2' (and their analogs, see US Pat. No.
  • Each of the aforementioned bicyclic sugar-bearing nucleosides can be prepared with one or more optically active sugar configurations, including, for example, ⁇ -L-ribofuranose and ⁇ -D-ribofuranose.
  • GuNA of nucleosides with bicyclic sugars has been reported as artificial nucleosides with guanidine crosslinks (see WO2014 / 046212, WO2017 / 047816).
  • the bicyclic nucleosides ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Trz] and ALNA [Oxz] have been reported as crosslinked artificial nucleic acid amino LNA (ALNA) (international application PCT / JP2019). / 044182 (WO2020 / 100826)).
  • a bridge containing two linking groups is included, in which X is 0, 1 or 2; n is 1, 2, 3 or 4; each Ra and R b are independently H, a protective group.
  • Hydroxyl C 1 to C 12 alkyl, substituted C 1 to C 12 alkyl, C 2 to C 12 alkenyl, substituted C 2 to C 12 alkenyl, C 2 to C 12 alkynyl, substituted C 2 to C 12 alkynyl, aromatic ring.
  • the cross-linking of the bicyclic sugar moiety is-[C (R a ) (R b )] n -,-[C (R a ) (R b )] n- O-, -C. (R a R b ) -N (R) -O- or C (R a R b ) -ON (R)-.
  • the crosslinks are 4'-CH 2 -2', 4'-(CH 2 ) 2 -2', 4'-(CH 2 ) 3 -2', 4'-CH 2- O.
  • the nucleoside having a bicyclic sugar in this case is also referred to as LNA
  • the cross-linking of the bicyclic sugar moiety is 4'-CH 2- O-2'-(LNA) or CH 2- N (R)-, where each R is independent.
  • -CO-NH-CH (CH 3 ) 2 (ALNA [ipU]), 5-methyl-1,2,4-oxadiazole-3-yl (ALNA [Oxz])
  • International application PCT / JP2019 / 044182 (WO2020 / 100826)).
  • nucleosides with bicyclic sugars are further defined by isomer configuration.
  • the nucleoside containing the 4'-(CH 2 ) -O-2'crosslink may be present in the ⁇ -L-configuration or the ⁇ -D-configuration.
  • nucleosides with bicyclic sugars include, but are not limited to, ⁇ -L-4'-(CH 2 ). -O-2', ⁇ -D-4'-CH 2- O-2', 4'-(CH 2 ) 2- O-2', 4'-CH 2 -ON (R) -2' 4, 4'-CH 2- N (R) -O-2', 4'-CH (CH 3 ) -O-2', 4'-CH 2- S-2', 4'-CH 2 -CH ( CH 3) -2 'and 4' - (CH 2) 3 -2 '( wherein, R, H, a protecting group, C 1 ⁇ C 12 alkyl, or C 1 in ⁇ C 12 alkyl which may be substituted Urea or guanidine).
  • nucleosides with bicyclic sugars have the following formula: During the ceremony Bx is the heterocyclic base portion;
  • the T a and T b are each independently a hydrogen atom, a hydroxyl protecting group, optionally phosphate groups be substituted, a covalent bond etc. to phosphorus moiety or support;
  • Z a is C 1 to C 6 alkyl, C 2 to C 6 alkenyl, C 2 to C 6 alkynyl, substituted C 1 to C 6 alkyl, substituted C 2 to C 6 alkenyl, substituted C 2 to C 6 alkynyl, acyl. , Substituted acyl, substituted amide, thiol or substituted thiol.
  • NJ c J d in the formula, each J c , J d and J e are independently H, C 1 to C 6 alkyl or substituted C 1 to C 6 alkyl, and X is O or NJ c ). It is mono- or poly-substituted with independently selected substituents.
