Compounds, methods and pharmaceutical compositions for modulating ataxin 3 expression

Abstract

A modified oligonucleotide that has an activity of inhibiting the expression of Ataxin 3 and has any of the following nucleic acid base sequences or a nucleic acid base sequence having 17 consecutive bases contained in the nucleic acid base sequences. 1) TCGGGTAAGTAGATTTTC (SEQ ID NO. 239), 2) GAAGTATCTGTAGGCCTA (SEQ ID NO. 240), 3) GGACTGTATAGGAGATTA (SEQ ID NO. 241), 4) GGTTATAGGATGCAGGTA (SEQ ID NO.242), 5) AGGTTATAGGATGCAGGT (SEQ ID NO.243), 6) GAAGCTAAGTAGGTGACT (SEQ ID NO. 244), 7) TGAAGCTAAGTAGGTGAC (SEQ ID NO. 245), 8) CCTAGTCACTTTGATAGA (SEQ ID NO. 246), 9) GGAACATCTTGAGTAGGT (SEQ ID NO. 247), 10) GGTGTTCAGGGTAGATGT (SEQ ID NO. 248), 11) GGATACTCTGCCCTGTTC (SEQ ID NO. 249), 12) GGTGTCAAACGTGTGGTT (SEQ ID NO. 250), 13) CCGTGTGCTAGTATTTGT (SEQ ID NO. 251), 14) TAGTAGAGTTTTGCTTGG (SEQ ID NO.252), 15) GATGTAGTAGAGTTTTGC (SEQ ID NO. 253), 16) TGATGTAGTAGAGTTTTG (SEQ ID NO. 254), 17) CTGATGTAGTAGAGTTTT (SEQ ID NO. 255), 19)GCAAGTTGGTTTGTGGTA (SEQ ID NO. 256), 20) TCTAGGCAATTGTGGTGG (SEQ ID NO. 257), 21) GTAACTCTGCACTTCCCA (SEQ ID NO. 258), 22) GTCATCCCTATGTCTTAT (SEQ ID NO. 259), 23) GTCATATGGTCAGGGTAT (SEQ ID NO. 260), 24) TGTCATATGGTCAGGGTA (SEQ ID NO. 261), 25) ATGTCATATGGTCAGGGT (SEQ ID NO. 262) and 26) TATGTCATATGGTCAGGG (SEQ ID NO. 263).

Description

[Technical Field]

[0001] The present invention relates to compounds for reducing at least one of pre-mRNA, mRNA, and protein levels of Ataxin 3 (ATXN3) in animals, methods of using the compounds, and pharmaceutical compositions containing the compounds. The methods of the present invention are useful for treating, preventing, or slowing the progression of ATXN3-related diseases, such as spinocerebellar ataxia type 3 (SCA3, also known as Machado-Joseph disease, MJD). [Background technology]

[0002] Spinocerebellar degeneration type 3 (SCD) is the most common autosomal dominant type of spinocerebellar degeneration. The disease develops at an average age of 36 years, with early symptoms including nystagmus, increased deep tendon reflexes, and dysarthria. The condition then progressively worsens, resulting in symptoms such as difficulty walking, dysphagia, ophthalmoplegia, and diplopia. Patients die within approximately 20 years from aspiration pneumonia or falls. There is no cure for the condition; only symptomatic treatments for motor dysfunction, extrapyramidal disorders, painful muscle spasms, depression, and fatigue, as well as rehabilitation treatments to slow the progression of the disease, are available.

[0003] The gene responsible for spinocerebellar ataxia type 3 was reported to be linked to 14q24.3-q32 in 1993 (Non-Patent Document 1: Takiyama et al., Nature Genetics, 1993, 4, 300-304). Subsequently, in 1994, a novel gene, ATXN3 (or MJD1), was identified as the responsible gene after CAG repeat expansion (Non-Patent Document 2: Kawaguchi et al., Nature Genetics, 1994, 8, 221-228). The CAG repeats in the normal allele range from 14 to 37, whereas those in the expanded allele range from 61 to 84 (Non-Patent Document 3: Takiyama et al., Neurology, 1997, 49, 604-606). It has been reported that there is a negative correlation between CAG repeat expansion and age of onset (Non-Patent Document 2: Kawaguchi et al., Nature Genetics, 1994, 8, 221-228). CAG repeat expansion is thought to cause motor dysfunction following the decline and loss of cerebellar Purkinje cells, as a result of various pathological hypotheses, including RNA toxicity of the CAG repeat itself, or mitochondrial damage due to its translation product, polyglutamine, transcriptional abnormalities, calcium homeostasis abnormalities, autophagy abnormalities, and axonal transport abnormalities (Non-Patent Document 4 MM Evers et al., Molecular Neurobiology, 2014, 49, 1513-1531).

[0004] Antisense oligonucleotides have been increasingly applied clinically as an effective means of regulating the expression of certain gene products. In recent years, drugs such as Spinraza have been shown to be highly effective in treating central nervous system genetic disorders (Aartsma-Rus, A. Nucleic Acid Ther. 2017, 27, 67). Therefore, antisense oligonucleotides may prove useful in several therapeutic, diagnostic, and research applications for regulating ATXN3 mRNA.

[0005] Previous attempts have been made to develop a radical treatment for spinocerebellar ataxia type 3 using antisense oligonucleotides. DE KIMPE et al. attempted to selectively reduce the expression of abnormal alleles using antisense oligonucleotides targeting the CAG repeat portion of the ATXN3 coding region (Patent Document 1, WO2008 / 018795, and Patent Document 2, WO2009 / 099326). UZCATEGUI et al. also prepared antisense oligonucleotides complementary to a region containing a single nucleotide polymorphism (G987C) common to some patients with spinocerebellar ataxia type 3, and attempted to selectively reduce the expression of abnormal alleles (Patent Document 3, WO2013 / 138353). VAN ROON-MOM et al. reported an antisense oligonucleotide complementary to ATXN3 mRNA that skips exons 10 and 9, which contain CAG repeats (Patent Document 4, WO2015 / 053624). However, these four reports only examined the effect of antisense oligonucleotides on a very limited region of ATXN3 mRNA, and no reports of efficacy in genetically modified animal models (animal models of spinocerebellar degeneration type 3) have been published. In recent years, antisense oligonucleotides have been explored that are non-allele-selective and target the entire ATXN3 mRNA region. For example, non-allele-selective antisense oligonucleotides have been reported (Patent Document 5, WO2018 / 089805; Patent Document 6, WO2019 / 217708; and Patent Document 7, WO2020 / 172559), and their efficacy in genetically modified animal models (animal models of spinocerebellar degeneration type 3) has been confirmed (Non-Patent Document 6, Hayley S. McLoughlin et al., Annals of Neurology, 2018, 84, 64-77). However, the effective dose in this case was 700 μg per mouse, which was not considered to have sufficient therapeutic potential for clinical application. As described above, although there has been vigorous development of antisense oligonucleotides aimed at fundamentally treating spinocerebellar degeneration type 3, these have not been sufficiently effective. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2008 / 018795 [Patent Document 2] WO2009 / 099326 [Patent Document 3] WO2013 / 138353 [Patent Document 4] WO2015 / 053624 [Patent Document 5] WO2018 / 089805 [Patent Document 6] WO2019 / 217708 [Patent Document 7] WO2020 / 172559 [Non-patent literature]

[0007] [Non-Patent Document 1] Takiyama et al., Nature Genetics 1993, 4, 300-304 [Non-patent document 2] Kawaguchi et al., Nature Genetics 1994,8,221-228. [Non-patent document 3] Takiyama et al., Neurology 1997, 49, 604-606 [Non-patent document 4] MM Evers et al., Molecular Neurobiology, 2014, 49, 1513-1531 [Non-patent document 5] Aartsma-Rus, A. Nucleic Acid Ther. 2017, 27, 67 [Non-patent document 6] Hayley S. McLoughlin et al., Annals of Neurology,2018,84,64-77 Summary of the Invention [Problem to be solved by the invention]

[0008] It is an object of the present invention to provide compounds, methods, and pharmaceutical compositions for inhibiting ATXN3 expression and / or for treating, preventing, delaying, or ameliorating ATXN3-associated diseases. [Means for solving the problem]

[0009] The present inventors have conducted extensive research and discovered modified oligonucleotides that potently inhibit ATXN3 expression, thereby completing the present invention.

[0010] The gist of the present invention is as follows. [1] A modified oligonucleotide having an activity of inhibiting the expression of Ataxin 3 (ATXN3), wherein the nucleic acid base sequence of the modified oligonucleotide is: 1) TCGGGTAAGTAGATTTTC (complementary sequence of 2159 to 2176 in SEQ ID NO: 1) (SEQ ID NO: 239 in the Sequence Listing) 2) GAAGTATCTGTAGGCCTA (complementary sequence of 2513 to 2530 in SEQ ID NO: 1) (SEQ ID NO: 240 in the Sequence Listing) 3) GGACTGTATAGGAGATTA (complementary sequence of 2646 to 2663 in SEQ ID NO: 1) (SEQ ID NO: 241 in the Sequence Listing) 4) GGTTATAGGATGCAGGTA (complementary sequence of 5844 to 5861 in SEQ ID NO: 1) (SEQ ID NO: 242 in the Sequence Listing) 5) AGGTTATAGGATGCAGGT (complementary sequence of 5845 to 5862 in SEQ ID NO: 1) (SEQ ID NO: 243 in the Sequence Listing) 6) GAAGCTAAGTAGGTGACT (complementary sequence of 15115 to 15132 in SEQ ID NO: 1) (SEQ ID NO: 244 in the Sequence Listing) 7) TGAAGCTAAGTAGGTGAC (complementary sequence of 15116 to 15133 in SEQ ID NO: 1) (SEQ ID NO: 245 in the Sequence Listing) 8) CCTAGTCACTTTGATAGA (complementary sequence of 19163 to 19180 in SEQ ID NO: 1) (SEQ ID NO: 246 in the Sequence Listing) 9) GGAACATCTTGAGTAGGT (complementary sequence of 19737 to 19754 in SEQ ID NO: 1) (SEQ ID NO: 247 in the Sequence Listing) 10) GGTGTTCAGGGTAGATGT (complementary sequence of 20835 to 20852 in SEQ ID NO: 1) (SEQ ID NO: 248 in the Sequence Listing) 11) GGATACTCTGCCCTGTTC (complementary sequence of 21482 to 21499 of SEQ ID NO: 1) (SEQ ID NO: 249 in the Sequence Listing) 12) GGTGTCAAACGTGTGGTT (complementary sequence of 22200 to 22217 in SEQ ID NO: 1) (SEQ ID NO: 250 in the Sequence Listing) 13) CCGTGTGCTAGTATTTGT (complementary sequence of 27389 to 27406 in SEQ ID NO: 1) (SEQ ID NO: 251 in the Sequence Listing) 14) TAGTAGAGTTTTGCTTGG (complementary sequence of 31570 to 31587 in SEQ ID NO: 1) (SEQ ID NO: 252 in the Sequence Listing) 15) GATGTAGTAGAGTTTTGC (complementary sequence of 31574 to 31591 in SEQ ID NO: 1) (SEQ ID NO: 253 in the Sequence Listing) 16) TGATGTAGTAGAGTTTTG (complementary sequence of 31575 to 31592 in SEQ ID NO: 1) (SEQ ID NO: 254 in the Sequence Listing) 17) CTGATGTAGTAGAGTTTT (complementary sequence of 31576 to 31593 in SEQ ID NO: 1) (SEQ ID NO: 255 in the Sequence Listing) 19) GCAAGTTGGTTTGTGGTA (complementary sequence of 32008 to 32025 in SEQ ID NO: 1) (SEQ ID NO: 256 in the Sequence Listing) 20) TCTAGGCAATTGTGGTGG (complementary sequence of 32127 to 32144 in SEQ ID NO: 1) (SEQ ID NO: 257 in the Sequence Listing) 21) GTAACTCTGCACTTCCCA (complementary sequence of 36411 to 36428 in SEQ ID NO: 1) (SEQ ID NO: 258 in the Sequence Listing) 22) GTCATCCCTATGTCTTAT (complementary sequence of 36607 to 36624 in SEQ ID NO: 1) (SEQ ID NO: 259 in the Sequence Listing) 23) GTCATATGGTCAGGGTAT (complementary sequence of 40453 to 40470 in SEQ ID NO: 1) (SEQ ID NO: 260 in the Sequence Listing) 24) TGTCATATGGTCAGGGTA (complementary sequence of 40454 to 40471 in SEQ ID NO: 1) (SEQ ID NO: 261 in the Sequence Listing) 25) ATGTCATATGGTCAGGGT (complementary sequence of 40455 to 40472 in SEQ ID NO: 1) (SEQ ID NO: 262 in the Sequence Listing) and 26) TATGTCATATGGTCAGGG (complementary sequence of 40456 to 40473 in SEQ ID NO: 1) (SEQ ID NO: 263 in the Sequence Listing) (In this specification, base sequences are written in 5' to 3' order.) or a nucleic acid base sequence of 17 consecutive bases contained in said nucleic acid base sequence selected from the group consisting of: [2] The modified oligonucleotide of [1], which is single-stranded. [3] The modified oligonucleotide according to [1] or [2], wherein at least one nucleoside constituting the modified oligonucleotide contains a modified sugar. [4] The modified oligonucleotide of [3], wherein the modified sugar is selected from the group consisting of bicyclic sugars, 2'-MOE (2'-O-methoxyethyl)-modified sugars, and 2'-OMe-modified sugars. [5] The modified oligonucleotide of [4], wherein the bicyclic sugar is selected from the sugar moieties of LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], or ALNA[Trz]. [6] The modified oligonucleotide according to any one of [1] to [5], wherein at least one nucleoside constituting the modified oligonucleotide contains a modified nucleic acid base. [7] The modified oligonucleotide of [6], wherein the modified nucleobase is 5-methylcytosine. [8] The modified oligonucleotide according to any one of [1] to [7], wherein at least one internucleoside bond constituting the modified oligonucleotide is a modified internucleoside bond. [9] The modified oligonucleotide of [8], wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

