COMPOUND, METHOD AND PHARMACEUTICAL COMPOSITION FOR REGULATING EXPRESSION OF ATAXIN 3

Abstract

A modified oligonucleotide having an activity of inhibiting Ataxin 3 expression and having any one of the following nucleobase sequences or a nucleobase sequence of 17 contiguous bases contained in the nucleobase sequences:

TABLE-US-00001 (SEQ ID NO: 239) (1) TCGGGTAAGTAGATTTTC, (SEQ ID NO: 240) (2) GAAGTATCTGTAGGCCTA, (SEQ ID NO: 241) (3) GGACTGTATAGGAGATTA, (SEQ ID NO: 242) (4) GGTTATAGGATGCAGGTA, (SEQ ID NO: 243) (5) AGGTTATAGGATGCAGGT, (SEQ ID NO: 244) (6) GAAGCTAAGTAGGTGACT, (SEQ ID NO: 245) (7) TGAAGCTAAGTAGGTGAC, (SEQ ID NO: 246) (8) CCTAGTCACTTTGATAGA, (SEQ ID NO: 247) (9) GGAACATCTTGAGTAGGT, (SEQ ID NO: 248) (10) GGTGTTCAGGGTAGATGT, (SEQ ID NO: 249) (11) GGATACTCTGCCCTGTTC, (SEQ ID NO: 250) (12) GGTGTCAAACGTGTGGTT, (SEQ ID NO: 251) (13) CCGTGTGCTAGTATTTGT, (SEQ ID NO: 252) (14) TAGTAGAGTTTTGCTTGG, (SEQ ID NO: 253) (15) GATGTAGTAGAGTTTTGC, (SEQ ID NO: 254) (16) TGATGTAGTAGAGTTTTG, (SEQ ID NO: 255) (17) CTGATGTAGTAGAGTTTT, (SEQ ID NO: 256) (19) GCAAGTTGGTTTGTGGTA, (SEQ ID NO: 257) (20) TCTAGGCAATTGTGGTGG, (SEQ ID NO: 258) (21) GTAACTCTGCACTTCCCA, (SEQ ID NO: 259) (22) GTCATCCCTATGTCTTAT, (SEQ ID NO: 260) (23) GTCATATGGTCAGGGTAT, (SEQ ID NO: 261) (24) TGTCATATGGTCAGGGTA, (SEQ ID NO: 262) (25) ATGTCATATGGTCAGGGT, and (SEQ ID NO: 263) (26) TATGTCATATGGTCAGGG.

Claims

1. A modified oligonucleotide having an activity of inhibiting Ataxin 3 (ATXN3) expression, wherein a nucleobase sequence of the modified oligonucleotide is a nucleobase sequence selected from the group consisting of (1) TCGGGTAAGTAGATTTTC (complementary sequence of 2159-2176 of SEQ ID NO: 1) (SEQ ID NO: 239 of the sequence listing), (2) GAAGTATCTGTAGGCCTA (complementary sequence of 2513-2530 of SEQ ID NO: 1) (SEQ ID NO: 240 of the sequence listing), (3) GGACTGTATAGGAGATTA (complementary sequence of 2646-2663 of SEQ ID NO: 1) (SEQ ID NO: 241 of the sequence listing), (4) GGTTATAGGATGCAGGTA (complementary sequence of 5844-5861 of SEQ ID NO: 1) (SEQ ID NO: 242 of the sequence listing), (5) AGGTTATAGGATGCAGGT (complementary sequence of 5845-5862 of SEQ ID NO: 1) (SEQ ID NO: 243 of the sequence listing), (6) GAAGCTAAGTAGGTGACT (complementary sequence of 15115-15132 of SEQ ID NO: 1) (SEQ ID NO: 244 of the sequence listing), (7) TGAAGCTAAGTAGGTGAC (complementary sequence 15116-15133 of SEQ ID NO: 1) (SEQ ID NO: 245 of the sequence listing), (8) CCTAGTCACTTTGATAGA (complementary sequence of 19163-19180 of SEQ ID NO: 1) (SEQ ID NO: 246 of the sequence listing), (9) GGAACATCTTGAGTAGGT (complementary sequence of 19737-19754 of SEQ ID NO: 1) (SEQ ID NO: 247 of the sequence listing), (10) GGTGTTCAGGGTAGATGT (complementary sequence of 20835-20852 of SEQ ID NO: 1) (SEQ ID NO: 248 of the sequence listing), (11) GGATACTCTGCCCTGTTC (complementary sequence of 21482-21499 of SEQ ID NO: 1) (SEQ ID NO: 249 of the sequence listing), (12) GGTGTCAAACGTGTGGTT (complementary sequence of 22200-22217 of SEQ ID NO: 1) (SEQ ID NO: 250 of the sequence listing), (13) CCGTGTGCTAGTATTTGT (complementary sequence of 27389-27406 of SEQ ID NO: 1) (SEQ ID NO: 251 of the sequence listing), (14) TAGTAGAGTTTTGCTTGG (complementary sequence of 31570-31587 of SEQ ID NO: 1) (SEQ ID NO: 252 of the sequence listing), (15) GATGTAGTAGAGTTTTGC (complementary sequence of 31574-31591 of SEQ ID NO: 1) (SEQ ID NO: 253 of the sequence listing), (16) TGATGTAGTAGAGTTTTG (complementary sequence of 31575-31592 of SEQ ID NO: 1) (SEQ ID NO: 254 of the sequence listing), (17) CTGATGTAGTAGAGTTTT (complementary sequence of 31576-31593 of SEQ ID NO: 1) (SEQ ID NO: 255 of the sequence listing), (19) GCAAGTTGGTTTGTGGTA (complementary sequence of 32008-32025 of SEQ ID NO: 1) (SEQ ID NO: 256 of the sequence listing), (20) TCTAGGCAATTGTGGTGG (complementary sequence of 32127-32144 of SEQ ID NO: 1) (SEQ ID NO: 257 of the sequence listing), (21) GTAACTCTGCACTTCCCA (complementary sequence of 36411-36428 of SEQ ID NO: 1) (SEQ ID NO: 258 of the sequence listing), (22) GTCATCCCTATGTCTTAT (complementary sequence of 36607-36624 of SEQ ID NO: 1) (SEQ ID NO: 259 of the sequence listing), (23) GTCATATGGTCAGGGTAT (complementary sequence of 40453-40470 of SEQ ID NO: 1) (SEQ ID NO: 260 of the sequence listing), (24) TGTCATATGGTCAGGGTA (complementary sequence of 40454-40471 of SEQ ID NO: 1) (SEQ ID NO: 261 of the sequence listing), (25) ATGTCATATGGTCAGGGT (complementary sequence of 40455-40472 of SEQ ID NO: 1) (SEQ ID NO: 262 of the sequence listing), and (26) TATGTCATATGGTCAGGG (complementary sequence of 40456-40473 of SEQ ID NO: 1) (SEQ ID NO: 263 of the sequence listing) or a nucleobase sequence of 17 contiguous bases contained in the nucleobase sequence.