  • nucleosides with bicyclic sugars have the following formula: During the ceremony Bx is the heterocyclic base portion;
  • the T a and T b are each independently a hydrogen atom, a hydroxyl protecting group, optionally phosphate groups be substituted, a covalent bond etc. to phosphorus moiety or support;
  • nucleosides with bicyclic sugars have the following formula: During the ceremony Bx is the heterocyclic base portion;
  • the T a and T b are each independently a hydrogen atom, a hydroxyl protecting group, optionally phosphate groups be substituted, a covalent bond etc. to phosphorus moiety or support;
  • R d is C 1 to C 6 alkyl, substituted C 1 to C 6 alkyl, C 2 to C 6 alkenyl, substituted C 2 to C 6 alkenyl, C 2 to C 6 alkynyl or substituted C 2 to C 6 alkynyl.
  • Each q a , q b , q c and q d are independently H, halogen, C 1 to C 6 alkyl, substituted C 1 to C 6 alkyl, C 2 to C 6 alkenyl, substituted C 2 to C 6 alkenyl, C 2 to C 6 alkynyl or substituted C 2 to C 6 alkynyl, C 1 to C 6 alkoxyl, substituted C 1 to C 6 alkoxyl, acyl, substituted acyl, C 1 to C 6 aminoalkyl or substituted C 1 to C 6 amino It is alkyl.
  • nucleosides with bicyclic sugars have the following formula: During the ceremony Bx is the heterocyclic base portion;
  • the T a and T b are each independently a hydrogen atom, a hydroxyl protecting group, optionally phosphate groups be substituted, a covalent bond etc. to phosphorus moiety or support;
  • q a , q b , q e and q f are independently hydrogen, halogen, C 1 to C 12 alkyl, substituted C 1 to C 12 alkyl, C 2 to C 12 alkoxy, substituted C 2 to C 12 alkoxy, respectively.
  • adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil bicyclic nucleosides are oligomerizations and nucleic acids thereof. It is described together with the recognition characteristics (Koshkin etal., Tetrahedron, 1998, 54, 3607-3630).
  • the synthesis of nucleosides with bicyclic sugars is also described in WO98 / 39352 and WO99 / 14226.
  • 2'-amino-LNA bicyclic nucleosides in this case also referred to as ALNA
  • ALNA a conformational oligonucleotide analog with constitutive limitations
  • nucleosides with bicyclic sugars have the following formula: During the ceremony Bx is the heterocyclic base portion;
  • the T a and T b are each independently a hydrogen atom, a hydroxyl protecting group, optionally phosphate groups be substituted, a covalent bond etc. to phosphorus moiety or support;
  • Each q i , q j , q k and q l are independently H, halogen, C 1 to C 12 alkyl, substituted C 1 to C 12 alkyl, C 2 to C 12 alkoxy, substituted C 2 to C 12 alkoxy, respectively.
  • nucleosides having bicyclic sugars include, but are not limited to, compounds as shown below.
  • Bx is the base moiety
  • R represents independently a protecting group, C 1 ⁇ C 6 alkyl or C 1 ⁇ C 6 alkoxy.
  • nucleosides having bicyclic sugars have the following general formula: [During the ceremony, B is a nucleobase; Each of X and Y can independently include a nucleoside represented by a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group which may be substituted, a covalent bond to a phosphorus moiety or a support, etc.] (WO98 / See 39352). A typical specific example is the following formula: The nucleotides indicated by can be mentioned.
  • nucleosides including bicyclics, have the following general formula: [In the formula, B is a nucleic acid base, and R 3 , R 4 , R 5 and R 6 are each independently a hydrogen atom or a C 1-6 alkyl group which may be substituted with one or more substituents. Yes, R 7 and R 8 are independently hydrogen atoms, hydroxyl group protective groups, optionally substituted phosphate groups, covalent bonds to phosphorus moieties or supports, etc., and R 9 , R 10 , R 11 is a C 1-6 alkyl group or amino group protective group, each of which may be independently substituted with a hydrogen atom or one or more substituents. ] It is a nucleoside represented by (see, for example, International Publication No. 2014/046212, International Publication No. 2017/047816).