[10] The modified oligonucleotide, 1) gap segment, 2) 5' wing segment and 3) a 3' wing segment, the gap segment is positioned between the 5' wing segment and the 3' wing segment; The modified oligonucleotide according to any one of [1] to [9], wherein the nucleosides constituting the 5' wing segment and the 3' wing segment contain modified sugars.

[11] A modified oligonucleotide consisting of 12 to 24 residues, having the activity of inhibiting ATXN3 expression, wherein the nucleic acid base sequence of the modified oligonucleotide is at least 85% complementary to an equal-length portion of the nucleic acid base sequence of SEQ ID NO: 1 in the Sequence Listing, and at least one of the nucleosides constituting the oligonucleotide has a modified sugar selected from the sugar moieties of ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], or ALNA[Trz].

[12] A pharmaceutical composition comprising the modified oligonucleotide according to any one of [1] to

[11] or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[13] The pharmaceutical composition of

[12] for treating, preventing, or delaying the progression of an ATXN3-associated disease.

[14] The pharmaceutical composition according to

[13] , wherein the ATXN3-associated disease is a neurodegenerative disease.

[15] The pharmaceutical composition according to

[14] , wherein the neurodegenerative disease is spinocerebellar ataxia type 3.

[16] A method for treating, preventing, or delaying the progression of an ATXN3-related disease in a subject, comprising administering an effective amount of any of the modified oligonucleotides [1] to

[13] to a subject in need thereof.

[17] Use of any of the modified oligonucleotides [1] to

[11] in the manufacture of a medicament for treating, preventing, or delaying the progression of an ATXN3-associated disease.

[18] A modified oligonucleotide according to any one of [1] to

[11] for treating, preventing, or delaying the progression of an ATXN3-associated disease. [Effects of the Invention]

[0011] The present invention provides a pharmaceutical agent that can improve the symptoms of ATXN3-related diseases, such as spinocerebellar ataxia type 3, and is effective in preventing or treating such diseases. The modified oligonucleotide of the present invention has excellent activity in inhibiting ATXN3 expression and few side effects such as toxicity, making it an excellent active ingredient for a drug for preventing or treating ATXN3-related diseases. DETAILED DESCRIPTION OF THE INVENTION

[0012] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. Herein, the use of the singular includes the plural unless expressly stated otherwise. Herein, the use of the term "including" as well as other forms, such as "includes" and "included," is not limiting. Furthermore, unless expressly stated otherwise, terms such as "element" encompass elements containing one unit and elements containing more than one subunit.

[0013] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are expressly incorporated herein by reference in their entirety and with respect to the portions of the documents discussed herein.

[0014] (definition) Unless specific definitions are provided, the nomenclatures utilized in connection with, and the procedures and techniques of, analytical chemistry, organic synthetic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for the chemical syntheses and chemical analyses used herein. Where permitted, all patents, applications, published applications, and other publications, GenBank accession numbers and associated sequence information and other data available through databases such as the National Center for Biotechnology Information (NCBI), referred to throughout this disclosure are incorporated by reference in their entirety and with respect to portions of the documents discussed herein. This specification is also filed together with a Sequence Listing in electronic format, the information in the Sequence Listing set forth in said electronic format being incorporated herein by reference in its entirety.

[0015] Unless otherwise indicated, the following terms have the following meanings:

[0016] "Nucleobase" means a heterocyclic moiety capable of pairing with a base of another nucleic acid.

[0017] "Nucleobase sequence" means the order of consecutive nucleobases that make up an oligonucleotide of the invention.

[0018] "Nucleoside" refers to a molecule comprising a linked sugar and a linked nucleobase. In certain embodiments, the nucleoside is linked to a phosphate group.

[0019] "Nucleotide" refers to a molecule in which a phosphate group is attached to the sugar moiety of a nucleoside. Naturally occurring nucleotides have ribose or deoxyribose sugar moieties, which are covalently linked via the phosphate group by a phosphodiester bond.

[0020] "Oligomeric compound" or "oligomer" means a polymer of linked monomeric subunits that is capable of hybridizing to at least a region of a nucleic acid molecule.

[0021] "Oligonucleotide" means a polymer of nucleosides in which each nucleoside and each internucleoside linkage is linked independently to each other.

[0022] "Internucleoside linkage" refers to the chemical bond between nucleosides.

[0023] "Naturally occurring internucleoside linkage" means a 3'-5' phosphodiester linkage.

[0024] A "modified internucleoside linkage" refers to a substitution or any change from a naturally occurring internucleoside linkage (i.e., a phosphodiester internucleoside linkage), such as, but not limited to, a phosphorothioate internucleoside linkage.

[0025] "Phosphorothioate internucleoside linkage" means a linkage between nucleosides in which the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is one example of such a modified internucleoside linkage.

[0026] "Modified base" refers to any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. For example, but not limited to, 5-methylcytosine. "Unmodified nucleobase" refers to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0027] "Modified oligonucleotide" means an oligonucleotide comprising at least one modified nucleoside and / or modified internucleoside linkage.

[0028] "Salt" is a general term for a compound in which one or more dissociable hydrogen ions contained in an acid are replaced with a cation such as a metal ion or ammonium ion. Examples of salts of modified oligonucleotides include, but are not limited to, salts (e.g., sodium salts, magnesium salts) formed with inorganic ions (e.g., sodium ions, magnesium ions) on phosphorothioates or phosphodiesters, or on functional groups (e.g., amino groups) within modified nucleic acid bases.

[0029] "Sugar" or "sugar moiety" means a naturally occurring sugar moiety or a modified sugar moiety.

[0030] "Modified sugars" refer to substitutions or variations from natural sugars. Modified sugars include, for example, substituted sugar moieties and bicyclic sugars.

[0031] "Substituted sugar moiety" means a furanosyl other than the natural sugars of RNA or DNA.

[0032] "Bicyclic sugar" means a furanosyl ring modified by bridging 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 contains a "bicyclic sugar."

[0033] "Single-stranded oligonucleotide" means an oligonucleotide that is not hybridized to a complementary strand.

[0034] "ATXN3" refers to a nucleic acid or protein also known as Ataxin 3. ATXN3 may include, for example, various splicing variants transcribed from the ATXN3 gene, single nucleotide polymorphisms (SNPs), or CAG repeat expansions.

[0035] "Complementary" refers to the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid. In certain embodiments, adenine is complementary to thymidine or uracil. In certain embodiments, cytosine is complementary to guanine. In certain embodiments, 5-methylcytosine is complementary to guanine.

[0036] "Fully complementary (also referred to as complementarity)" or "100% complementary (also referred to as complementarity)" means that every nucleobase of a nucleobase sequence of a first nucleic acid has a complementary nucleobase in a second nucleobase sequence of a second nucleic acid. In certain embodiments, the first nucleic acid is a modified oligonucleotide and the target nucleic acid is the second nucleic acid.

[0037] A "mismatch" or "non-complementary nucleobase" refers to the case where a nucleobase of a first nucleic acid fails to pair with the corresponding nucleobase of a second or target nucleic acid.

[0038] "Target nucleic acid," "target RNA," and "target RNA transcript" all refer to a nucleic acid that can be targeted by a modified oligonucleotide. In certain embodiments, the target nucleic acid comprises a region of ATXN3 mRNA or ATXN3 pre-mRNA.

[0039] "Motif" refers to a combination of chemically heterogeneous regions in a modified oligonucleotide.

[0040] "Immediately adjacent" means that there are no intervening elements between the immediately adjacent elements.

[0041] "Pharmaceutically acceptable salts" means physiologically and pharmaceutically acceptable salts of modified oligonucleotides of the present invention, i.e., salts that retain the desired biological activity of the modified oligonucleotide and do not impart undesired toxicological effects thereto.

[0042] "Administering" means giving a drug to an animal, and includes, but is not limited to, administration by a medical professional or family member and self-administration.

[0043] "Amelioration" refers to a reduction in at least one indicator, sign, or symptom of the associated disease, disorder, or condition. The severity of the indicator can be determined by subjective or objective measures known to those skilled in the art.

[0044] "Animal" refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

[0045] "Effective amount" refers to an amount of a modified oligonucleotide of the present invention that is sufficient to achieve a desired physiological outcome in an individual in need of the drug. The effective amount may vary from individual to individual, depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated, the formulation of the composition, an evaluation of the individual's medical condition, and other relevant factors.

[0046] "Individual" means a human or non-human animal selected for treatment or therapy.

[0047] "Prevent" means to delay or forestall the onset or occurrence of a disease, disorder, or adverse health condition, or one or more symptoms associated with that disease, disorder, or adverse health condition, for a period of time ranging from minutes to indefinite. Prevent can also mean reducing the risk of developing a disease, disorder, or adverse health condition.

[0048] "Treating" means alleviating, eliminating, or inhibiting the progression of a disease, disorder, or adverse condition, or one or more symptoms associated with the disease, disorder, or adverse condition, or partially eliminating or eradicating one or more causes of the disease, disorder, or adverse condition itself.

[0049] (Specific embodiment) Certain specific embodiments, including but not limited to those set forth below, provide compounds for inhibiting the expression of ATXN3, methods of using the compounds, and pharmaceutical compositions containing the compounds.