2. The modified oligonucleotide according to claim 1, wherein the modified oligonucleotide is single-stranded.

3. The modified oligonucleotide according to claim 1, wherein at least one nucleoside forming the modified oligonucleotide includes a modified sugar.

4. The modified oligonucleotide according to claim 3, wherein the modified sugar is selected from the group consisting of a bicyclic sugar, a 2′-MOE (2′-O-methoxyethyl) modified sugar, and a 2′-OMe modified sugar.

5. The modified oligonucleotide according to claim 4, wherein the bicyclic sugar is selected from sugar moieties of LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], or ALNA[Trz].

6. The modified oligonucleotide according to claim 1, wherein at least one nucleoside forming the modified oligonucleotide contains includes a modified nucleobase.

7. The modified oligonucleotide according to claim 6, wherein the modified nucleobase is 5-methylcytosine.

8. The modified oligonucleotide according to claim 1, wherein at least one internucleoside linkage forming the modified oligonucleotide is a modified internucleoside linkage.

9. The modified oligonucleotide according to claim 8, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

10. The modified oligonucleotide according to claim 1, comprising: a gap segment; a 5′ wing segment; and a 3′ wing segment, wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment, and nucleosides forming the 5′ wing segment and the 3′ wing segment each include a modified sugar.

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

12. A pharmaceutical composition, comprising: the modified oligonucleotide of claim 1 or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

13. A method for treating, preventing, or delaying progression of an ATXN3-related disease, comprising: administering an effective amount of the pharmaceutical composition of claim 12 to a subject in need thereof.

14. The pharmacological composition according to claim 13, wherein the ATXN3-related disease is a neurodegenerative disease.

15. The pharmacological composition according to claim 14, wherein the neurodegenerative disease is spinocerebellar ataxia type 3.

16. A method for treating, preventing or delaying progression of an ATXN3-related disease, comprising: administering an effective amount of the modified oligonucleotide of claim 1 or a pharmaceutically acceptable salt thereof to a subject in need thereof.

17. (canceled)

18. (canceled)

19. The method according to claim 16, wherein the ATXN3-related disease is a neurodegenerative disease.

20. The method according to claim 16, wherein the neurodegenerative disease is spinocerebellar ataxia type 3.

21. The modified oligonucleotide according to claim 2, wherein at least one nucleoside forming the modified oligonucleotide includes a modified sugar.

22. The modified oligonucleotide according to claim 21, wherein the modified sugar is selected from the group consisting of a bicyclic sugar, a 2′-MOE (2′-O-methoxyethyl) modified sugar, and a 2′-OMe modified sugar.

Description

MODE FOR CARRYING OUT THE INVENTION

[0070] It is to be understood that both the foregoing summary and the following detailed description are exemplary and explanatory only and are not restrictive of the present invention as claimed. In the present specification, unless specifically stated otherwise, the use of a singular form includes a plural form. As used herein, the use of the term “including” and its other forms, such as “includes” and “included,” is not limiting. Further, unless specifically stated otherwise, the term “element” or the like includes an element that includes one unit and an element that includes more than one subunit.

[0071] 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, reports, books and articles, are expressly incorporated herein by reference, with respect to the portions of the document discussed herein, and in their entirety.

Definitions

[0072] Unless a specific definition is given, the nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medical chemistry and pharmaceutical chemistry, and their procedures and techniques described herein are those that are well known in the art and are commonly used. Standard techniques can be used for chemical synthesis and chemical analysis as used herein. Where permitted, all patents, patent applications, published patent applications and other publications referred to throughout the disclosure of this specification, and GenBank accession numbers and relevant sequence information as well as other data available through databases such as the National Center for Biotechnology Information (NCBI), are incorporated by reference with respect to portions of the documents discussed herein, and in its entirety.

[0073] Further, this specification is filed together with a sequence listing in electronic format. However, the information of the sequence listing described in the electronic format is incorporated herein by reference in its entirety.

[0074] Unless otherwise indicated, the following terms have the following meanings.

[0075] A “nucleobase” means a heterocyclic moiety that can pair with a base of another nucleic acid.

[0076] A “nucleobase sequence” means an order of contiguous nucleobases forming the oligonucleotide of the present invention.

[0077] A “nucleoside” means a molecule in which a sugar and a nucleobase are linked. In certain embodiments, a nucleoside is linked to a phosphate group.

[0078] A “nucleotide” means a molecule in which a phosphate group is linked to a sugar moiety of a nucleoside. In a naturally occurring nucleotide, a sugar moiety is a ribose or deoxyribose and is covalently linked by a phosphodiester bond via a phosphate group.

[0079] An “oligomer compound” or “oligomer” refers to a polymer of linked monomeric subunits that is capable of hybridizing to at least one region of a nucleic acid molecule.

[0080] An “oligonucleotide” means a polymer of nucleosides in which nucleosides and internucleoside linkages are linked independently of each other.

[0081] An “internucleoside linkage” refers to a chemical bond between nucleosides.

[0082] A “naturally occurring internucleoside linkage” means a 3′-5′phosphodiester linkage.

[0083] A “modified internucleoside linkage” refers to a substitution or any change from a naturally occurring internucleoside linkage (that is, a phosphodiester internucleoside linkage). For example, there is a phosphorothioate internucleoside linkage, but it is not limited to this.

[0084] A “phosphorothioate internucleoside linkage” means an internucleoside linkage in which a phosphodiester linkage is modified by replacing one of non-crosslinked oxygen atoms with a sulfur atom. A phosphorothioate linkage is one example of the modified internucleoside linkage.

[0085] A “modified nucleobase” refers to any nucleobase other than adenine, cytosine, guanine, thymidine or uracil. For example, there is a 5-methylcytosine, but it is not limited to this. “Unmodified nucleobases” means purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C) and uracil (U).

[0086] A “modified oligonucleotide” means an oligonucleotide that contains at least one of the modified nucleoside and/or the modified internucleoside linkage.

[0087] “Salt” is a generic term for compounds in which one or more dissociable hydrogen ions contained in an acid are replaced by cations such as metal ions and ammonium ions, and examples of salts of a modified oligonucleotide include, but not limited to, salts (for example, a sodium salt, and a magnesium salt) formed with inorganic ions (for example, sodium ions, and magnesium ions) on phosphorothioates or phosphodiesters or on functional groups (for example, an amino group) in a modified nucleobase.

[0088] A “sugar” or a “sugar moiety” means a natural sugar moiety or a modified sugar moiety.

[0089] A “modified sugar” refers to a substitution or change from a natural sugar. Examples of modified sugars include a substituted sugar moiety and a bicyclic sugar.