  • nucleosides comprising bicyclic sugars are represented by the following general formula (I):
  • B is a nucleobase
  • R 1 , R 2 , R 3 and R 4 are C 1-6 alkyl groups, each independently of which may be substituted with a hydrogen atom or one or more substituents
  • R 5 and R 6 are each independently a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group which may be substituted, a covalent bond to a phosphorus moiety or a support, and the like
  • m is 1 or 2
  • X is the following formula (II-1): It is a group indicated by; Symbols described in formula (II-1): Indicates the binding point with the 2'-amino group;
  • One of R 7 and R 8 is a hydrogen atom and the other is a methyl group which may be substituted with one or more substituents.
  • a typical embodiment is a nucleoside in which one of R 7 and R 8 is a hydrogen atom and the other is an unsubstituted methyl group.
  • a nucleoside comprising a bicyclic sugar is a nucleoside having the general formula (I) as defined in ALNA [mU] above, wherein the nucleoside comprises the above formula.
  • X is the following formula (II-1): It is a group indicated by; One of R 7 and R 8 is a hydrogen atom and the other is an isopropyl group which may be substituted with one or more substituents (see, for example, international application PCT / JP2019 / 0441182 (WO2020 / 100826)). ..
  • a typical embodiment is a nucleoside in which one of R 7 and R 8 is a hydrogen atom and the other is an unsubstituted isopropyl group.
  • the nucleoside comprising the bicyclic formula is a nucleoside having the above general formula (I), wherein X is the following formula (II-2) :. It is a group indicated by; A is a triazolyl group which may be substituted with one or more substituents (see, for example, international application PCT / JP2019 / 044182 (WO2020 / 100826)).
  • a typical embodiment of ALNA [Trz] is a triazolyl group in which A may have one or more methyl groups, more specifically 1,5-dimethyl-1,2,4-. It is a nucleoside, which is a triazole-3-yl group.
  • X is the following formula (II-2): It is a group indicated by; A is an oxadiazolyl group which may be substituted with one or more substituents (see, for example, international application PCT / JP2019 / 044182 (WO2020 / 100826)).
  • a typical embodiment is an oxadiazolyl group in which A may have one or more methyl groups, more specifically a 5-methyl-1,2,4-oxadiazole-3-yl group. Is a nucleoside or nucleotide.
  • the nucleoside comprising the bicyclic formula is a nucleoside having the above general formula (I), wherein X is the following general formula (II-3) :. It is a group indicated by; M is a sulfonyl group substituted with a methyl group, which may be substituted with one or more substituents (see, eg, International Application PCT / JP2019 / 044182 (WO2020 / 100826)).
  • a typical embodiment of ALNA [Ms] is a nucleoside, which is a sulfonyl group in which M is substituted with an unsubstituted methyl group.
  • the nucleoside is modified by replacing the ribosyl ring with a sugar substitute.
  • modifications include, but are not limited to, substitute ring systems (sometimes referred to as DNA analogs), such as morpholino rings, cyclohexenyl rings, cyclohexyl rings or tetrahydropyranyl rings, such as:
  • substitute ring systems sometimes referred to as DNA analogs
  • morpholino rings such as morpholino rings, cyclohexenyl rings, cyclohexyl rings or tetrahydropyranyl rings
  • morpholino rings such as morpholino rings, cyclohexenyl rings, cyclohexyl rings or tetrahydropyranyl rings, such as:
  • sugar substitutes having the following formula are selected: During the ceremony Bx is the heterocyclic base portion; T 3 and T 4 independently link a tetrahydropyran nucleoside analog to an oligomer compound, or one of T 3 and T 4 links a tetrahydropyran nucleoside analog to an oligomer compound or oligonucleotide.
  • q 1 , q 2 , q 3 , q 4 , q 5 , q 6 and q 7 are independently H, C 1 to C 6 alkyl, substituted C 1 to C 6 alkyl, C 2 to C 6 alkenyl, substituted, respectively.
  • q 1 , q 2 , q 3 , q 4 , q 5 , q 6 and q 7 are H, respectively. In certain embodiments, at least one of q 1 , q 2 , q 3 , q 4 , q 5 , q 6 and q 7 is other than H. In certain embodiments, at least one of q 1 , q 2 , q 3 , q 4 , q 5 , q 6 and q 7 is methyl. In certain embodiments, a THP nucleoside in which one of R 1 and R 2 is F is provided. In certain embodiments, R 1 is fluoro and R 2 is H; R 1 is methoxy and R 2 is H, and R 1 is methoxyethoxy and R 2 is. It is H.