[0050] (1) Modified oligonucleotides The modified oligonucleotide of the present invention (hereinafter sometimes referred to as the "compound of the present invention" or the "modified oligonucleotide of the present invention") is an antisense oligonucleotide of ATXN3 that has the activity of inhibiting ATXN3 expression. In the present invention, inhibiting ATXN3 expression (level) means suppressing at least one of the level of pre-mRNA transcribed from the ATXN3 gene, the level of mRNA spliced ​​from the pre-mRNA, and the level of ATXN3 protein translated from the mRNA. The ATXN3 pre-mRNA may contain a SNP and / or a CAG repeat expansion found in patients with spinocerebellar ataxia type 3, and examples thereof include the sequence shown in NC_000014.9:c92106621-92058552 Homo sapiens chromosome 14, GRCh38.p13 Primary Assembly (SEQ ID NO: 1 in the Sequence Listing). The ATXN3 mRNA may contain at least one of a variant, a SNP, and a CAG repeat expansion found in patients with spinocerebellar ataxia type 3, and examples thereof include the sequence shown in GenBank accession number NM_004993.5 (SEQ ID NO: 2 in the Sequence Listing). Note that while SEQ ID NOs: 1 and 2 show DNA base sequences, in the case of RNA sequences, T should be read as U.

[0051] The degree of inhibition of ATXN3 expression by the compound of the present invention may be any degree so long as at least one of the ATXN3 pre-mRNA level, mRNA level, and protein level is reduced compared to when the compound is not administered, resulting in prevention and / or improvement of symptoms associated with ATXN3-related diseases.Specifically, for example, in the in vitro ATXN3 expression measurement method described below, the compound of the present invention is used such that, after contacting cells with the compound, the ATXN3 expression level is at least 70% or less, preferably 50% or less, more preferably 40% or less, even more preferably 30% or less, and particularly preferably 20% or less, compared to when not contacted or when contacted with a negative control substance.

[0052] 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, which has an activity of inhibiting ATXN3 expression, and which is located at 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 2177, 2502 to 2530, 2646 to 2667, 4011 to 4029, 403 8~4063rd, 4259~4292nd, 5179~5196th, 5762~5777th, 5843~5870th, 7203~7225th, 7535~7553rd, 7602~7617th, 7741~7758th, 7788~7810th, 10273~ 10288th, 10997~11014th, 11642~11672nd, 11734~11759th, 11799~11825th, 12710~12727th, 13136~13156th, 14325~14340th, 15114~15139th, 15 703~15723rd, 18108~18123rd, 18749~18766th, 19061~19078th, 19163~19181st, 19355~19372nd, 19736~19756th, 20833~20854th, 21059~21077 21482~21502, 22109~22126, 22192~22220, 22913~22930, 22998~23013, 23046~23074, 24651~24670, 26470~26485, 27388~2 7412nd, 30559~30582nd, 31570~31595th, 31841~31858th, 32006~32029th, 32127~32144th, 33630~33647th, 35710~35729th, 36411~36441st, 366 07~36624th, 37690~37705, 38556~38576, 38587~38624, 40452~40492, 40539~40557, 41281~41300, 41392~41410, 42805~42820,Any nucleic acid sequence containing at least 8 consecutive nucleic acid bases (hereinafter referred to as "ATXN3 complementary nucleic acid base sequence") complementary to an equal-length portion of either positions 43091 to 43106 or positions 45892 to 45909 may be used. Furthermore, the ATXN3 complementary nucleic acid base sequence is preferably a sequence of 8 to 18 consecutive nucleic acid bases, more preferably a sequence of 16, 17, or 18 consecutive nucleic acid bases.

[0053] Alternatively, the ATXN3 complementary nucleic acid base sequence may be any of the following from the 5' end of the nucleic acid base sequence of SEQ ID NO: 1 in the Sequence Listing: positions 1512 to 1536, 1568 to 1583, 3931 to 3946, 5668 to 5683, 7817 to 7832, 9925 to 9940, 10409 to 10424, 10556 to 10571, 10572 to 10573, 10574 to 10576, 10578 to 10579, 10579 to 10582, 10583 to 10584, 10585 to 10586, 10587 to 10589, 10590 to 1059 584~10599th, 11002~11017th, 11617~11632nd, 12076~12091st, 14074~14089th, 14213~ 14228th, 16211~16226, 16740~16755, 18015~18030, 18033~18048, 20832~20847 20848~20863, 22116~22131, 22194~22225, 27062~27077, 27271~27286, 29 393~29408th, 30535~30550, 30556~30571, 30930~30945, 33237~33252, 34900~3 It may also be a sequence of 16 or more consecutive nucleic acid bases complementary to an equal-length portion of any of positions 4915, 35841 to 35856, 41294 to 41309, 42110 to 42125, 43182 to 43197, 43209 to 43233, 43933 to 43948, or 44050 to 44065.

[0054] Here, the modified oligonucleotide of the present invention may have an additional sequence on the 5'-end and / or 3'-end, in addition to the above-mentioned ATXN3-complementary nucleobase sequence, so long as its total length is 12 to 24 residues, preferably 16 to 18 residues, and more preferably 18 residues, and the full-length nucleobase sequence is at least 85% complementary to the isolength portion of the nucleobase sequence of SEQ ID NO: 1 in the Sequence Listing. Furthermore, the additional sequence may be any, as long as the modified oligonucleotide of the present invention has the activity of inhibiting ATXN3 expression.

[0055] The full-length nucleobase sequence of the modified oligonucleotide has at least 85% complementarity to the isolength portion of SEQ ID NO: 1 in the Sequence Listing, with this complementarity preferably being 90%, more preferably 95%, and even more preferably 100%. Here, the isolength portion refers to a portion that is detected as having homology when the nucleobase sequence of the modified oligonucleotide of the present invention is aligned with the nucleobase sequence of ATXN3 pre-mRNA or mRNA using software such as BLAST or Genetyx software (GENETYX CORPORATION). In other words, the nucleobase sequence of the oligonucleotide of the present invention is preferably completely complementary to the isolength portion of the nucleobase sequence of ATXN3 pre-mRNA or mRNA, but may contain one or more mismatched nucleobases. The nucleobase sequence of the oligonucleotide of the present invention has a complementarity of 85% or more, 90% or more, and preferably 95% or more.

[0056] The mismatched nucleobases may be consecutive or may be flanked by ATXN3 complementary nucleobase sequences. The percent complementarity of the antisense oligonucleotide of the present invention with ATXN3 pre-mRNA or mRNA can be routinely determined, for example, by using the BLAST program (basic local alignment search tools) and PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) known in the art, or Genetyx software (GENETYX CORPORATION). Percent homology, sequence identity, or complementarity can be determined, for example, by using the GAP program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison, Wis.) with default settings using the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489) in Genetyx software (GENETYX CORPORATION). For example, when 18 of the 20 nucleobases of a modified oligonucleotide are complementary to and hybridize with an equal length portion of ATXN3 pre-mRNA, the modified oligonucleotide has 90% complementarity.

[0057] The activity of the compound of the present invention can be verified by any method that can verify that the compound of the present invention inhibits ATXN3 expression. Specifically, for example, the in vitro ATXN3 expression measurement method described below can be used.

[0058] The modified oligonucleotides of the present invention selected in this manner include those at positions 256 to 273, 404 to 421, 405 to 422, 407 to 424, 408 to 425, 409 to 426, 410 to 427, 541 to 558, 647 to 664, 648 to 665, 697 to 714, 698 to 715, 699 to 716, 767 to 784, 1512 to 1527, 1521 to 1536, 1568 to 1583, 2043 to 2060, 2045 to 2062, 2046 to 2063, 2047 to 2048, 2048 to 2049, 2050 to 2051, 2052 to 2053, 2054 to 2055, 2056 to 2057, 2058 to 2059, 2060 to 2061, 2061 to 2062, 2062 to 2063, 2063 to 2064, 2064 to 2065, 2065 to 2066, 2066 to 2067, 2067 to 2068, 2068 to 2070, 2071 to 2072, 2072 to 2073, 2073 to 2074, 2074 to 2075, 2075 to 2076, 2076 to 2077, 2077 to 2078, 2078 to 2064th place, 2048~2065th place, 2051~2066th place, 2092~2109th place, 2159~2176th place, 2162~2177th place, 2 502~2519th, 2513~2530th, 2646~2663rd, 2650~2667th, 3931~3946th, 4011~4028 4012~4029, 4038~4053, 4041~4058, 4046~4063, 4259~4274, 4267~ 4284th, 4268~4285, 4269~4286, 4277~4292, 5179~5196, 5668~5683, 57 62~5777th, 5843~5860, 5844~5861, 5845~5862, 5853~5870, 7203~7220 7208~7225, 7535~7552, 7536~7553, 7602~7617, 7741~7758, 7788~ 7805th, 7792~7809, 7793~7810, 7817~7832, 9925~9940, 10273~10288 , 10409~10424th, 10556~10571st, 10584~10599th, 10997~11014th, 11002~1101 7th place, 11617~11632nd place, 11642~11659th place, 11643~11660th place, 11644~11661st place, 11645~1 1662nd, 11646~11663rd, 11648~11665th, 11649~11666th, 11650~11665th, 11650 ~11667th, 11651~11668, 11654~11669, 11655~11672, 11734~11751, 11 735~11752nd, 11738~11753rd, 11741~11758th, 11744~11759th, 11799~11816th,Bits 11800-11817, 11810-11825, 12076-12091, 12710-12727, 13136-13153, 13141-13156, 14074-14089, 14213-14228, 14325-14340, 15114-15 129 bits, 15115~15132 bits, 15116~15133 bits, 15124~15139 bits, 15703~15720 bits, 15704~15721 bits, 15705~15722 bits, 15706~15723 bits, 16211~16226 bits, 16740~16755 bits, 180 15~18030, 18033~18048, 18108~18123, 18749~18766, 19061~19078, 19163~19180, 19166~19181, 19355~19372, 19736~19753, 19737~19754 Bits 19738-19755, 19739-19756, 20832-20847, 20833-20850, 20835-20852, 20836-20853, 20837-20854, 20838-20855, 20839-20854, 20848~ 20863, 21059~21076, 21060~21077, 21482~21499, 21483~21500, 21487~21502, 22109~22126, 22116~22131, 22192~22209, 22193~22210, 2 Bits 2194~22209, 22194~22211, 22195~22212, 22196~22213, 22197~22214, 22197~22214, 22198~22215, 22198~22215, 22199~22214, 22199~222 16 bits, 22200~22217 bits, 22201~22218 bits, 22202~22219 bits, 22203~22220 bits, 22203~22218 bits, 22210~22225 bits, 22913~22930 bits, 22998~23013 bits, 23046~23063 bits, 2304 Bits 7-23064, 23048-23065, 23049-23066, 23050-23067, 23055-23072, 23056-23073, 23057-23074, 24651-24668, 24652-24669, 24653-24670Bits 26470-26485, 27062-27077, 27271-27286, 27388-27405, 27389-27406, 27391-27408, 27392-27409, 27393-27410, 27394-27411, 27395-27 412 bits, 29393~29408 bits, 30535~30550 bits, 30556~30571 bits, 30559~30576 bits, 30560~30577 bits, 30561~30578 bits, 30564~30581 bits, 30565~30582 bits, 30930~30945 bits, 315 Bits 70-31587, 31574-31591, 31575-31592, 31576-31591, 31576-31593, 31577-31594, 31578-31595, 31580-31595, 31841-31858, 32006-32021 Bits 32006-32023, 32007-32024, 32008-32025, 32009-32026, 32010-32027, 32011-32028, 32012-32029, 32127-32144, 33237-33252, 33630~ 33647th bit, 34900~34915th bit, 35710~35725th bit, 35711~35728th bit, 35712~35729th bit, 35841~35856th bit, 36411~36428th bit, 36412~36429th bit, 36413~36428th bit, 36413~36430th bit, 3 Bits 6417~36432, 36426~36441, 36607~36624, 36609~36624, 37690~37705, 38556~38571, 38559~38576, 38587~38604, 38588~38605, 38589~386 06th bit, 38590~38607th bit, 38593~38610th bit, 38594~38611th bit, 38596~38613th bit, 38597~38614th bit, 38598~38615th bit, 38599~38616th bit, 38600~38617th bit, 38601~38618th bit, 3860th bit Bits 4-38621, 38606-38623, 38607-38624, 40452-40467, 40453-40470, 40454-40471, 40455-40472, 40456-40473, 40470-40485, 40475-4049240539~40556th, 40540~40557th, 41281~41298th, 41285~41300th, 41294~41309th, 41392~41409th, 41393~41410th, 42110~4212 5th place, 42805~42820th, 43091~43106th, 43182~43197th, 43209~43224th, 43218~43233rd, 43933~43948th, 44050~44065th or 45892~ a nucleic acid base sequence selected from any of positions 2159 to 2176, 5668 to 5683, 5845 to 5862, 11650 to 11665, 12076 to 12091, 14213 to 14228, 15115 to 15132, 16740 to 16755, 18033 to 18048, 22199 to 22214, 22203 to 22218, 27062 to 27077, 31575 to 31592, 31576 to 31593, a nucleic acid base sequence selected from positions 591, 31576 to 31593, 32006 to 32021, 35841 to 35856, 36607 to 36624, 36609 to 36624, 37690 to 37705, 40453 to 40470, 40454 to 40471, 40455 to 40472, 40456 to 40473, 40470 to 40485, or 41294 to 41309, more preferably positions 12076 to 12091, Examples of such modified oligonucleotides include those consisting of a sequence complementary to a nucleic acid base sequence selected from positions 22199 to 22214, 22203 to 22218, 27062 to 27077, 31576 to 31591, 32006 to 32021, 35841 to 35856, 36609 to 36624, 40453 to 40470, and 41294 to 41309, or those consisting of a sequence complementary to a nucleic acid base sequence of 16 or 17 consecutive bases contained in such a nucleic acid base sequence. Here, as long as the full-length nucleic acid base sequence of the modified oligonucleotide is 100% complementary to the isolength portion of the nucleic acid base sequence of SEQ ID NO: 1 in the Sequence Listing, it may have an additional sequence of 1 or 2 residues on the 5'-end and / or 3'-end thereof.