[0090] A “substituted sugar moiety” means a furanosyl other than a natural sugar of RNA or DNA.

[0091] A “bicyclic sugar” means a furanosyl ring modified by crosslinking of two different carbon atoms present on the same ring. A “bicyclic nucleic acid” refers to a nucleoside or nucleotide in which a furanose moiety of the nucleoside or nucleotide contains a “bicyclic sugar.”

[0092] A “single-stranded oligonucleotide” means an oligonucleotide that is not hybridized to a complementary strand.

[0093] “ATXN3” means a nucleic acid or protein that is also known as Ataxin 3. ATXN3 may include, for example, various splicing variants transcribed from the ATXN3 gene, single nucleotide polymorphism (SNP) or CAG repeat expansion.

[0094] “Complementary” means an ability with respect to 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.

[0095] “Fully complementary (also called complementarity)” or “100% complementary (also called complementarity)” means that all nucleobases in a nucleobase sequence of a first nucleic acid have complementary nucleobases in a second nucleobase sequence of a second nucleic acid. In certain embodiments, a first nucleic acid is a modified oligonucleotide and a target nucleic acid is a second nucleic acid.

[0096] “Mismatch” or “non-complementary nucleobase” refers to a case where a nucleobase of a first nucleic acid cannot pair with a corresponding nucleobase of a second nucleic acid or a target nucleic acid.

[0097] A “target nucleic acid,” a “target RNA” and a “target RNA transcript” all refer to a nucleic acid that can be targeted by a modified oligonucleotide. In certain embodiments, a target nucleic acid includes a region of an ATXN3 mRNA or an ATXN3 pre-mRNA.

[0098] “Motif” means a combination of chemically distinct regions in a modified oligonucleotide.

[0099] “Immediately adjacent” means that there is no intervening element between immediately adjacent elements.

[0100] A “pharmaceutically acceptable salt” means a physiologically and pharmaceutically acceptable salt of the modified oligonucleotide of the present invention, that is, a salt that retains a desired biological activity of a modified oligonucleotide and does not impart an undesired toxic effect to the modified oligonucleotide.

[0101] “Administering” means providing a drug to an animal, and examples thereof include, but are not limited to, administering by a medical professional or family member, and self-administering.

[0102] “Amelioration” refers to a lessening of at least one indicator, sign, or symptom of a related disease, disorder, or condition. Severity of an indicator can be determined by a subjective or objective measure that is known to a person skilled in the art.

[0103] “Animals” refers to humans or non-human animals, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

[0104] An “effective amount” means an amount of the 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 can vary from individual to individual depending on the health and physical conditions of the individual to be treated, the taxonomic group of the individual to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.

[0105] An “individual” means a human or non-human animal selected for treatment or therapy.

[0106] “Preventing” means delaying or preventing the onset or development of a disease, disorder, unfavorable health condition, or one or more symptoms associated with the disease, disorder or undesired health condition for a period of time from minutes to indefinitely. Preventing also means reducing a risk of developing a disease, disorder, or undesirable health condition.

[0107] “Treating” means alleviating or eliminating a disease, disorder, or unfavorable health condition, or one or more symptoms associated with the disease, disorder, or unfavorable condition, or partially eliminating or eradicating one or more causes of the disease, disorder, or unfavorable health condition itself.

Specific Embodiments

[0108] Certain specific embodiments described below provide, but are not limited to, a compound for inhibiting expression of ATXN3, a method using the compound, and a pharmaceutical composition containing the compound.

[0109] (1) Modified Oligonucleotide

[0110] A modified oligonucleotide of the present invention (hereinafter, may be referred to as “the compound of the present invention” or “the modified oligonucleotide of the present invention”) is an antisense oligonucleotide of ATXN3 having an activity of inhibiting ATXN3 expression. Inhibiting ATXN3 expression (level) in the present invention means suppressing at least one of a pre-mRNA level transcribed from the ATXN3 gene, an mRNA level spliced from pre-mRNA, and an ATXN3 protein level translated from mRNA. ATXN3 pre-mRNA may contain CAG repeat expansion found in patients with SNP and/or spinocerebellar ataxia type 3. However, an example thereof is that represented by a sequence (SEQ ID NO: 1 in the sequence listing) described in NC_000014.9: c92106621-92058552 Homo sapiens chromosome 14, GRCH.sub.38.p13 Primary Assembly. ATXN3 mRNA may contain at least one of CAG repeat expansions found in patients with variant, SNP and spinocerebellar ataxia type 3. However, an example thereof is that represented by a sequence (SEQ ID NO: 2 in the sequence listing) described in GenBank accession number NM 004993.5. SEQ ID NOs: 1 and 2 show DNA base sequences.

[0111] However, in the case of RNA sequences, T is replaced with U.

[0112] A degree of inhibition of ATXN3 expression of the compound of the present invention may be any degree as long as at least one of the pre-mRNA level, the pre-mRNA level and the protein level of ATXN3 is lower than when the compound is not administered, and as a result, prevention and/or amelioration of symptoms associated with an ATXN3-related disease is observed. However, specifically, for example, in an in vitro ATXN3 expression measurement method to be described later, after the compound of the present invention is brought into contact with cells, 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 as compared to a case of non-contact or contact with a negative control substance.

[0113] The compound of the present invention is a modified oligonucleotide consisting of 12-24 residues, preferably 16-18 residues, and more preferably 18 residues and having an activity of inhibiting ATXN3 expression, and may be any compound as long as the compound includes at least 8 contiguous nucleobase sequences complementary to an equal length portion of any one of positions 256-273, positions 404-427, positions 541-558, positions 647-665, positions 697-716, positions 767-784, positions 2043-2066, positions 2092-2109, positions 2159-2177, positions 2502-2530, positions 2646-2667, positions 4011-4029, positions 4038-4063, positions 4259-4292, positions 5179-5196, positions 5762-5777, positions 5843-5870, positions 7203-7225, positions 7535-7553, positions 7602-7617, positions 7741-7758, positions 7788-7810, positions 10273-10288, positions 10997-11014, positions 11642-11672, positions 11734-11759, positions 11799-11825, positions 12710-12727, positions 13136-13156, positions 14325-14340, positions 15114-15139, positions 15703-15723, positions 18108-18123, positions 18749-18766, positions 19061-19078, positions 19163-19181, positions 19355-19372, positions 19736-19756, positions 20833-20854, positions 21059-21077, positions 21482-21502, positions 22109-22126, positions 22192-22220, positions 22913-22930, positions 22998-23013, positions 23046-23074, positions 24651-24670, positions 26470-26485, positions 27388-27412, positions 30559-30582, positions 31570-31595, positions 31841-31858, positions 32006-32029, positions 32127-32144, positions 33630-33647, positions 35710-35729, positions 36411-36441, positions 36607-36624, positions 37690-37705, positions 38556-38576, positions 38587-38624, positions 40452-40492, positions 40539-40557, positions 41281-41300, positions 41392-41410, positions 42805-42820, positions 43091-43106, or positions 45892-45909 from the 5′ end of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing (hereinafter this is referred to as an “ATXN3 complementary nucleobase sequence”). Further, the above ATXN3 complementary nucleobase sequence is preferably 8-18 contiguous nucleobase sequences, and more preferably 16, 17 or 18 contiguous nucleobase sequences.