  • Such sugar substitutes include, but are not limited to, those referred to in the art as hexitol nucleic acid (HNA), altritor nucleic acid (ANA) and mannitol nucleic acid (MNA) (Leumann, C.J. , Bioorg. & Med. Chem., 2002, 10, 841-854).
  • HNA hexitol nucleic acid
  • ANA altritor nucleic acid
  • MNA mannitol nucleic acid
  • the sugar substitute comprises a ring having more than 5 atoms and more than 1 heteroatom.
  • a morpholino sugar moiety has been reported (eg, Braach et al., Biochemistry, 2002, 41, 4503-4510; and US Pat. No. 5,698,685; 5,166. , 315; 5,185,444; and 5,034,506).
  • morpholino means a sugar substitute having the following structure:
  • the morpholino can be modified, for example, by adding or modifying various substituents from the morpholino structure described above.
  • Such sugar substitutes are referred to herein as "modified morpholino".
  • the oligonucleotide comprises one or more modified cyclohexenyl nucleosides, which are nucleosides having a 6-membered cyclohexenyl in place of the pentoflanosyl residue of the naturally occurring nucleoside.
  • Modified cyclohexenyl nucleosides include, but are not limited to, those described in the art (eg, WO2010 / 036696, published April 10, 2010, Robeyns et al., Concerning sharing. J. Am. Chem.
  • Certain modified cyclohexenyl nucleosides have the following formula: During the ceremony Bx is the heterocyclic base portion; T 3 and T 4 are each independently nucleoside interlinking groups that link the cyclohexenyl nucleoside analog to the oligonucleotide compound, or one of T 3 and T 4 ligates the tetrahydropyran nucleoside analog to the oligonucleotide compound.
  • T 3 and T 4 is an H, hydroxyl protecting group, linked conjugate group or 5'-or 3'-terminal group; q 1 , q 2 , q 3 , q 4 , q 5 , q 6 , q 7 , q 8 and q 9 are independently H, C 1 to C 6 alkyl, substituted C 1 to C 6 alkyl, C 2 respectively. ⁇ C 6 alkenyl, substituted C 2 to C 6 alkenyl, C 2 to C 6 alkynyl, substituted C 2 to C 6 alkynyl or other sugar substituents.
  • nucleobase moiety (natural, modified or a combination thereof) is maintained during hybridization with a suitable nucleic acid target.
  • modified sugars are the sugar moieties of ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Oxz], or ALNA [Trz], of which the sugar moiety of ALNA [Ms] is more preferred.
  • modified sugars are novel, and the modified oligonucleotide for ATXN3 containing these modified sugars may hybridize to any region of ATXN3. That is, in certain embodiments, the modified oligonucleotide of the present invention is a modified oligonucleotide consisting of 12 to 24 residues, preferably 16, 17 or 18 bases, having an activity of inhibiting ATXN3 expression, as described above.
  • the nucleic acid base sequence of the modified oligonucleotide has at least 85%, at least 90%, or at least 95% complementarity to the equal length portion of the nucleic acid base sequence of SEQ ID NO: 1 in the sequence listing, and is a nucleoside constituting the oligonucleotide. At least one of them is a modified oligonucleotide having a modified sugar selected from the sugar moiety of ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Oxz], or ALNA [Trz].
  • Modifications or substitutions of nucleobases are structurally distinguishable from naturally occurring or synthetic unmodified nucleobases and are more functionally compatible with such unmodified nucleobases. Both natural and modified nucleobases can be involved in hydrogen bonding. Such nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological properties to the oligonucleotide compound.
  • the modified oligonucleotide of the present invention one in which at least one nucleoside constituting the oligonucleotide contains a modified nucleobase is preferably used.
  • the modified nucleobase include 5-methylcytosine (5-me-C).
  • 5-Methylcytosine means cytosine modified with a methyl group attached to the 5-position. Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of oligonucleotides. For example, 5-methylcytosine substitution has been shown to increase double-stranded stability of nucleic acids by 0.6-1.2 ° C (Sanghvi, YS, CRC, ST and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).