[0059] Among the modified oligonucleotides selected in this manner, when added to SH-SY5Y cells by the gymnosis method to a final concentration of 3 μM, modified oligonucleotides that inhibit ATXN3 mRNA levels to 70% or less compared to when not contacted with the modified oligonucleotide include, for example, nucleic acid base sequences of modified oligonucleotides that are located at positions 698 to 715, 699 to 716, 2043 to 2060, 2046 to 2063, 2047 to 2064, 2092 to 2109, 2159 to 2176, 2513 to 2530, 2646 to 2663, 2650 to 2667, 4041 to 4058, 5843 to 5860, 5844 to 5861, 5845 to 5862, 5846 to 5863, 5847 to 5864, 5848 to 5849, 5849 to 5850, 5851 to 5852, 5853 to 5854, 5855 to 5856, 5857 to 5858, 5859 to 5860, 5859 to 5861, 5859 to 5862, 5859 to 5863, 5859 to 5864, 5859 to 5865, 5859 to 5866, 5859 to 5867, 5859 to 5868, 5859 to 5869, 5860 to 5861, 5861, 5861, 5862, 5862, 5863, 586 5~5862nd place, 7792~7809th place, 11642~11659th place, 11643~11660th place, 11644~11661st place, 11645~11662nd, 11649~11666th, 11650~11667th, 11655~11672nd, 11734~ 11751st, 11735~11752nd, 12710~12727th, 13136~13153rd, 15115~15132nd , 15116~15133rd, 15704~15721st, 15706~15723rd, 19163~19180th, 19737 ~19754th, 20835~20852nd, 20836~20853rd, 20837~20854th, 21482~21499th, 21483~21500th, 22192~22209th, 22194~22211th, 22199~22216th, 222 00~22217th place, 22201~22218th place, 22202~22219th place, 23056~23073rd place, 27388~274 05th place, 27389~27406th place, 27392~27409th place, 27393~27410th place, 27394~27411th place, 30 559~30576th, 30560~30577th, 30564~30581st, 31570~31587th, 31574~31 591st, 31575~31592, 31576~31593, 31577~31594, 31578~31595, 3 1841~31858th, 32006~32023rd, 32007~32024th, 32008~32025th, 32009~3 2026th place, 32011~32028th place, 32127~32144th place, 35711~35728th place, 35712~35729th place,36411~36428, 36412~36429, 36607~36624, 38559~38576, 38587~38604, 38588~38605, 38589~38606 38590~38607, 38593~38610, 38594~38611, 38604~38621, 38607~38624, 40453~40470, 40454~404 Examples of the nucleic acid base sequence include those consisting of a sequence complementary to a nucleic acid base sequence selected from positions 71, 40455 to 40472, 40456 to 40473, 40475 to 40492, 41281 to 41298, 41392 to 41409, 41393 to 41410, and 45892 to 45909, or those consisting of a sequence complementary to a nucleic acid base sequence of 16 or 17 consecutive bases contained in the nucleic acid base sequence.

[0060] Specific examples of the nucleic acid base sequences of the modified oligonucleotides of the present invention include: TCGGGTAAGTAGATTTTC (complementary sequence of 2159 to 2176 in SEQ ID NO: 1) (SEQ ID NO: 239 in the Sequence Listing), GAAGTATCTGTAGGCCTA (complementary sequence of 2513 to 2530 in SEQ ID NO: 1) (SEQ ID NO: 240 in the Sequence Listing), GGACTGTATAGGAGATTA (complementary sequence of 2646 to 2663 in SEQ ID NO: 1) (SEQ ID NO: 241 in the Sequence Listing), GGTTATAGGATGCAGGTA (complementary sequence of 5844 to 5861 in SEQ ID NO: 1) (SEQ ID NO: 242 in the Sequence Listing), AGGTTATAGGATGCAGGT (complementary sequence of 5845 to 5862 in SEQ ID NO: 1) (SEQ ID NO: 243 in the Sequence Listing), GAAGCTAAGTAGGTGACT (complementary sequence of 15115 to 15132 in SEQ ID NO: 1) (SEQ ID NO: 244 in the Sequence Listing), TGAAGCTAAGTAGGTGAC (complementary sequence of 15116 to 15133 in SEQ ID NO: 1) (SEQ ID NO: 245 in the Sequence Listing), CCTAGTCACTTTGATAGA (complementary sequence of 19163 to 19180 in SEQ ID NO: 1) (SEQ ID NO: 246 in the Sequence Listing), GGAACATCTTGAGTAGGT (complementary sequence of 19737 to 19754 in SEQ ID NO: 1) (SEQ ID NO: 247 in the Sequence Listing), GGTGTTCAGGGTAGATGT (complementary sequence of 20835 to 20852 in SEQ ID NO: 1) (SEQ ID NO: 248 in the Sequence Listing), GGATACTCTGCCCTGTTC (complementary sequence of 21482 to 21499 in SEQ ID NO: 1) (SEQ ID NO: 249 in the Sequence Listing), GGTGTCAAACGTGTGGTT (complementary sequence of 22200 to 22217 in SEQ ID NO: 1) (SEQ ID NO: 250 in the Sequence Listing), CCGTGTGCTAGTATTTGT (complementary sequence of 27389 to 27406 in SEQ ID NO: 1) (SEQ ID NO: 251 in the Sequence Listing), TAGTAGAGTTTTGCTTGG (complementary sequence of 31570 to 31587 in SEQ ID NO: 1) (SEQ ID NO: 252 in the Sequence Listing), GATGTAGTAGAGTTTTGC (complementary sequence of 31574 to 31591 in SEQ ID NO: 1) (SEQ ID NO: 253 in the Sequence Listing), TGATGTAGTAGAGTTTTG (complementary sequence of 31575 to 31592 in SEQ ID NO: 1) (SEQ ID NO: 254 in the Sequence Listing), CTGATGTAGTAGAGTTTT (complementary sequence of 31576 to 31593 in SEQ ID NO: 1) (SEQ ID NO: 255 in the Sequence Listing), GCAAGTTGGTTTGTGGTA (complementary sequence of 32008 to 32025 in SEQ ID NO: 1) (SEQ ID NO: 256 in the Sequence Listing), TCTAGGCAATTGTGGTGG (complementary sequence of 32127 to 32144 in SEQ ID NO: 1) (SEQ ID NO: 257 in the Sequence Listing), GTAACTCTGCACTTCCCA (complementary sequence of 36411 to 36428 in SEQ ID NO: 1) (SEQ ID NO: 258 in the Sequence Listing), GTCATCCCTATGTCTTAT (complementary sequence of 36607 to 36624 in SEQ ID NO: 1) (SEQ ID NO: 259 in the Sequence Listing), GTCATATGGTCAGGGTAT (complementary sequence of 40453 to 40470 in SEQ ID NO: 1) (SEQ ID NO: 260 in the Sequence Listing), TGTCATATGGTCAGGGTA (complementary sequence of 40454 to 40471 in SEQ ID NO: 1) (SEQ ID NO: 261 in the Sequence Listing), ATGTCATATGGTCAGGGT (complementary sequence of 40455 to 40472 in SEQ ID NO: 1) (SEQ ID NO: 262 in the Sequence Listing), or TATGTCATATGGTCAGGG (complementary sequence of 40456 to 40473 in SEQ ID NO: 1) (SEQ ID NO: 263 in the Sequence Listing) or a nucleic acid base sequence of 17 consecutive bases contained in said nucleic acid base sequence.

[0061] Specific nucleic acid base sequences of the modified oligonucleotide of the present invention are preferably: TCGGGTAAGTAGATTTTC (complementary sequence of 2159 to 2176 in SEQ ID NO: 1) (SEQ ID NO: 239 in the Sequence Listing), AGGTTATAGGATGCAGGT (complementary sequence of 5845 to 5862 in SEQ ID NO: 1) (SEQ ID NO: 243 in the Sequence Listing), GAAGCTAAGTAGGTGACT (complementary sequence of 15115 to 15132 in SEQ ID NO: 1) (SEQ ID NO: 244 in the Sequence Listing), GTCATCCCTATGTCTTAT (complementary sequence of 36607 to 36624 in SEQ ID NO: 1) (SEQ ID NO: 259 in the Sequence Listing), GTCATATGGTCAGGGTAT (complementary sequence of 40453 to 40470 in SEQ ID NO: 1) (SEQ ID NO: 260 in the Sequence Listing), TGTCATATGGTCAGGGTA (complementary sequence of 40454 to 40471 in SEQ ID NO: 1) (SEQ ID NO: 261 in the Sequence Listing), ATGTCATATGGTCAGGGT (complementary sequence of 40455 to 40472 in SEQ ID NO: 1) (SEQ ID NO: 262 in the Sequence Listing), or TATGTCATATGGTCAGGG (complementary sequence of 40456 to 40473 in SEQ ID NO: 1) (SEQ ID NO: 263 in the Sequence Listing) or a nucleic acid base sequence of 17 consecutive bases contained in the nucleic acid base sequence, more preferably GTCATATGGTCAGGGTAT (complementary sequence of 40453 to 40470 in SEQ ID NO: 1) (SEQ ID NO: 260 in the Sequence Listing) or a nucleic acid base sequence of 17 consecutive bases contained in said nucleic acid base sequence.