[0114] Or, the ATXN3 complementary nucleobase sequence may be 16 or more contiguous nucleobase sequences complementary to an equal length portion of any one of positions 1512-1536, positions 1568-1583, positions 3931-3946, positions 5668-5683, positions 7817-7832, positions 9925-9940, positions 10409-10424, positions 10556-10571, positions 10584-10599, positions 11002-11017, positions 11617-11632, positions 12076-12091, positions 14074-14089, positions 14213-14228, positions 16211-16226, positions 16740-16755, positions 18015-18030, positions 18033-18048, positions 20832-20847, positions 20848-20863, positions 22116-22131, positions 22194-22225, positions 27062-27077, positions 27271-27286, positions 29393-29408, positions 30535-30550, positions 30556-30571, positions 30930-30945, positions 33237-33252, positions 34900-34915, positions 35841-35856, positions 41294-41309, positions 42110-42125, positions 43182-43197, positions 43209-43233, positions 43933-43948, or positions 44050-44065 from the 5′ end of the sequence of SEQ ID NO: 1 in the sequence listing.

[0115] Here, the modified oligonucleotide of the present invention may have, in addition to the above ATXN3 complementary nucleobase sequence, an additional sequence on the 5′ end side and/or the 3′ end side as long as its total length is in a range of 12-24 residues, preferably 16-18 residues, and more preferably 18 residues and the full-length nucleobase sequence has a complementarity of at least 85% to an equal length portion of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing. Further, the addition sequence may be any sequence as long as the modified oligonucleotide of the present invention has an activity of inhibiting ATXN3 expression.

[0116] The full-length nucleobase sequence of the modified oligonucleotide has a complementarity of at least 85% to an equal length portion of SEQ ID NO: 1 in the sequence listing. However, the complementarity is preferably 90%, more preferably 95%, and even more preferably 100%. Here, an equal length portion means a portion detected as a portion having homology between the nucleobase sequence of the modified oligonucleotide of the present invention and a nucleobase sequence of ATXN3 pre-mRNA or mRNA when the nucleobase sequences are aligned, for example, using software such as BLAST, Genetyx software (GENETYX CORPORATION). That is, the nucleobase sequence of the oligonucleotide of the present invention is desirably fully complementary to an equal length portion of a nucleobase sequence of ATXN3 pre-mRNA or mRNA, but may have one or more mismatched nucleobases, and those having a complementarity of 85% or more, 90% or more, and preferably 95% or more are used.

[0117] The mismatched nucleobases may be contiguous or may be interspersed with an ATXN3 complementary nucleobase sequence. Percent complementarity of the antisense oligonucleotide of the present invention with respect to ATXN3 pre-mRNA or mRNA can be determined conventionally, for example, using the BLAST program (basic local alignment search tools) and the PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656), and the Genetyx software (GENETYX CORPORATION), which are known in the art. Percent homology, sequence identity, or complementarity, can be determined, for example, by the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), by using default settings using the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489), and by using the Genetyx software (GENETYX CORPORATION). For example, 18 of 20 nucleobases of a modified oligonucleotide are complementary to an equal length portion of ATXN3 pre-mRNA, and when hybridized, the modified oligonucleotide has a 90 percent complementarity.

[0118] A method for verifying the activity of the compound of the present invention may be any method as long as the method can verify that the compound of the present invention inhibits ATXN3 expression. However, specifically, for example, the in vitro ATXN3 expression measurement method to be described later is used.