  • nucleic acid bases include 5-hydroxymethylcytosine, xanthin, hypoxanthin, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2 -Thiouracil, 2-thiothymine and 2-thiocitosine, 5-halouracil and cytosine, 5-propynyl (-C ⁇ C-CH 3 ) uracil and citocin and other alkynyl derivatives of pyrimidine base, 6-azouracil, cytosine and chimin, 5 -Uracil (Pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halos, especially 5-bromo, 5 -Trifluoromethyl
  • the heterocyclic base moiety can also include those in which the purine or pyrimidine base is replaced with another heterocycle, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
  • Nucleobases particularly useful for increasing the binding affinity of modified oligonucleotides include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines (2-aminopropyladenine, Includes 5-propynyluracil and 5-propynylcytosine).
  • modified nucleoside bond between RNA and DNA is a 3'-5'phosphodiester bond.
  • modified, i.e., non-naturally occurring, oligonucleotides with nucleoside linkages include, for example, enhanced cell uptake, enhanced affinity for target nucleic acids and increased stability in the presence of nucleases. Often preferred over naturally occurring oligonucleotides with internucleoside linkages because of their properties.
  • Oligonucleotides with modified nucleoside bonds include nucleoside bonds that retain a phosphorus atom and nucleoside bonds that do not have a phosphorus atom.
  • Representative phosphorus-containing nucleoside bonds include, but are not limited to, one or more of phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing bonds are well known.
  • the nucleoside linkage of the modified oligonucleotide of the present invention may be any as long as the modified oligonucleotide has an activity of inhibiting ATXN3 expression, but one in which at least one nucleoside linkage contains a phosphorothioate nucleoside linkage is preferably used.
  • all nucleoside linkages may be phosphorothioate nucleoside linkages.
  • the modified oligonucleotides of the invention obtain increased resistance to degradation by nucleases, increased cell uptake, increased binding affinity for target nucleic acids and / or increased ATXN3 expression inhibitory activity. Can have a gapmer motif.
  • Gapmer means a modified oligonucleotide in which an internal region with multiple nucleosides that assists cleavage by RNase H is located between external regions with one or more nucleosides.
  • the inner region can be referred to as a "gap segment” and the outer region can be referred to as a "wing segment”.
  • a wing segment existing on the 5'side of the gap segment can be called a "5'wing segment”
  • a wing segment existing on the 3'side of the gap segment can be called a "3'wing segment”.
  • the sugar portion of each wing's nucleoside approaching the gap is the sugar portion of the adjacent gap nucleoside.
  • the sugar moiety of the most 3'side nucleoside of the 5'wing and the most 5'side nucleoside of the 3'wing are modified sugars, and the sugar moiety of the adjacent gap nucleoside is the sugar moiety of natural DNA. ..
  • the wing-gap-wing motif can be described as "XYZ", where "X” represents the sequence length of the 5'wing region, "Y” represents the sequence length of the gap region, and "Z”. "Represents the array length of the 3'wing region.
  • the modified oligonucleotide of the invention comprises 1) a gap segment, 2) a 5'wing segment and 3) a 3'wing segment, wherein the gap segment comprises the 5'wing segment and the 3'. It is a modified oligonucleotide positioned between the wing segment and containing the modified sugar in the nucleosides of the 5'wing segment and the 3'wing segment.
  • the sugar moiety of the nucleoside in the gap segment may be only the sugar moiety of natural DNA or may contain one or more modified sugars.
  • the modified sugar is preferably selected from the group consisting of bicyclic sugars, sugars modified with 2'-MOE, and sugars modified with 2'-OMe, and the bicyclic sugars include, for example, At least one sugar moiety of LNA, GuNA, ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Oxz] and ALNA [Trz] can be mentioned.
  • the number of nucleosides contained in the gap segment, 5'wing segment and 3'wing segment may be anything as long as it has ATXN3 expression inhibitory activity, and for example, 12 gap segments, 3 5'wing segments and the like. There are 3 3'wing segments, or 10 gap segments, 3 5'wing segments and 3 3'wing segments, 12 gap segments, 3 5'wing segments and 3 'Three wing segments are more preferred.