[0062] Among these sequences, one or more cytosines may be 5-methylcytosine, a modified nucleic acid base described below, and specific examples include the sequences shown in SEQ ID NO: 23, SEQ ID NO: 38, SEQ ID NO: 63, SEQ ID NO: 131, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150 or SEQ ID NO: 151, and preferably the sequence shown in SEQ ID NO: 148.

[0063] Furthermore, examples of the 16-base nucleic acid base sequences of the modified oligonucleotides of the present invention include the sequences shown in 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, and preferably the sequences shown in SEQ ID NO: 265, 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 or SEQ ID NO: 230.

[0064] Among these sequences, one or more cytosines may be 5-methylcytosine, a modified nucleic acid base described below, and specific examples include the sequences shown in SEQ ID NO: 168, SEQ ID NO: 184, SEQ ID NO: 187, SEQ ID NO: 194, SEQ ID NO: 203, SEQ ID NO: 208, SEQ ID NO: 224, or SEQ ID NO: 225, and 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.

[0065] The modified oligonucleotide of the present invention may be a double-stranded modified oligonucleotide, but a single-stranded modified oligonucleotide is preferably used.

[0066] (modified sugar) The modified oligonucleotide of the present invention preferably contains a modified sugar in at least one nucleoside. In the present invention, a modified sugar refers to a sugar moiety that has been modified. Modified oligonucleotides containing one or more such modified sugars have advantageous characteristics such as enhanced nuclease stability and increased binding affinity. Preferably, at least one of the modified sugars is selected from the group consisting of bicyclic sugars, 2'-MOE-modified sugars, and 2'-OMe-modified sugars. Examples of bicyclic sugars include the sugar moieties of LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], and ALNA[Trz], as shown below. The sugar moiety of ALNA[Ms] is preferred.

[0067] Examples of substituted sugar moieties include, but are not limited to, nucleosides containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3 (2'-OMe), 2'-OCH2CH3, 2'-OCH2CH2F, and 2'-O(CH2)2OCH3 (2'-MOE) substituents. Substituents at the 2' position include allyl, amino, azido, thio, O-allyl, O-C1-C2 10 Alkyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ), O-CH2-C(=O)-N(R m )(R n ) and O-CH2-C(=O)-N(R l )-(CH2)2-N(R m )(R n )(In the formula, each R l , R m and R n are independently H or substituted or unsubstituted C1 to C 10 alkyl).

[0068] Examples of nucleosides having a bicyclic sugar include, but are not limited to, nucleosides comprising a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the oligonucleotides provided herein comprise one or more nucleosides having a bicyclic sugar, wherein the bridge comprises one of the following formulas: 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' and 4'-CH(CHOCH3)-O-2' (and analogs thereof, see U.S. Patent No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and analogs thereof, see WO2009 / 023109). 006478); 4'-CH2-N(OCH3)-2' (and analogs thereof, see WO2008 / 150729); 4'-CH2-ON(CH3)-2' (and analogs thereof, see US2004-0171570); 4'-CH2-N(R)-O-2' (wherein R is H, C1-C12 alkyl, or a protecting group) (see U.S. Patent No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (and analogs thereof, see Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogs thereof, see WO2008 / 154401).

[0069] Nucleosides with additional bicyclic sugars have been reported in the published literature (e.g., Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638; Singh et al. al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al.,; U.S. Patents US 7,399,845; 6,770,748; 6,525,191; 6,268,490; U.S. Patents US 2008-0039618; US 2007-0287831; US ​​2004-0171570; US 2009-0012281; WO2010 / 036698; WO (See WO 2009 / 067647; WO 2009 / 067647; WO 2007 / 134181; WO 2005 / 021570; WO 2004 / 106356; WO 94 / 14226; WO 2009 / 006478; WO 2008 / 154401; and WO 2008 / 150729.) Each of the foregoing bicyclic sugar-containing nucleosides can be prepared with one or more optically active sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose.

[0070] The bicyclic sugar nucleoside GuNA has been reported as an artificial nucleoside with a guanidine bridge (see WO2014 / 046212 and WO2017 / 047816). The bicyclic nucleosides ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Trz], and ALNA[Oxz] have been reported as bridged artificial nucleic acid amino LNAs (ALNAs) (see International Application PCT / JP2019 / 044182 (WO2020 / 100826)).

[0071] In certain embodiments, nucleosides having a bicyclic sugar include a bridge between the 4' and 2' carbon atoms of the pentofuranosyl sugar moiety, including but not limited to, -[C(R a )(R b )] n -, -C(R a )=C(R b )-, -C(R a )=N-, -C(=NR a )-, -C(=O)-, -C(=S)-, -N(R a )-, -O-, -Si(R a )2- and -S(=O) x -, wherein X is 0, 1, or 2; n is 1, 2, 3, or 4; and each R a and R b are independently H, a protecting group, hydroxyl, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 alkynyl, aromatic ring group, substituted aromatic ring group, heterocyclic group, substituted heterocyclic group, C5-C7 alicyclic group, substituted C5-C7 alicyclic group, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1) or sulfoxyl (S(=O)-J1); Each of J1 and J2 is independently H, C1 to C 12 Alkyl, substituted C1-C12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, aromatic ring group, substituted aromatic ring group, acyl (C(=O)-H), substituted acyl, heterocyclic group, substituted heterocyclic group, C1-C 12 Aminoalkyl, substituted C1-C 12 It is an aminoalkyl or a protecting group.

[0072] In certain embodiments, the bridge 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 In certain embodiments, the bridge is 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2' (in which case the nucleoside with a bicyclic sugar is also referred to as LNA), 4'-(CH2)2-O-2', 4'-CH2-ON(R)-2', and 4'-CH2-N(R)-O-2'-, where each R is independently H, a protecting group, or a C1-C 12 It is alkyl.

[0073] In certain embodiments, the bridge of the bicyclic sugar moiety is 4'-CH2-O-2'-(LNA) or CH2-N(R)-, where each R is independently -SO2-CH3 (ALNA[Ms]), -CO-NH-CH3 (ALNA[mU]), 1,5-dimethyl-1,2,4-triazol-3-yl (ALNA[Trz]), -CO-NH-CH(CH3)2 (ALNA[ipU]), or 5-methyl-1,2,4-oxadiazol-3-yl (ALNA[Oxz]) (International Application No. PCT / JP2019 / 044182 (WO2020 / 100826)).

[0074] In certain embodiments, nucleosides having bicyclic sugars are further defined by their isomeric configuration. For example, nucleosides containing a 4'-(CH2)-O-2' bridge can exist in either the α-L- or β-D-configuration.

[0075] In certain embodiments, nucleosides having a bicyclic sugar include those having a 4'-2' bridge, including, but not limited to, α-L-4'-(CH)-O-2', β-D-4'-CH-O-2', 4'-(CH)-O-2', 4'-CH-ON(R)-2', 4'-CH-N(R)-O-2', 4'-CH(CH)-O-2', 4'-CH-S-2', 4'-CH-CH(CH)-2', and 4'-(CH)-2', where R is H, a protecting group, C1-C 12 Alkyl or C1-C 12 and urea or guanidine which may be substituted with alkyl.

[0076] In certain embodiments, the nucleoside having a bicyclic sugar has the formula: [ka] During the ceremony, Bx is a heterocyclic base moiety; T a and T b are each independently a hydrogen atom, a protecting group for a hydroxyl group, an optionally substituted phosphate group, a covalent bond to a phosphorus moiety or a support, or the like; Z a is C1 to C6 alkyl, C2 to C6 alkenyl, C2 to C6 alkynyl, substituted C1 to C6 alkyl, substituted C2 to C6 alkenyl, substituted C2 to C6 alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thiol.

[0077] In certain embodiments, each of the substituents is independently selected from halogen, oxo, hydroxyl, OJ, c , N.J. c J d, S.J. c , N3, OC(=X)J c and N.J. e C(=X)NJ c J d (In the formula, each J c , J d and J e are independently H, C1-C6 alkyl or substituted C1-C6 alkyl, and X is O or NJ c and is mono- or polysubstituted with substituents independently selected from:

[0078] In certain embodiments, the nucleoside having a bicyclic sugar has the formula: [ka] During the ceremony, Bx is a heterocyclic base moiety; T a and T b are each independently a hydrogen atom, a protecting group for a hydroxyl group, an optionally substituted phosphate group, a covalent bond to a phosphorus moiety or a support, or the like; Z b is C1 to C6 alkyl, C2 to C6 alkenyl, C2 to C6 alkynyl, substituted C1 to C6 alkyl, substituted C2 to C6 alkenyl, substituted C2 to C6 alkynyl or substituted acyl (C(=O)-).

[0079] In certain embodiments, the nucleoside having a bicyclic sugar has the formula: [ka] During the ceremony, Bx is a heterocyclic base moiety; T a and T b are each independently a hydrogen atom, a protecting group for a hydroxyl group, an optionally substituted phosphate group, a covalent bond to a phosphorus moiety or a support, or the like; R dis C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; each q a , q b , q c and q d are independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, C1-C6 alkoxyl, substituted C1-C6 alkoxyl, acyl, substituted acyl, C1-C6 aminoalkyl or substituted C1-C6 aminoalkyl.

[0080] In certain embodiments, the nucleoside having a bicyclic sugar has the formula: [ka] During the ceremony, Bx is a heterocyclic base moiety; T a and T b are each independently a hydrogen atom, a protecting group for a hydroxyl group, an optionally substituted phosphate group, a covalent bond to a phosphorus moiety or a support, or the like; q a , q b , q e and q f are each independently hydrogen, halogen, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C1-C 12 Alkoxy, substituted C1-C 12 Alkoxy, OJ j , S.J. j , SOJ j , SO2J j , N.J. j J k , N3, CN, C(=O)OJ j, C(=O)NJ j J k , C(=O)J j , OC(=O)NJ j J k , N(H)C(=NH)NJ j J k , N(H)C(=O)-NJ j J k Or N(H)C(=S)NJ j J k and; Or, q e and q f are both =C(q g )(q h ) and; q g and q h are each independently H, halogen, C1-C 12 Alkyl or substituted C1-C 12 It is alkyl.

[0081] The synthesis and preparation of adenine, cytosine, guanine, 5-methyl-cytosine, thymine, and uracil bicyclic nucleosides (also known as LNAs) bearing a 4'-CH2-O-2' bridge have been described, along with their oligomerization and nucleic acid recognition properties (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). The synthesis of nucleosides bearing bicyclic sugars has also been described in WO98 / 39352 and WO99 / 14226.

[0082] Various bicyclic nucleoside analogs with 4'-2' bridging groups, such as 4'-CH2-O-2' (the bicyclic nucleoside is also referred to as LNA) and 4'-CH2-S-2', have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of oligodeoxyribonucleotide duplexes containing bicyclic nucleosides for use as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99 / 14226). Furthermore, the synthesis of 2'-amino-LNA (the bicyclic nucleoside is also referred to as ALNA), a conformationally restricted high-affinity oligonucleotide analog, has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Additionally, 2'-amino- and 2'-methylamino-LNAs have been prepared and the thermal stability of duplexes with complementary RNA and DNA strands has been previously reported.