[0119] Examples of thus selected modified oligonucleotide of the present invention include those consisting of a sequence complementary to a nucleobase sequence selected from any one of positions 256-273, positions 404-421, positions 405-422, positions, 407-424, positions 408-425, positions 409-426, positions 410-427, positions 541-558, positions 647-664, positions 648-665, positions 697-714, positions 698-715, positions 699-716, positions 767-784, positions 1512-1527, positions 1521-1536, positions 1568-1583, positions 2043-2060, positions, 2045-2062, positions 2046-2063, positions 2047-2064, positions, 2048-2065, positions 2051-2066, positions 2092-2109, positions 2159-2176, positions, 2162-2177, positions 2502-2519, positions 2513-2530, positions, 2646-2663, positions 2650-2667, positions 3931-3946, positions 4011-4028, positions 4012-4029, positions 4038-4053, positions 4041-4058, positions 4046-4063, positions 4259-4274, positions 4267-4284, positions 4268-4285, positions 4269-4286, positions 4277-4292, positions 5179-5196, positions 5668-5683, positions 5762-5777, positions 5843-5860, positions 5844-5861, positions, 5845-5862, positions 5853-5870, positions 7203-7220, positions 7208-7225, positions 7535-7552, positions 7536-7553, positions 7602-7617, positions 7741-7758, positions 7788-7805, positions 7792-7809, positions 7793-7810, positions 7817-7832, positions 9925-9940, positions 10273-10288, positions 10409-10424, positions 10556-10571, positions 10584-10599, positions 10997-11014, positions 11002-11017, positions 11617-11632, positions 11642-11659, positions 11643-11660, positions 11644-11661, positions 11645-11662, positions 11646-11663, positions 11648-11665, positions 11649-11666, positions 11650-11665, positions 11650-11667, positions 11651-11668, positions 11654-11669, positions 11655-11672, positions 11734-11751, positions 11735-11752, positions 11738-11753, positions 11741-11758, positions 11744-11759, positions 11799-11816, positions 11800-11817, positions 11810-11825, positions 12076-12091, positions 12710-12727, positions 13136-13153, positions 13141-13156, positions 14074-14089, positions 14213-14228, positions 14325-14340, positions 15114-15129, positions 15115-15132, positions 15116-15133, positions 15124-15139, positions 15703-15720, positions 15704-15721, positions 15705-15722, positions, 15706-15723, positions 16211-16226, positions 16740-16755, positions, 18015-18030, positions 18033-18048, positions 18108-18123, positions 18749-18766, positions 19061-19078, positions 19163-19180, positions 19166-19181, positions 19355-19372, positions 19736-19753, positions 19737-19754, positions 19738-19755, positions 19739-19756, positions 20832-20847, positions 20833-20850, positions 20835-20852, positions 20836-20853, positions 20837-20854, positions 20838-20855, positions 20839-20854, positions 20848-20863, positions 21059-21076, positions 21060-21077, positions 21482-21499, positions 21483-21500, positions 21487-21502, positions 22109-22126, positions 22116-22131, positions 22192-22209, positions 22193-22210, positions 22194-22209, positions 22194-22211, positions 22195-22212, positions 22196-22213, positions 22197-22214, positions 22197-22214, positions 22198-22215, positions 22198-22215, positions 22199-22214, positions 22199-22216, positions 22200-22217, positions 22201-22218, positions 22202-22219, positions 22203-22220, positions 22203-22218, positions 22210-22225, positions 22913-22930, positions 22998-23013, positions 23046-23063, positions 23047-23064, positions 23048-23065, positions 23049-23066, positions, 23050-23067, positions 23055-23072, positions 23056-23073, positions, 23057-23074, positions 24651-24668, positions 24652-24669, positions 24653-24670, positions 26470-26485, positions 27062-27077, positions 27271-27286, positions 27388-27405, positions 27389-27406, positions 27391-27408, positions 27392-27409, positions 27393-27410, positions 27394-27411, positions 27395-27412, positions 29393-29408, positions 30535-30550, positions 30556-30571, positions 30559-30576, positions 30560-30577, positions 30561-30578, positions 30564-30581, positions 30565-30582, positions 30930-30945, positions 31570-31587, positions 31574-31591, positions 31575-31592, positions 31576-31591, positions 31576-31593, positions 31577-31594, positions 31578-31595, positions 31580-31595, positions 31841-31858, positions 32006-32021, positions 32006-32023, positions 32007-32024, positions 32008-32025, positions 32009-32026, positions 32010-32027, positions 32011-32028, positions 32012-32029, positions 32127-32144, positions 33237-33252, positions 33630-33647, positions 34900-34915, positions 35710-35725, positions 35711-35728, positions 35712-35729, positions 35841-35856, positions 36411-36428, positions 36412-36429, positions 36413-36428, positions 36413-36430, positions 36417-36432, positions 36426-36441, positions 36607-36624, positions 36609-36624, positions 37690-37705, positions 38556-38571, positions 38559-38576, positions 38587-38604, positions 38588-38605, positions 38589-38606, positions 38590-38607, positions 38593-38610, positions 38594-38611, positions 38596-38613, positions 38597-38614, positions 38598-38615, positions 38599-38616, positions 38600-38617, positions 38601-38618, positions, 38604-38621, positions 38606-38623, positions 38607-38624, positions 40452-40467, positions 40453-40470, positions 40454-40471, positions 40455-40472, positions 40456-40473, positions 40470-40485, positions 40475-40492, positions 40539-40556, positions 40540-40557, positions 41281-41298, positions 41285-41300, positions 41294-41309, positions 41392-41409, positions 41393-41410, positions 42110-42125, positions 42805-42820, positions 43091-43106, positions 43182-43197, positions 43209-43224, positions 43218-43233, positions 43933-43948, positions 44050-44065 or positions 45892-45909, preferably a nucleobase sequence selected from any one of positions 2159-2176, positions 5668-5683, positions 5845-5862, positions 11650-11665, positions 12076-12091, positions 14213-14228, positions 15115-15132, positions 16740-16755, positions 18033-18048, positions 22199-22214, positions 22203-22218, positions 27062-27077, positions 31575-31592, positions 31576-31591, positions 31576-31593, positions 32006-32021, positions 35841-35856, positions 36607-36624, positions 36609-36624, positions 37690-37705, positions 40453-40470, positions 40454-40471, positions 40455-40472, positions 40456-40473, positions 40470-40485 or positions 41294-41309, more preferably a nucleobase sequence selected from any one of positions 12076-12091, positions 22199-22214, positions, 22203-22218, positions 27062-27077, positions, 31576-31591, positions 32006-32021, positions 35841-35856, positions 36609-36624, positions 40453-40470 or positions 41294-41309, from the 5′ end of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing, or those consisting of a sequence complementary to a nucleobase sequence of 16 or 17 contiguous bases contained in the nucleobase sequence. Here, when a full-length nucleobase sequence of a modified oligonucleotide has 100% complementarity to an equal-length portion of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing, it may have an additional sequence of 1 or 2 residues on a 5′ end side and/or a 3′ end side thereof.

[0120] Examples of nucleobase sequences of modified oligonucleotides, which are among the thus selected modified oligonucleotides and inhibit an ATXN3 mRNA level to 70% or less when the modified oligonucleotides are added to SH-SY5Y cells so as to have a final concentration of 3 μM using a gymnosis method as compared to a case of non-contact with the modified oligonucleotides, include those consisting of a sequence complementary to a nucleobase sequence selected from any one of positions 698-715, positions 699-716, positions 2043-2060, positions 2046-2063, positions 2047-2064, positions 2092-2109, positions 2159-2176, positions 2513-2530, positions 2646-2663, positions 2650-2667, positions 4041-4058, positions 5843-5860, positions 5844-5861, positions 5845-5862, positions 7792-7809, positions 11642-11659, positions 11643-11660, positions 11644-11661, positions 11645-11662, positions 11649-11666, positions 11650-11667, positions 11655-11672, positions 11734-11751, positions 11735-11752, positions 12710-12727, positions 13136-13153, positions 15115-15132, positions 15116-15133, positions 15704-15721, positions 15706-15723, positions 19163-19180, positions 19737-19754, positions 20835-20852, positions 20836-20853, positions 20837-20854, positions 21482-21499, positions 21483-21500, positions 22192-22209, positions 22194-22211, positions 22199-22216, positions 22200-22217, positions 22201-22218, positions 22202-22219, positions 23056-23073, positions 27388-27405, positions 27389-27406, positions 27392-27409, positions 27393-27410, positions 27394-27411, positions 30559-30576, positions 30560-30577, positions 30564-30581, positions 31570-31587, positions 31574-31591, positions 31575-31592, positions 31576-31593, positions 31577-31594, positions 31578-31595, positions 31841-31858, positions 32006-32023, positions 32007-32024, positions 32008-32025, positions 32009-32026, positions 32011-32028, positions 32127-32144, positions 35711-35728, positions 35712-35729, positions 36411-36428, positions 36412-36429, positions 36607-36624, positions 38559-38576, positions 38587-38604, positions 38588-38605, positions 38589-38606, positions 38590-38607, positions 38593-38610, positions 38594-38611, positions 38604-38621, positions 38607-38624, positions 40453-40470, positions 40454-40471, positions 40455-40472, positions 40456-40473, positions 40475-40492, positions 41281-41298, positions 41392-41409, positions 41393-41410, positions or positions 45892-45909 from the 5′ end of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing, or those consisting of a sequence complementary to a nucleobase sequence of 16 or 17 contiguous bases contained in the nucleobase sequence.