  • cap structures are included at the ends of one or both of the modified oligonucleotides to enhance properties such as nuclease stability.
  • Suitable cap structures include 4', 5'-methylene nucleotides, 1- ( ⁇ -D-erythrofuranosyl) nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol.
  • the method for evaluating the compound of the present invention may be any method as long as the compound of the present invention can verify the inhibition of the expression level of ATXN3 in cells, and specifically, for example, in vitro and in vivo shown below.
  • the ATXN3 expression measurement method is used.
  • the in vitro ATXN3 expression measuring method for evaluating the inhibition of ATXN3 expression in cells of the compound of the present invention can be any cell expressing ATXN3 (hereinafter, may be referred to as "ATXN3 expressing cell”).
  • ATXN3 expressing cell can also be used, and examples thereof include SH-SY5Y cells (human neuroblastoma cells, for example, ATCC CRL-2266).
  • the method of contacting the compound of the present invention with ATXN3-expressing cells is also not particularly limited, but a method generally used for introducing nucleic acid into cells can be mentioned. Specifically, for example, a lipofection method, an electroporation method, a Gymnosis method, or the like. In the Gymnosis method, the compound of the present invention can be treated, for example, at a final concentration of 0.3, 1, 3, 10 or 30 ⁇ M.
  • Intracellular mRNA levels of ATXN3 can be assayed by a variety of methods known in the art. Specific examples include Northern blot analysis, competitive polymerase chain reaction (PCR), quantitative real-time PCR, and the like.
  • PCR competitive polymerase chain reaction
  • Intracellular protein levels of ATXN3 can be assayed by a variety of methods known in the art. Specifically, for example, immunoprecipitation, Western blotting (immun blot), enzyme-linked immunosorbent assay (ELISA), quantitative protein assay, protein activity assay (eg, caspase activity assay), immunohistochemistry. , Immunocytochemistry or fluorescence activated cell sorting (FACS) and the like.
  • the in vivo ATXN3 expression measuring method for evaluating the inhibition of ATXN3 expression in cells of the present invention is, for example, to administer the compound of the present invention to an animal expressing ATXN3 and to express the above-mentioned ATXN3 in the cells. There is a method of performing level analysis.
  • the compound of the present invention can be synthesized by the phosphoramidite method using a commercially available amidite for DNA / RNA synthesis (including LNA).
  • the artificial nucleic acids ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Oxz] and ALNA [Trz] synthesize oligomers by the method described in the international application PCT / JP2019 / 0441182 (WO2020 / 100826). (See the reference example below).
  • the compound of the present invention can treat ATXN3-related diseases by inhibiting the expression of ATXN3.
  • the ATXN3-related disease is not particularly limited as long as it is a disease caused by an abnormality of ATXN3, and examples thereof include neurodegenerative diseases.
  • the compound of the present invention can ameliorate one or more symptoms of neurodegenerative diseases, and the symptoms include, for example, ataxia, neuropathy and aggregate formation. Examples of neurodegenerative diseases include spinocerebellar degeneration type 3.
  • RNA toxicity or mitochondrial disorder due to its translation product polyglutamine, transcription abnormality, calcium constancy abnormality, autophagy abnormality, axonal transport abnormality, etc. Is caused, and cerebellar Purkinje cells are dysfunctional and shed, followed by motor dysfunction. It is known that motor dysfunction is suppressed by administration of antisense oligonucleotide of ATXN3, which causes inhibition of expression of ATXN3 mRNA, to spinocerebellar degeneration type 3 model animals expressing CAG repeat elongation of ATXN3. Therefore, the modified oligonucleotide of the present invention that strongly inhibits ATXN3 expression can treat, prevent, or delay the progression of spinocerebellar degeneration type 3.