[0083] In certain embodiments, the nucleoside having a bicyclic sugar has the formula: [ka] During the ceremony, Bx is a heterocyclic base moiety; T a and T b are each independently a hydrogen atom, a protecting group for a hydroxyl group, an optionally substituted phosphate group, a covalent bond to a phosphorus moiety or a support, or the like; each q i , q j , q k and q l are independently H, halogen, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C1-C 12 Alkoxyl, substituted C1-C12 Alkoxyl, OJ j , S.J. j , SOJ j , SO2J j , N.J. j J k , N3, CN, C(=O)OJ j , C(=O)NJ j J k , C(=O)J j , OC(=O)NJ j J k , N(H)C(=NH)NJ j J k , N(H)C(=O)NJ j J k or N(H)C(=S)NJ j J k and; q i and q j or q l and q k are both =C(q g )(q h ) where q g and q h are each independently H, halogen, C1-C 12 Alkyl or substituted C1-C 12 It is alkyl.

[0084] One carbocyclic bicyclic nucleoside with a 4'-(CH2)3-2' bridge and an alkenyl analog bridge 4'-CH=CH-CH2-2' has been described (Frier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides have also been described, along with their oligomerization and biochemical studies (Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

[0085] In certain embodiments, nucleosides having bicyclic sugars include, but are not limited to, compounds such as those shown below.

[0086] [ka] In the formula, Bx is a base moiety, and R is independently a protecting group, C1-C6 alkyl, or C1-C6 alkoxy.

[0087] In certain embodiments (LNA), the nucleoside having a bicyclic sugar has the following general formula: [ka] [In the formula, B is a nucleobase; X and Y each independently represent a hydrogen atom, a protecting group for a hydroxyl group, an optionally substituted phosphate group, a covalent bond to a phosphorus moiety or a support, or the like (see WO98 / 39352). Typical specific examples include nucleosides represented by the following formula: [ka] Examples of nucleotides include those represented by the following formula:

[0088] In certain embodiments (GuNA), the bicyclic nucleoside has the following general formula: [ka] wherein B is a nucleic acid base, and R3, R4, R5, and R6 each independently represent a hydrogen atom or a C 1-6 R7 and R8 are each independently a hydrogen atom, a protecting group for a hydroxyl group, an optionally substituted phosphate group, a covalent bond to a phosphorus moiety or a support, and R9 and R 10 , R 11 each independently represents a hydrogen atom, or C optionally substituted with one or more substituents 1-6 It is a protecting group for an alkyl group or an amino group. (See, for example, International Publication Nos. WO 2014 / 046212 and WO 2017 / 047816).

[0089] In certain embodiments (ALNA[mU]), the nucleoside containing a bicyclic sugar has the following general formula (I): [ka] [In the formula, B is a nucleobase; R1, R2, R3 and R4 each independently represent a hydrogen atom or a C group optionally substituted with one or more substituents. 1-6 is an alkyl group; R5 and R6 are each independently a hydrogen atom, a hydroxyl protecting group, an optionally substituted phosphate group, a phosphorus moiety, or a covalent bond to a support, or the like; m is 1 or 2; X is represented by the following formula (II-1): [ka] is a group represented by the formula: Symbols shown in formula (II-1): [ka] indicates the point of attachment to the 2'-amino group; One of R7 and R8 is a hydrogen atom, and the other is a methyl group which may be substituted with one or more substituents.] (See, for example, International Application No. PCT / JP2019 / 044182 (WO2020 / 100826)). A typical example is a nucleoside in which one of R7 and R8 is a hydrogen atom and the other is an unsubstituted methyl group.

[0090] In certain embodiments (ALNA[ipU]), the nucleoside containing a bicyclic sugar is a nucleoside having the general formula (I) as defined above in ALNA[mU], wherein: X is the following formula (II-1): [ka] is a group represented by the formula: One of R7 and R8 is a hydrogen atom, and the other is an isopropyl group optionally substituted with one or more substituents (see, for example, International Application No. PCT / JP2019 / 044182 (WO2020 / 100826)). A typical example is a nucleoside in which one of R7 and R8 is a hydrogen atom, and the other is an unsubstituted isopropyl group.

[0091] In certain embodiments (ALNA[Trz]), the bicyclic nucleoside is a nucleoside having the general formula (I) above, wherein X is a group represented by the following formula (II-2): [ka] is a group represented by the formula: A is a triazolyl group optionally substituted with one or more substituents (see, for example, International Application No. PCT / JP2019 / 044182 (WO2020 / 100826)). A typical example of ALNA[Trz] is a nucleoside in which A is a triazolyl group optionally having one or more methyl groups, more specifically, a 1,5-dimethyl-1,2,4-triazol-3-yl group.

[0092] In certain embodiments (ALNA[Oxz]), nucleosides having the general formula (I) as defined above in ALNA[mU], wherein: X is the following formula (II-2): [ka] is a group represented by the formula: A is an oxadiazolyl group optionally substituted with one or more substituents (see, for example, International Application No. PCT / JP2019 / 044182 (WO2020 / 100826)). A typical example is a nucleoside or nucleotide in which A is an oxadiazolyl group optionally having one or more methyl groups, more specifically, a 5-methyl-1,2,4-oxadiazol-3-yl group.

[0093] In certain embodiments (ALNA[Ms]), the bicyclic nucleoside is a nucleoside having the general formula (I) above, wherein X is a nucleoside having the following general formula (II-3): [ka] is a group represented by the formula: M is a sulfonyl group substituted with a methyl group, which may be substituted with one or more substituents (see, for example, International Application No. PCT / JP2019 / 044182 (WO2020 / 100826)). A typical example of ALNA[Ms] is a nucleoside in which M is a sulfonyl group substituted with an unsubstituted methyl group.

[0094] In certain embodiments, nucleosides are modified by replacement of the ribosyl ring with a sugar surrogate, including, but not limited to, replacement of the ribosyl ring with a surrogate ring system (sometimes referred to as a DNA analog), such as a morpholino ring, a cyclohexenyl ring, a cyclohexyl ring, or a tetrahydropyranyl ring, such as one of the following formulas: [ka]

[0095] In certain embodiments, a sugar substitute is selected having the following formula: [ka] During the ceremony, Bx is a heterocyclic base moiety; T3 and T4 are each independently an internucleoside linking group linking a tetrahydropyran nucleoside analog to an oligomeric compound, or one of T3 and T4 is an internucleoside linking group linking a tetrahydropyran nucleoside analog to an oligomeric compound or oligonucleotide, and the other of T3 and T4 is H, a hydroxyl protecting group, a linking conjugate group, or a 5'- or 3'-terminal group; q1, q2, q3, q4, q5, q6 and q7 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; One of R1 and R2 is hydrogen, and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2, and CN (wherein X is O, S, or NJ1, and each J1, J2, and J3 is independently H or C1-C6 alkyl).

[0096] In certain embodiments, q1, q2, q3, q4, q5, q6, and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is other than H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is methyl. In certain embodiments, THP nucleosides are provided wherein one of R1 and R2 is F. In certain embodiments, R1 is fluoro and R2 is H; R1 is methoxy and R2 is H, and R1 is methoxyethoxy and R2 is H.

[0097] Such sugar substitutes include, but are not limited to, those referred to in the art as hexitol nucleic acids (HNA), altritol nucleic acids (ANA), and mannitol nucleic acids (MNA) (see Leumann, CJ, Bioorg. & Med. Chem., 2002, 10, 841-854).

[0098] In certain embodiments, sugar surrogates contain rings with more than five atoms and more than one heteroatom. For example, their use in nucleosides and oligomeric compounds containing morpholino sugar moieties has been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Patents 5,698,685; 5,166,315; 5,185,444; and 5,034,506).

[0099] As used herein, the term "morpholino" refers to a sugar surrogate having the following structure: [ka]

[0100] In certain embodiments, morpholinos can be modified, for example, by adding or varying various substituents from the morpholino structures shown above. Such sugar surrogates are referred to herein as "modified morpholinos."

[0101] In certain embodiments, the oligonucleotide comprises one or more modified cyclohexenyl nucleosides, which are nucleosides having a six-membered cyclohexenyl in place of the pentofuranosyl residue of naturally occurring nucleosides.Modified cyclohexenyl nucleosides include, but are not limited to, those described in the art (e.g., co-owned WO2010 / 036696 published April 10, 2010; Robeyns et al., J. Am. Chem. Soc., 2008, 130(6), 1979-1984; Horvath et al., Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30), 9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research,2005,33(8),2452-2463;Robeyns et al.,Acta Crystallographica,Section F:Structural Biology and Crystallization Communications,2005,F61(6),585-586;Gu et al.,Tetrahedron,2004,60(9),2111-2123;Gu et al. al.,Oligonucleotides,2003,13(6),479-489;Wang et al.,J.Org.Chem.,2003,68,4499-4505;Verbeure et al.,Nucleic AcidsResearch,2001,29(24),4941-4947;Wang etal.,J.Org.Chem.,2001,66,8478-82;Wang et al. al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7), 785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; WO06 / 047842; and WO01 / 049687; the texts of each of which are incorporated herein by reference in their entirety.

[0102] Certain modified cyclohexenyl nucleosides have the formula: [ka] During the ceremony, Bx is a heterocyclic base moiety; T3 and T4 are each independently an internucleoside linking group linking a cyclohexenyl nucleoside analog to an oligonucleotide compound, or one of T3 and T4 is an internucleoside linking group linking a tetrahydropyran nucleoside analog to an oligonucleotide compound, and the other of T3 and T4 is H, a hydroxyl protecting group, a linking conjugate group, or a 5'- or 3'-terminal group; q1, q2, q3, q4, q5, q6, q7, q8, and q9 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, or other sugar substituent.

[0103] Many other bicyclic and tricyclic sugar surrogate ring systems are known in the art that can be used to modify nucleosides for incorporation into oligonucleotides (see, for example, review: Leumann, Christian J., Bioorg. & Med. Chem., 2002, 10, 841-854). These ring systems can undergo various further substitutions to enhance activity.

[0104] Methods for preparing modified sugars are well known to those skilled in the art. Some representative US patents that teach the preparation of such modified sugars include, but are not limited to, US: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,639,873; 5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032 and WO2005 / 121371, each of which is incorporated herein by reference in its entirety.

[0105] In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified, or a combination thereof) are maintained upon hybridization with an appropriate nucleic acid target.

[0106] Preferred modified sugars are the sugar moieties of ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], or ALNA[Trz], with the sugar moiety of ALNA[Ms] being more preferred. These modified sugars are novel, and modified oligonucleotides against 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, which has the activity of inhibiting ATXN3 expression, wherein the nucleic acid base sequence of the modified oligonucleotide is at least 85%, at least 90%, or at least 95% complementary to an equal-length portion of the nucleic acid base sequence of SEQ ID NO: 1 in the Sequence Listing, and at least one of the nucleosides constituting the oligonucleotide has a modified sugar selected from the sugar moieties of ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], or ALNA[Trz].

[0107] (modified nucleobases) Nucleobase (or base) modifications or substitutions are structurally distinguishable from naturally occurring or synthetic unmodified nucleobases, yet functionally compatible with such unmodified nucleobases. Both natural and modified nucleobases can participate in hydrogen bonding. Such nucleobase modifications can impart nuclease stability, binding affinity, or some other beneficial biological properties to oligonucleotide compounds. Modified oligonucleotides of the present invention are preferably used in which at least one nucleoside comprises a modified nucleobase. An example of a modified nucleobase is 5-methylcytosine (5-me-C). 5-methylcytosine refers to a 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 nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

[0108] Further modified nucleobases include 5-hydroxymethylcytosine, xanthine, hypoxanthine, 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-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine. Heterocyclic base moieties can also include those in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Particularly useful nucleobases for increasing the binding affinity of modified oligonucleotides include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.