[0121] A specific example of the nucleobase sequence of the modified oligonucleotide of the present invention is a nucleobase sequence described in

[0122] TCGGGTAAGTAGATTTTC (complementary sequence of 2159-2176 of SEQ ID NO: 1) (SEQ ID NO: 239 of the sequence listing),

[0123] GAAGTATCTGTAGGCCTA (complementary sequence of 2513-2530 of SEQ ID NO: 1) (SEQ ID NO: 240 of the sequence listing),

[0124] GGACTGTATAGGAGATTA (complementary sequence of 2646-2663 of SEQ ID NO: 1) (SEQ ID NO: 241 of the sequence listing),

[0125] GGTTATAGGATGCAGGTA (complementary sequence of 5844-5861 of SEQ ID NO: 1) (SEQ ID NO: 242 of the sequence listing),

[0126] AGGTTATAGGATGCAGGT (complementary sequence of 5845-5862 of SEQ ID NO: 1) (SEQ ID NO: 243 of the sequence listing),

[0127] GAAGCTAAGTAGGTGACT (complementary sequence of 15115-15132 of SEQ ID NO: 1) (SEQ ID NO: 244 of the sequence listing),

[0128] TGAAGCTAAGTAGGTGAC (complementary sequence 15116-15133 of SEQ ID NO: 1) (SEQ ID NO: 245 of the sequence listing),

[0129] CCTAGTCACTTTGATAGA (complementary sequence of 19163-19180 of SEQ ID NO: 1) (SEQ ID NO: 246 of the sequence listing),

[0130] GGAACATCTTGAGTAGGT (complementary sequence of 19737-19754 of SEQ ID NO: 1) (SEQ ID NO: 247 of the sequence listing),

[0131] GGTGTTCAGGGTAGATGT (complementary sequence of 20835-20852 of SEQ ID NO: 1) (SEQ ID NO: 248 of the sequence listing),

[0132] GGATACTCTGCCCTGTTC (complementary sequence of 21482-21499 of SEQ ID NO: 1) (SEQ ID NO: 249 of the sequence listing),

[0133] GGTGTCAAACGTGTGGTT (complementary sequence of 22200-22217 of SEQ ID NO: 1) (SEQ ID NO: 250 of the sequence listing),

[0134] CCGTGTGCTAGTATTTGT (complementary sequence of 27389-27406 of SEQ ID NO: 1) (SEQ ID NO: 251 of the sequence listing),

[0135] TAGTAGAGTTTTGCTTGG (complementary sequence of 31570-31587 of SEQ ID NO: 1) (SEQ ID NO: 252 of the sequence listing),

[0136] GATGTAGTAGAGTTTTGC (complementary sequence of 31574-31591 of SEQ ID NO: 1) (SEQ ID NO: 253 of the sequence listing),

[0137] TGATGTAGTAGAGTTTTG (complementary sequence of 31575-31592 of SEQ ID NO: 1) (SEQ ID NO: 254 of the sequence listing),

[0138] CTGATGTAGTAGAGTTTT (complementary sequence of 31576-31593 of SEQ ID NO: 1) (SEQ ID NO: 255 of the sequence listing),

[0139] GCAAGTTGGTTTGTGGTA (complementary sequence of 32008-32025 of SEQ ID NO: 1) (SEQ ID NO: 256 of the sequence listing),

[0140] TCTAGGCAATTGTGGTGG (complementary sequence of 32127-32144 of SEQ ID NO: 1) (SEQ ID NO: 257 of the sequence listing),

[0141] GTAACTCTGCACTTCCCA (complementary sequence of 36411-36428 of SEQ ID NO: 1) (SEQ ID NO: 258 of the sequence listing),

[0142] GTCATCCCTATGTCTTAT (complementary sequence of 36607-36624 of SEQ ID NO: 1) (SEQ ID NO: 259 of the sequence listing),

[0143] GTCATATGGTCAGGGTAT (complementary sequence of 40453-40470 of SEQ ID NO: 1) (SEQ ID NO: 260 of the sequence listing),

[0144] TGTCATATGGTCAGGGTA (complementary sequence of 40454-40471 of SEQ ID NO: 1) (SEQ ID NO: 261 of the sequence listing),

[0145] ATGTCATATGGTCAGGGT (complementary sequence of 40455-40472 of SEQ ID NO: 1) (SEQ ID NO: 262 of the sequence listing), or

[0146] TATGTCATATGGTCAGGG (complementary sequence of 40456-40473 of SEQ ID NO: 1) (SEQ ID NO: 263 of the sequence listing)

[0147] or a nucleobase sequence of 17 contiguous bases contained in the nucleobase sequence.

[0148] A specific example of the nucleobase sequence of the modified oligonucleotide of the present invention is preferably a nucleobase sequence described in

[0149] TCGGGTAAGTAGATTTTC (complementary sequence of 2159-2176 of SEQ ID NO: 1) (SEQ ID NO: 239 of the sequence listing),

[0150] AGGTTATAGGATGCAGGT (complementary sequence of 5845-5862 of SEQ ID NO: 1) (SEQ ID NO: 243 of the sequence listing),

[0151] GAAGCTAAGTAGGTGACT (complementary sequence of 15115-15132 of SEQ ID NO: 1) (SEQ ID NO: 244 of the sequence listing),

[0152] GTCATCCCTATGTCTTAT (complementary sequence of 36607-36624 of SEQ ID NO: 1) (SEQ ID NO: 259 of the sequence listing),

[0153] GTCATATGGTCAGGGTAT (complementary sequence of 40453-40470 of SEQ ID NO: 1) (SEQ ID NO: 260 of the sequence listing),

[0154] TGTCATATGGTCAGGGTA (complementary sequence of 40454-40471 of SEQ ID NO: 1) (SEQ ID NO: 261 of the sequence listing),

[0155] ATGTCATATGGTCAGGGT (complementary sequence of 40455-40472 of SEQ ID NO: 1) (SEQ ID NO: 262 of the sequence listing), or

[0156] TATGTCATATGGTCAGGG (complementary sequence of 40456-40473 of SEQ ID NO: 1) (SEQ ID NO: 263 of the sequence listing)

[0157] or a nucleobase sequence of 17 contiguous bases contained in the nucleobase sequence, and is more preferably a nucleobase sequence described in GTCATATGGTCAGGGTAT (complementary sequence of 40453-40470 of SEQ ID NO: 1) (SEQ ID NO: 260 of the sequence listing),

[0158] or a nucleobase sequence of 17 contiguous bases contained in the nucleobase sequence.

[0159] Of these sequences, one or more cytosines may be 5-methylcytosine of a modified nucleobase to be described later. Specific examples include 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 and SEQ ID NO: 151, and a preferred example is the sequence shown in SEQ ID NO: 148.

[0160] Further, examples of 16-base nucleobase sequences of the modified oligonucleotide of the present invention include 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 and SEQ ID NO: 230, and preferred examples thereof include 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 and SEQ ID NO: 230.