  • the present invention comprises modified oligonucleotides for use in the treatment, prevention or delay of progression of ATXN3-related diseases; pharmaceutical compositions for use in the treatment, prevention or delay of progression of ATXN3-related diseases; Use of modified oligonucleotides for treatment, prevention or delay of progression; Use of modified oligonucleotides in the manufacture of drugs for the treatment, prevention or delay of progression of ATXN3-related diseases; for treatment, prevention or delay of progression of ATXN3-related diseases Provided are modified oligonucleotides for use in the manufacture of a pharmaceutical; a method for treating, preventing or delaying progression of an ATXN3-related disease, comprising administering an effective amount of the modified oligonucleotide to a subject in need thereof.
  • composition containing a modified oligonucleotide The compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier thereof can be used as a pharmaceutical composition.
  • modified oligonucleotides can be mixed with one or more pharmaceutically acceptable active or inert substances.
  • the composition and method for formulating the pharmaceutical composition can be selected according to several criteria including the route of administration, the degree of disease or the dose to be administered. For example, as a composition for parenteral administration, for example, an injection is used.
  • Such injections are prepared according to methods known per se, for example, by dissolving, suspending or emulsifying the modified oligonucleotide in a sterile aqueous or oily solution usually used for injections.
  • Aqueous solutions for injection include, for example, phosphate buffered saline, saline, isotonic solutions containing glucose and other adjuvants, and suitable solubilizing agents such as alcohol (eg, ethanol). , Polyalcohol (eg, propylene glycol, polyethylene glycol), nonionic surfactant [eg, Polysolvate 80, HCO-50 (polyoxyethylene (50mo1) conducted of hydrogenated castor oil)] and the like.
  • a buffering agent, a pH adjusting agent, an tonicity agent, a soothing agent, a preservative, a stabilizer and the like can be included.
  • Such compositions are produced by known methods.
  • Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, intracranial administration, intrathecal administration, and intracerebroventricular administration. Administration may be continuous or long-term, short-term or intermittent.
  • compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups. Agents, emulsions, suspending agents and the like.
  • Such compositions are produced by known methods and contain carriers, diluents or excipients commonly used in the pharmaceutical field.
  • carriers, diluents or excipients commonly used in the pharmaceutical field.
  • the carrier and excipient for tablets for example, lactose, starch, sucrose, magnesium stearate and the like are used, and as the diluent, for example, physiological saline is used.
  • the pharmaceutical composition of the present invention can contain a reagent for introducing nucleic acid.
  • nucleic acid introduction reagent include liposomes, lipofectin, lipofectamine, DOGS (transferase), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, and cationic lipids such as poly (ethyleneimine) (PEI).
  • PES poly (ethyleneimine)
  • the modified oligonucleotide contained in the pharmaceutical composition of the present invention is biotin, a protein having affinity for in vivo molecules such as fatty acids, cholesterol, sugars, phospholipids, and antibodies at one or more locations.
  • Phenazine vitamins, peptides, folates, phenanthridines, anthraquinones, acridines, fluorescein, rhodamine, coumarins and dyes conjugated with conjugate groups are preferably used.
  • the conjugated modified oligonucleotide is produced by a known method, and one that enhances its activity, tissue distribution, cell distribution or cell uptake can be selected.
  • the conjugate group is directly attached to the modified oligonucleotide, or the conjugate group is amino, hydroxyl, carboxylic acid, thiol, unsaturated moiety (eg, double or triple bond), 8-amino-3. , 6-Dioxaoctanoic acid (ADO), succinimidyl 4- (N-maleimidemethyl) cyclohexane-1-carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA), azide, substituted C1-C10 alkyl, substituted Alternatively, it is bound to the modified oligonucleotide by a linking moiety selected from the unsubstituted C2-C10 alkenyl and the substituted or unsubstituted C2-C10 alkynyl.
  • ADO 6-Dioxaoctanoic acid
  • SCC succinimidyl 4- (N-maleimidemethyl) cyclohexane-1-carboxy
  • the substituent is selected from amino, alkoxy, carboxy, azide, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
  • the administration form of the pharmaceutical composition of the present invention may be systemic administration such as oral administration, intravenous administration or intraarterial administration, or local administration such as intracranial administration, intrathecal administration or intracerebral administration. There may be, but it is not limited to these.