[0109] (modified internucleoside linkage) The naturally occurring internucleoside linkage in RNA and DNA is a 3'-5' phosphodiester linkage. Oligonucleotides having one or more modified, i.e., non-naturally occurring, internucleoside linkages are often preferred over oligonucleotides having naturally occurring internucleoside linkages due to properties such as, for example, enhanced cellular uptake, increased affinity for target nucleic acids, and increased stability in the presence of nucleases.

[0110] Oligonucleotides with modified internucleoside linkages include those that retain a phosphorus atom and those that do not have a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, one or more of phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known.

[0111] The internucleoside linkage of the modified oligonucleotide of the present invention may be any, as long as the modified oligonucleotide has the activity of inhibiting ATXN3 expression. However, it is preferred to use one in which at least one internucleoside linkage contains a phosphorothioate internucleoside linkage, and for example, all internucleoside linkages may be phosphorothioate internucleoside linkages.

[0112] (Modified Oligonucleotide Motif) In certain embodiments, the modified oligonucleotides of the present invention may have a gapmer motif to achieve increased resistance to degradation by nucleases, increased cellular uptake, increased binding affinity for target nucleic acids, and / or increased ATXN3 expression inhibitory activity.

[0113] A "gapmer" refers to a modified oligonucleotide in which an internal region having multiple nucleosides that supports cleavage by RNase H is located between external regions having one or more nucleosides. The internal region can be referred to as a "gap segment," and the external region can be referred to as a "wing segment." The wing segment located 5' from the gap segment can be referred to as a "5' wing segment," and the wing segment located 3' from the gap segment can be referred to as a "3' wing segment." In a gapmer, the sugar moiety of each wing nucleoside adjacent to the gap (the 3'-most nucleoside of the 5' wing and the 5'-most nucleoside of the 3' wing) is different from the sugar moiety of the adjacent gap nucleoside. For example, the sugar moiety of the 3'-most nucleoside of the 5' wing and the 5'-most nucleoside of the 3' wing is a modified sugar, and the sugar moiety of the adjacent gap nucleoside is a naturally occurring DNA sugar moiety.

[0114] The wing-gap-wing motif can be written 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 sequence length of the 3' wing region.

[0115] In certain embodiments, the modified oligonucleotide of the present invention comprises 1) a gap segment, 2) a 5' wing segment, and 3) a 3' wing segment, wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, and the nucleosides of the 5' wing segment and the 3' wing segment comprise modified sugars. The sugar moieties of the nucleosides of the gap segment may be solely natural DNA sugar moieties or may include one or more modified sugars. The modified sugars are preferably selected from the group consisting of bicyclic sugars, 2'-MOE-modified sugars, and 2'-OMe-modified sugars, and examples of bicyclic sugars include at least one sugar moiety of LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], and ALNA[Trz].

[0116] The number of nucleosides contained in the gap segment, 5' wing segment, and 3' wing segment may be any number as long as it has ATXN3 expression inhibitory activity, but examples include 12 gap segments, 3 5' wing segments, and 3 3' wing segments, or 10 gap segments, 3 5' wing segments, and 3 3' wing segments, with 12 gap segments, 3 5' wing segments, and 3 3' wing segments being more preferred.

[0117] In certain embodiments, modified oligonucleotides include cap structures at one or both ends to enhance properties such as nuclease stability. Suitable cap structures include 4',5'-methylene nucleotides, 1-(β-D-erythrofuranosyl) nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, phosphorodithioate linkages, threo-pentofuranosyl nucleotides, acyclic 3',4'-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3'-3'-inverted nucleotide moieties, 3'-3'-inverted abasic moieties, 3'-2'-inverted nucleotide moieties, 3'-2'-inverted abasic moieties, 1,4-butanediol phosphate, 3'-phosphoramido nucleotides ... phosphate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3'-phosphorothioate, phosphorodithioate, bridged and unbridged methyl phosphonate moieties, 5'-amino-alkyl phosphate, 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate, hydroxypropyl phosphate, 5'-5'-inverted nucleotide moieties, 5'-5'-inverted abasic moieties, 5'-phosphoramidate, 5'-phosphorothioate, 5'-amino, bridged and / or unbridged 5'-phosphoramidate, phosphorothioate, and 5'-mercapto moieties.

[0118] (Method for evaluating the compound of the present invention) The method for evaluating the compound of the present invention may be any method that can verify that the compound of the present invention inhibits the expression level of ATXN3 in cells. Specifically, for example, the in vitro and in vivo ATXN3 expression measurement methods shown below may be used.

[0119] In an in vitro ATXN3 expression measurement method for evaluating the inhibition of ATXN3 expression in cells by the compound of the present invention, any cells expressing ATXN3 (hereinafter, sometimes referred to as "ATXN3-expressing cells") can be used, for example, SH-SY5Y cells (human neuroblastoma cells, e.g., ATCC CRL-2266).

[0120] The method for contacting the compound of the present invention with ATXN3-expressing cells is not particularly limited, and includes methods commonly used for introducing nucleic acids into cells. Specific examples include lipofection, electroporation, and Gymnosis. In Gymnosis, the compound of the present invention can be used at a final concentration of, for example, 0.3, 1, 3, 10, or 30 μM.

[0121] The intracellular mRNA level of ATXN3 can be assayed by various methods known in the art, such as Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitative real-time PCR.

[0122] Intracellular ATXN3 protein levels can be assayed by various methods known in the art, including, for example, immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assay, protein activity assay (e.g., caspase activity assay), immunohistochemistry, immunocytochemistry, or fluorescence-activated cell sorting (FACS).

[0123] An in vivo ATXN3 expression measurement method for evaluating the inhibition of ATXN3 expression in cells by the compound of the present invention includes, for example, a method in which the compound of the present invention is administered to an animal expressing ATXN3 and the ATXN3 expression level in the cells is analyzed as described above.

[0124] The compounds of the present invention can be synthesized by the phosphoramidite method using commercially available amidites (including LNA) for DNA and RNA synthesis. The artificial nucleic acids ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], and ALNA[Trz] can be synthesized as oligomers by the method described in International Application No. PCT / JP2019 / 044182 (WO2020 / 100826) (see the Reference Examples below).

[0125] (Treatment of ATXN3-related diseases with the compounds of the present invention) The compound of the present invention can treat ATXN3-associated diseases by inhibiting ATXN3 expression. The ATXN3-associated diseases are not particularly limited as long as they are diseases caused by abnormalities in ATXN3, and examples thereof include neurodegenerative diseases. The compounds of the present invention can also improve one or more symptoms of neurodegenerative diseases, such as ataxia, neuropathy, and aggregate formation, including spinocerebellar ataxia type 3. In spinocerebellar ataxia type 3, CAG repeat expansion in ATXN3 causes mitochondrial damage, transcriptional abnormalities, calcium homeostasis abnormalities, autophagy abnormalities, axonal transport abnormalities, etc. due to RNA toxicity or its translation product, polyglutamine, resulting in decreased function and loss of cerebellar Purkinje cells, followed by motor dysfunction, etc. It is known that motor dysfunction is suppressed by administering an ATXN3 antisense oligonucleotide that inhibits ATXN3 mRNA expression to a spinocerebellar ataxia type 3 model animal in which a CAG repeat expansion in ATXN3 has been expressed. Therefore, the modified oligonucleotide of the present invention, which potently inhibits ATXN3 expression, can treat, prevent, or slow the progression of spinocerebellar ataxia type 3.

[0126] Thus, the present invention provides 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 treating, preventing, or delaying progression of ATXN3-related diseases; use of modified oligonucleotides in the manufacture of medicaments for treating, preventing, or delaying progression of ATXN3-related diseases; modified oligonucleotides for use in the manufacture of medicaments for treating, preventing, or delaying progression of ATXN3-related diseases; and a method for treating, preventing, or delaying progression of ATXN3-related diseases, comprising administering an effective amount of a modified oligonucleotide to a subject in need thereof.

[0127] (2) Pharmaceutical composition containing modified oligonucleotide The compound of the present invention, or its pharmaceutically acceptable salt, and the compound of the present invention, including a pharmaceutically acceptable carrier, can be used as a pharmaceutical composition.To prepare a pharmaceutical composition, the modified oligonucleotide can be mixed with one or more pharmaceutically acceptable active or inactive substances.The composition and method for formulating a pharmaceutical composition can be selected according to several criteria, including the route of administration, the degree of disease, or the dosage to be administered. For example, compositions for parenteral administration include injections. Such injections are prepared according to known methods, for example, by dissolving, suspending, or emulsifying the modified oligonucleotide in a sterile aqueous or oily liquid typically used for injections. Examples of aqueous solutions for injection include phosphate-buffered saline, physiological saline, and isotonic solutions containing glucose and other adjuvants. These solutions may contain appropriate solubilizers, such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene(50mol) adduct of hydrogenated castor oil)). These solutions may also contain buffers, pH adjusters, isotonicity agents, soothing agents, preservatives, stabilizers, and the like. Such compositions are manufactured by known methods.

[0128] Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, intracranial administration, intrathecal administration, and intraventricular administration. Administration may be continuous or long-term, or short-term or intermittent.

[0129] 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), syrups, emulsions, suspensions, etc. Such compositions are produced by known methods and contain carriers, diluents, or excipients commonly used in the pharmaceutical field. Examples of carriers and excipients for tablets include lactose, starch, sucrose, magnesium stearate, etc., and examples of diluents include physiological saline.

[0130] The pharmaceutical composition of the present invention may also contain a nucleic acid transfer reagent, such as liposome, lipofectin, lipofectamine, DOGS (transfectam), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, or a cationic lipid such as poly(ethyleneimine) (PEI).

[0131] Furthermore, the modified oligonucleotide contained in the pharmaceutical composition of the present invention is preferably conjugated at one or more sites with a conjugate group of a protein, biotin, phenazine, vitamin, peptide, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, or dye that has affinity for biological molecules such as fatty acids, cholesterol, carbohydrates, phospholipids, and antibodies. The conjugated modified oligonucleotide is produced by known methods, and can be selected to enhance its activity, tissue distribution, cellular distribution, or cellular uptake.

[0132] The conjugate group is either directly attached to the modified oligonucleotide, or the conjugate group is attached to the modified oligonucleotide through a linking moiety selected from amino, hydroxyl, carboxylic acid, thiol, unsaturated moiety (e.g., double or triple bond), 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA), azide, substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10 alkynyl, where the substituents are selected from amino, alkoxy, carboxy, azido, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0133] The administration route of the pharmaceutical composition of the present invention may be, but is not limited to, systemic administration such as oral administration, intravenous administration, or intra-arterial administration, or local administration such as intracranial administration, intrathecal administration, or intraventricular administration. The dosage of the pharmaceutical composition of the present invention may be varied as appropriate depending on the purpose of use, severity of the disease, and the age, weight, and sex of the patient, but can usually be selected from the range of 0.1 ng to 100 mg / kg / day, preferably 1 ng to 10 mg / kg / day, in terms of the amount of modified oligonucleotide.

[0134] Non-limiting disclosure and incorporation by reference While certain compounds, compositions, and methods described herein are specifically described according to certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to be limiting thereof. Each of the references described in this application is incorporated herein by reference in its entirety.