[0161] Of these sequences, one or more cytosines may be 5-methylcytosine of a modified nucleobase to be described later. Specific examples include 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, and SEQ ID NO: 225, and preferred examples include the sequences shown in SEQ ID NO: 184, SEQ ID NO: 203, SEQ ID NO: 208, SEQ ID NO: 224 and SEQ ID NO: 225.

[0162] The modified oligonucleotide of the present invention may be a double-stranded modified oligonucleotide, but a single-stranded modified oligonucleotide is preferably used.

[0163] (Modified Sugar)

[0164] As the modified oligonucleotide of the present invention, a modified oligonucleotide in which at least one nucleoside forming the modified oligonucleotide contains a modified sugar is preferably used. In the present invention, the term “modified sugar” refers to a sugar in which a sugar moiety is modified, and a modified oligonucleotide containing one or more such modified sugars has advantageous characteristics such as enhanced nuclease stability and increased binding affinity. At least one of modified sugars is preferably selected from a group consisting of a bicyclic sugar, a 2′-MOE modified sugar, and a 2′-OMe modified sugar. Examples of bicyclic sugars include, as illustrated below, sugar moieties of LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], or ALNA[Trz], and the sugar moiety of ALNA[Ms] is preferably used.

[0165] 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′-OCH.sub.3(2′-OMe), 2′-OCH.sub.2CH.sub.3, 2′-OCH.sub.2CH.sub.2F, and 2′-O (CH.sub.2).sub.2OCH.sub.3(2′-MOE) substituent groups. The substituent at the 2′ position can be selected from allyl, amino, azido, thio, O-allyl, O—C.sub.1-C.sub.10 alkyl, OCF.sub.3, OCH.sub.2F, O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2—O—N(R.sub.m)(R.sub.n), O—CH.sub.2—C(═O)—N(R.sub.m)(R.sub.n) and O—CH.sub.2—C(═O)—N(R.sub.l)—(CH.sub.2).sub.2—N(R.sub.m)(R.sub.n) (wherein R.sub.l, R.sub.m and R.sub.n are each independently H or substituted or unsubstituted C.sub.1-C.sub.10 alkyl).

[0166] Examples of nucleosides having a bicyclic sugar include, but are not limited to, nucleosides that contain a crosslink between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, an oligonucleotide provided herein includes a nucleoside having one or more bicyclic sugars, in which a crosslink includes one of the following formulas: 4′-(CH.sub.2)—O-2′ (LNA); 4′-(CH.sub.2)—S-2; 4′-(CH.sub.2).sub.2—O-2′ (ENA); 4′-CH(CH.sub.3)—O-2′ and 4′-CH(CH.sub.2OCH.sub.3)—O-2′ (and analogs thereof, see U.S. Pat. No. 7,399,845); 4′-C(CH.sub.3)(CH.sub.3)—O-2′ (and analogs thereof, see WO 2009/006478); 4′-CH.sub.2—N(OCH.sub.3)-2′ (and analogs thereof; see WO 2008/150729); 4′-CH.sub.2—O—N(CH.sub.3)-2′ (and analogs thereof; see US 2004-0171570); 4′-CH.sub.2—N(R)—O-2′ (wherein R is H, C.sub.1-C.sub.12 alkyl or a protecting group) (see U.S. Pat. No. 7,427,672); 4′-CH.sub.2—C(H)(CH.sub.3)-2′ (and analogs thereof; see Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH.sub.2—C(═CH.sub.2)-2′ (and analogues thereof; see WO 2008/154401).

[0167] Additional nucleosides having a bicyclic sugar are reported in published literatures (see, for example: 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, 558-61; 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. U.S.A., 2000, 97, 5633-5638; Singh et 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. Pat. Nos. 7,399,845; 6,770,748; 6,525,191; 6,268,490; US 2008-0039618; US 2007-0287831; US 2004-0171570; US 2009-0012281; WO 2010/036698; 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). The above-described nucleosides having a bicyclic sugar each can be prepared having one or more optically active sugar configurations including, for example, an α-L-ribofuranose and a β-D-ribofuranose.

[0168] GuNA, a nucleoside having a bicyclic sugar, has been reported as an artificial nucleoside having a guanidine crosslink (see WO 2014/046212 and WO 2017/047816). Bicyclic nucleosides ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Trz] and ALNA[Oxz] have been reported as crosslinked artificial nucleic acid amino LNA (ALNA) (see International Application PCT/JP 2019/044182 (WO 2020/100826)).

[0169] In certain embodiments, a nucleoside having a bicyclic sugar includes a crosslink between 4′ and 2′ carbon atoms of a pentofuranosyl sugar moiety, and a crosslink is included that contains 1 or 1 to 4 linking groups that are each independently selected from, but are not limited to, —[C(R.sub.a)(R.sub.b)].sub.n—, —C(R.sub.a)═C(R.sub.b)—, —C(R.sub.a)═N—, —C(═NR.sub.a)—, —C(═O)—, —C(═S)—, —N(R.sub.a)—, —O—, —Si(R.sub.a).sub.2— and —S(═O).sub.x—, wherein: X is 0, 1, or 2; n is 1, 2, 3, or 4; R.sub.a and R.sub.b are each, independently, H, a protecting group, hydroxyl, C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl, an aromatic ring group, a substituted aromatic ring group, a heterocyclic group, a substituted heterocyclic group, a C.sub.5-C.sub.7 alicyclic group, a substituted C.sub.5-C.sub.7 alicyclic group, halogen, OJ.sub.1, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, COOJ.sub.1, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O).sub.2-J.sub.1) or sulfoxyl (S(═O)-J.sub.1), J.sub.1 and J.sub.2 are each independently H, C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl, an aromatic ring group, a substituted aromatic ring group, acyl (C(═O)—H), substituted acyl, a heterocyclic group, a substituted heterocyclic group, C.sub.1-C.sub.12 aminoalkyl, substituted C.sub.1-C.sub.12 aminoalkyl or a protecting group.

[0170] In certain embodiments, the crosslink of the bicyclic sugar moiety is [C(R.sub.a)(R.sub.b)].sub.n—, —[CR.sub.a) (R.sub.b)].sub.n—O—, —C(R.sub.aR.sub.b)—N(R)—O— or —C(R.sub.aR.sub.b)—O—N(R)—. In certain embodiments, the crosslink is 4′-CH.sub.2-2′, 4′-(CH.sub.2).sub.2-2′, 4′-(CH.sub.2).sub.3-2′, 4′-CH.sub.2—O-2′ (the nucleoside having a bicyclic sugar in this case is also referred to as LNA), 4′-(CH.sub.2).sub.2—O-2′, 4′-CH.sub.2—O—N(R)-2′ and 4′-CH.sub.2—N(R)—O-2′-, wherein each R is, independently, H, a protecting group or C.sub.1-C.sub.12 alkyl.