  • the dose of the pharmaceutical composition of the present invention may be appropriately changed depending on the purpose of use, the severity of the disease, the age, body weight, sex, etc. of the patient, but usually, the amount of the modified oligonucleotide is 0.1 ng to 100 mg / kg / kg /. It can be selected from the range of 1 ng to 10 mg / kg / day, preferably 1 ng to 10 mg / kg / day.
  • Example 1 Synthesis and purification of modified oligonucleotide compounds for in vitro evaluation Using ALNA [Ms] amidite (synthesized by the method described in international application PCT / JP2019 / 0441826 (WO2020 / 100826)), the modified oligonucleotide compound was subjected to DNA / RNA oligonucleotide automatic synthesizer nS-8II (stock). Synthesized using CPG or a polystyrene carrier on a 0.5 ⁇ mol scale by GeneDesign, Inc.).
  • the synthesized modified oligonucleotide compounds are shown in Table 1 (18-residue modified oligonucleotide) and Table 2 (16-residue modified oligonucleotide).
  • Table 1 18-residue modified oligonucleotide
  • Table 2 16-residue modified oligonucleotide.
  • each nucleotide is represented by three letters.
  • the nucleotide at the 3'end is represented by two letters because there is no nucleoside bond.
  • the target start position indicates the ATXN3 pre-mRNA5'target site of the modified oligonucleotide (the position of SEQ ID NO: 1 in the sequence table corresponding to the 3'end of the modified oligonucleotide), and the target end position is the ATXN3 pre of the modified oligonucleotide.
  • -MRNA3'target site position of SEQ ID NO: 1 in the sequence listing corresponding to the 5'end of the modified oligonucleotide is shown.
  • Example 3 In vitro ATXN3 expression ratio test (Gymnosis method) At the same time as seeding 3 ⁇ 10 3 SH-SY5Y cells per well, the synthesized modified oligonucleotide was added at a final concentration of 3 or 10 ⁇ mol / L and incubated in a CO 2 incubator for 3 days. After extracting RNA from the cells, reverse transcription reaction was performed to obtain cDNA. Using the obtained cDNA, quantitative real-time PCR with primers and probes for ATXN3 or GAPDH was performed. ATXN3 mRNA levels were obtained by calculating the volume ratio to GAPDH.
  • the ATXN3 expression ratio was calculated as a percentage of the ATXN3 mRNA level in cells supplemented with the modified oligonucleotide compared to the ATXN3 mRNA level in cells not supplemented with the modified oligonucleotide.
  • the results of the ATXN3 expression ratio when the modified oligonucleotide of 18 residues were added at a final concentration of 3 ⁇ mol / L are shown in Tables 1 and 3, and the ATXN3 expression was expressed when the modified oligonucleotide of 16 residues was added at a final concentration of 10 ⁇ mol / L.
  • the results of the ratio are shown in Table 2.
  • Example 4 In vitro ATXN3 expression ratio test (Gymnosis method) 650528 which is the representative modified oligonucleotide described in WO2018 / 089805, 1100673 which is the representative modified oligonucleotide described in WO2019 / 217708, or 1287095 which is the representative modified oligonucleotide described in WO2020 / 1725559 was synthesized.
  • the target start positions and target end positions of these modified oligonucleotides are as shown in Table 4.
  • the synthesis was carried out using 2'-MOE (2'-O-methoxyethyl) amidite in the same manner as in Example 1 and Example 2.
  • the synthesized modified oligonucleotide was added at a final concentration of 3 or 10 ⁇ mol / L and incubated in a CO 2 incubator for 3 days. After extracting RNA from the cells, reverse transcription reaction was performed to obtain cDNA. Using the obtained cDNA, quantitative real-time PCR with primers and probes for ATXN3 or GAPDH was performed. ATXN3 mRNA levels were obtained by calculating the volume ratio to GAPDH.
  • the ATXN3 expression ratio was calculated as a percentage of the ATXN3 mRNA level in cells supplemented with the modified oligonucleotide compared to the ATXN3 mRNA level in cells not supplemented with the modified oligonucleotide. The results are shown in Table 4.

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