[0135] Example 1 Synthesis and purification of modified oligonucleotide compounds for in vitro evaluation Modified oligonucleotide compounds were synthesized using ALNA[Ms] amidite (synthesized according to the method described in International Application PCT / JP2019 / 044182 (WO2020 / 100826)) on a 0.5 μmol scale using a DNA / RNA oligonucleotide automated synthesizer nS-8II (Gene Design, Inc.) on a CPG or polystyrene support. All amidites were prepared in 0.1 M acetonitrile solution. Coupling time for unnatural nucleosides was 10 minutes; all other steps were performed under standard nS-8II conditions. Activator 42 (Sigma-Aldrich) was used as the activator, Sulfurizing Reagent II (Gren Research Corporation) was used for thiolation, and Oxidizer (Sigma-Aldrich) was used for oxidation. The synthesized oligonucleotides were cleaved from the support and deprotected by adding 28% aqueous ammonia and reacting at 85°C for 8 hours. After the ammonia was concentrated and distilled off, the residue was subjected to simple purification using NAP10, followed by reversed-phase HPLC purification.

[0136] The synthesized modified oligonucleotide compounds are shown in Table 1 (18-residue modified oligonucleotide) and Table 2 (16-residue modified oligonucleotide). Each nucleotide in the compound is represented by three letters. However, the 3'-terminal nucleotide is represented by two letters because it does not have an internucleoside bond. 1) The first letter is capitalized and represents the following nucleobase: A = adenine, T = thymine, G = guanine, C = cytosine, U = uracil, M = 5-methylcytosine, 2) The second letter represents the sugar moiety: m = ALNA [Ms], d = 2'-deoxyribose, 3) The third letter indicates the internucleoside bond: s = phosphorothioate, p = phosphodiester. The target start position indicates the ATXN3 pre-mRNA 5' target site of the modified oligonucleotide (the position of SEQ ID NO: 1 in the Sequence Listing corresponding to the 3' end of the modified oligonucleotide), and the target end position indicates the ATXN3 pre-mRNA 3' target site of the modified oligonucleotide (the position of SEQ ID NO: 1 in the Sequence Listing corresponding to the 5' end of the modified oligonucleotide).

[0137] Example 2 Purification and purity check of modified oligonucleotide compounds The synthesized modified oligonucleotide compounds were purified and their purity was confirmed by reverse phase HPLC under the following conditions. Reverse phase HPLC (purification) Mobile phase: Solution A: 400 mM hexafluoroisopropanol, 15 mM triethylamine Solution B: Methanol Gradient: A:B = 83:17 → 70:30 (10 min) Columns used: Preparative Waters XBridge@ Oligonucleotide BEH C18 OBDTM Prep Column,130Å,2.5μm,10mm*50mm Flow rate: Preparative 5mL / min Column temperature: 60℃ Detection: UV (260 nm)

[0138] Reverse phase HPLC (purity confirmation) Mobile phase: Solution A: 400 mM hexafluoroisopropanol, 15 mM triethylamine aqueous solution Solution B: Methanol Gradient: A:B = 82:18 → 73.5:26.5 (5 min) Columns used: Waters XBridge@ Oligonucleotide BEH C18 OBDTM Prep Column,130Å,2.5μm,4.6mm*50mm Flow rate: 1mL / min Column temperature: 60℃ Detection: UV (260 nm)

[0139] Example 3 In vitro ATXN3 expression ratio test (Gymnosis method) 3 × 10 SH-SY5Y cells per well 3 Simultaneously with seeding, synthesized modified oligonucleotides were added at a final concentration of 3 or 10 μmol / L and incubated in a CO2 incubator for 3 days. RNA was extracted from the cells and reverse-transcribed to obtain cDNA. Quantitative real-time PCR was performed using the resulting cDNA with primers and probes for ATXN3 or GAPDH. ATXN3 mRNA levels were determined by calculating the ratio of ATXN3 mRNA to GAPDH. The ATXN3 expression ratio was calculated as a percentage of the ATXN3 mRNA level in cells treated with modified oligonucleotides compared to that in cells without modified oligonucleotides. Tables 1 and 3 show the ATXN3 expression ratio when an 18-residue modified oligonucleotide was added at a final concentration of 3 μmol / L, and Table 2 shows the ATXN3 expression ratio when a 16-residue modified oligonucleotide was added at a final concentration of 10 μmol / L.

[0140] Example 4 In vitro ATXN3 expression ratio test (Gymnosis method) Representative modified oligonucleotides 650528 described in WO2018 / 089805, 1100673 described in WO2019 / 217708, and 1287095 described in WO2020 / 172559 were synthesized. The target start and end positions of these modified oligonucleotides are shown in Table 4. Synthesis was performed in the same manner as in Examples 1 and 2 using 2'-MOE (2'-O-methoxyethyl) amidite. 3 x 10 SH-SY5Y cells were cultured per well. 3Simultaneously with seeding, synthesized modified oligonucleotides were added at a final concentration of 3 or 10 μmol / L and incubated in a CO2 incubator for 3 days. RNA was extracted from the cells, and reverse transcription was performed to obtain cDNA. Quantitative real-time PCR was performed using the resulting cDNA with primers and probes for ATXN3 or GAPDH. ATXN3 mRNA levels were determined by calculating the ratio to GAPDH levels. The ATXN3 expression ratio was calculated as a percentage of the ATXN3 mRNA levels in cells with and without modified oligonucleotides. The results are shown in Table 4.

[0141] [Table 1-1]

[0142] [Table 1-2]

[0143] [Table 1-3]

[0144] [Table 2-1]

[0145] [Table 2-2]

[0146] [Table 3]

[0147] [Table 4]

[0148] Reference example The synthesis schemes for ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, ALNA[ipU]-containing nucleosides, ALNA[Trz]-containing nucleosides, and ALNA[Oxz]-containing nucleosides are shown below. Note that starting compounds 1a, 1d, and 1g can be synthesized by the method described in WO 2017 / 047816.

[0149] Synthesis of ALNA[Ms]-T [ka]

[0150] Synthesis of ALNA[Ms]-mC [ka]

[0151] Synthesis of ALNA[Ms]-G [ka]

[0152] Synthesis of ALNA[Ms]-A [ka]

[0153] Synthesis of ALNA[mU]-T [ka]

[0154] Synthesis of ALNA[mU]-mC [ka]

[0155] Synthesis of ALNA[mU]-G [ka]

[0156] Synthesis of ALNA[mU]-A [ka]

[0157] Synthesis of ALNA[ipU]-T [ka]

[0158] Synthesis of ALNA[ipU]-mC [ka]

[0159] Synthesis of ALNA[ipU]-G [ka]

[0160] Synthesis of ALNA[ipU]-A [ka]

[0161] Synthesis of ALNA[Trz]-T [ka]

[0162] Synthesis of ALNA[Trz]-mC [ka]

[0163] Synthesis of ALNA[Trz]-G [ka]

[0164] Synthesis of ALNA[Trz]-A [ka]

[0165] Synthesis of ALNA[Oxz]-T [ka]

[0166] Synthesis of ALNA[Oxz]-mC [ka]

[0167] As used herein, the use of the singular includes the plural unless stated otherwise. As used herein, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms such as "includes" and "included" is not limiting. Furthermore, unless stated otherwise, terms such as "element" encompass elements that contain one unit and elements that contain more than one subunit.

[0168] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are expressly incorporated herein by reference in their entirety and with respect to the portions of the documents discussed herein.

[0169] Unless specific definitions are provided, the nomenclatures utilized in connection with, and the procedures and techniques of, analytical chemistry, organic synthetic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for the chemical syntheses and chemical analyses used herein. Where permitted, all patents, applications, published applications, and other publications, GenBank accession numbers and associated sequence information and other data available through databases such as the National Center for Biotechnology Information (NCBI), referred to throughout this disclosure are incorporated by reference in their entirety and with respect to portions of the documents discussed herein. This specification is also filed together with a Sequence Listing in electronic format, the information in the Sequence Listing set forth in said electronic format being incorporated herein by reference in its entirety.

Claims

1. A modified oligonucleotide having an activity of inhibiting the expression of Ataxin 3 (ATXN3), wherein the nucleic acid base sequence of the modified oligonucleotide is: 1) TCGGGTAAGTAGATTTTC (complementary sequence of 2159 to 2176 in SEQ ID NO: 1) (SEQ ID NO: 239 in the Sequence Listing) 2) AGGTTATAGGATGCAGGT (complementary sequence of 5845 to 5862 in SEQ ID NO: 1) (SEQ ID NO: 243 in the Sequence Listing) 3) GAAGCTAAGTAGGTGACT (complementary sequence of 15115 to 15132 in SEQ ID NO: 1) (SEQ ID NO: 244 in the Sequence Listing) 4) GTCATCCCTATGTCTTAT (complementary sequence of 36607 to 36624 in SEQ ID NO: 1) (SEQ ID NO: 259 in the Sequence Listing) 5) GTCATATGGTCAGGGTAT (complementary sequence of 40453 to 40470 in SEQ ID NO: 1) (SEQ ID NO: 260 in the Sequence Listing) 6) TGTCATATGGTCAGGGTA (complementary sequence of 40454 to 40471 in SEQ ID NO: 1) (SEQ ID NO: 261 in the Sequence Listing) 7) ATGTCATATGGTCAGGGT (complementary sequence of 40455 to 40472 in SEQ ID NO: 1) (SEQ ID NO: 262 in the Sequence Listing) and 8) TATGTCATATGGTCAGGG (complementary sequence of 40456 to 40473 in SEQ ID NO: 1) (SEQ ID NO: 263 in the Sequence Listing) or a nucleic acid base sequence of 17 consecutive bases contained in said nucleic acid base sequence, wherein at least one of the nucleosides constituting the oligonucleotide has a modified sugar selected from the group consisting of a bicyclic sugar, a sugar modified with 2'-MOE, and a sugar modified with 2'-OMe.

2. The modified oligonucleotide of claim 1 which is single-stranded.

3. 2. The modified oligonucleotide of claim 1, wherein the bicyclic sugar is selected from the group consisting of an LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], or ALNA[Trz] sugar moiety.

4. At least one nucleoside constituting the modified oligonucleotide has a modified nucleic acid base The modified oligonucleotide according to any one of claims 1 to 3, comprising:

5. The modified oligonucleotide of claim 4, wherein the modified nucleobase is 5-methylcytosine.

6. The modified oligonucleotide according to any one of claims 1 to 5, wherein at least one internucleoside bond constituting the modified oligonucleotide is a modified internucleoside bond.

7. 7. The modified oligonucleotide of claim 6, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

8. the modified oligonucleotide is 1) gap segments, 2) a 5' wing segment and 3) a 3' wing segment; the gap segment is positioned between the 5' wing segment and the 3' wing segment; The modified oligonucleotide according to any one of claims 1 to 7, wherein the nucleosides constituting the 5' wing segment and the 3' wing segment contain modified sugars.

9. A modified oligonucleotide consisting of 16 to 24 residues, which has activity of inhibiting ATXN3 expression, wherein the nucleic acid base sequence of the modified oligonucleotide is 100% complementary to an equal-length portion of the nucleic acid base sequence of SEQ ID NO: 1 in the Sequence Listing, and at least one of the nucleosides constituting the oligonucleotide has a modified sugar selected from the sugar moieties of ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], or ALNA[Trz].

10. A pharmaceutical composition comprising the modified oligonucleotide according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

11. The pharmaceutical composition of claim 10 for treating, preventing, or delaying the progression of an ATXN3-associated disease.

12. The pharmaceutical composition of claim 11, wherein the ATXN3-associated disease is a neurodegenerative disease.

13. The pharmaceutical composition of claim 12, wherein the neurodegenerative disease is spinocerebellar ataxia type 3.

14. 10. Use of the modified oligonucleotide of any one of claims 1 to 9 in the manufacture of a medicament for treating, preventing or slowing the progression of an ATXN3-related disease.

15. The modified oligonucleotide of any one of claims 1 to 9 for treating, preventing or slowing the progression of an ATXN3-related disease.

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