[0171] In certain embodiments, the crosslink of the bicyclic sugar moiety is 4′-CH.sub.2—O-2′-(LNA) or —CH.sub.2—N(R)—, wherein each R is independently —SO.sub.2—CH.sub.3 (ALNA[Ms]), —CO—NH—CH.sub.3 (ALNA[mU]), 1,5-dimethyl-1,2,4-triazol-3-yl (ALNA[Trz]), —CO—NH—CH(CH.sub.3).sub.2 (ALNA[ipU]), or 5-methyl-2,4-oxadiazol-3-yl (ALNA[Oxz]) (International Application PCT/JP 2019/044182 (WO 2020/100826)).

[0172] In certain embodiments, a nucleoside having a bicyclic sugar is further defined by an isomeric steric configuration. For example, a nucleoside containing a 4′-(CH.sub.2)—O-2′ crosslink may exist in an α-L-steric configuration or a β-D-steric configuration.

[0173] In certain embodiments, nucleosides having a bicyclic sugar include those having a 4′-2′ crosslink, and examples of such crosslinks include, but are not limited to, α-L-4′-(CH.sub.2)—O-2′, β-D-4′-CH.sub.2—O-2′, 4′-(CH.sub.2).sub.2—O-2′, 4′-CH.sub.2—O—N(R)-2′, 4′-CH.sub.2—N(R)—O-2′, 4′-CH(CH.sub.3)—O-2′, 4′-CH.sub.2—S-2′, 4′-CH.sub.2—CH(CH.sub.3)-2′, and 4′-(CH.sub.2).sub.3-2′ (wherein, R is H, a protecting group, C.sub.1-C.sub.12 alkyl, or urea or guanidine that may be substituted with C.sub.1-C.sub.12 alkyl).

[0174] In certain embodiments, a nucleoside having a bicyclic sugar has the following formula:

##STR00001##

[0175] wherein

[0176] Bx is a heterocyclic base moiety;

[0177] T.sub.a and T.sub.b are each independently a hydrogen atom, a protecting group of a hydroxyl group, a phosphate group that may be substituted, a phosphorus moiety, a covalent attachment to a support, or the like; and

[0178] Z.sub.a is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, substituted C.sub.1-C.sub.6 alkyl, substituted C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thiol.

[0179] In certain embodiments, the substituents are each independently monosubstituted or polysubstituted with a substituent independently selected from halogen, oxo, hydroxyl, OJ.sub.c, NJ.sub.cJ.sub.d, SJ.sub.c, N.sub.3, OC(═X)J.sub.c and NJ.sub.eC(═X)NJ.sub.cJ.sub.d (wherein J.sub.c, J.sub.d and J.sub.e are each independently H, C.sub.1-C.sub.6 alkyl or substituted C.sub.1-C.sub.6 alkyl; and X is O or NJ.sub.c).

[0180] In certain embodiments, a nucleoside having a bicyclic sugar has the following formula:

##STR00002##

[0181] wherein

[0182] Bx is a heterocyclic base moiety;

[0183] T.sub.a and T.sub.b are each independently a hydrogen atom, a protecting group of a hydroxyl group, a phosphate group that may be substituted, a phosphorus moiety, a covalent attachment to a support, or the like; and

[0184] Z.sub.b is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, substituted C.sub.1-C.sub.6 alkyl, substituted C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkynyl or substituted acyl (C(═O)—).

[0185] In certain embodiments, a nucleoside having a bicyclic sugar has the following formula:

##STR00003##

[0186] wherein

[0187] Bx is a heterocyclic base moiety;

[0188] T.sub.a and T.sub.b are each independently a hydrogen atom, a protecting group of a hydroxyl group, a phosphate group that may be substituted, a phosphorus moiety, a covalent attachment to a support, or the like;

[0189] R.sub.d is C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted C.sub.2-C.sub.6 alkynyl; and

[0190] q.sub.a, q.sub.b, q.sub.c and q.sub.d are each independently H, halogen, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6 alkoxyl, substituted C.sub.1-C.sub.6 alkoxyl, acyl, substituted acyl, C.sub.1-C.sub.6 aminoalkyl or substituted C.sub.1-C.sub.6 aminoalkyl.

[0191] In certain embodiments, a nucleoside having a bicyclic sugar has the following formula:

##STR00004##

[0192] wherein

[0193] Bx is a heterocyclic base moiety;

[0194] T.sub.a and T.sub.b are each independently a hydrogen atom, a protecting group of a hydroxyl group, a phosphate group that may be substituted, a phosphorus moiety, a covalent attachment to a support, or the like; and

[0195] q.sub.a, q.sub.b, q.sub.e and q.sub.f are each independently hydrogen, halogen, C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.1-C.sub.12 alkoxy, substituted C.sub.1-C.sub.12 alkoxy, OJ.sub.j, SJ.sub.j, SOJ.sub.j, SO.sub.2J.sub.j, NJ.sub.jJ.sub.k, N.sub.3, CN, C(═O)OJ.sub.j, C(═O)NJ.sub.jJ.sub.k, C(═O)J.sub.j, O—C(═O)NJ.sub.jJ.sub.k, N(H)C(═NH)NJ.sub.jJ.sub.k, N(H)C(═O)—NJ.sub.jJ.sub.k or N(H)C(═S)NJ.sub.jJ.sub.k;

[0196] or q.sub.e and q.sub.f are both ═C(q.sub.g)(q.sub.h); and

[0197] q.sub.g and q.sub.h are each independently H, halogen, C.sub.1-C.sub.12 alkyl or substituted C.sub.1-C.sub.12 alkyl.

[0198] Synthesis and preparation of adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil bicyclic nucleoside (also referred to as LNA), which have 4′-CH.sub.2—O-2′ bridges, are described along with their oligomerization and nucleic acid recognition properties (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). Synthesis of a nucleoside having a bicyclic sugar is also described in WO 98/39352 and WO 99/14226.

[0199] Various analogs of bicyclic nucleosides having 4′-2′ bridging groups such as 4′-CH.sub.2—O-2′ (the bicyclic nucleoside in this case is also referred to as LNA) and 4′-CH.sub.2—S-2′ have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). 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). Further, synthesis of 2′-amino-LNA (the bicyclic nucleoside in this case is also referred to as ALNA), that is, a conformationally restricted high-affinity oligonucleotide analog, has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Further, 2′-amino- and 2′-methylamino-LNAs have been prepared and thermal stability of duplexes with complementary RNA and DNA strands has been previously reported.

[0200] In certain embodiments, a nucleoside having a bicyclic sugar has the following formula:

##STR00005##

[0201] wherein

[0202] Bx is a heterocyclic base moiety;

[0203] T.sub.a and T.sub.b are each independently a hydrogen atom, a protecting group of a hydroxyl group, a phosphate group that may be substituted, a phosphorus moiety, a covalent attachment to a support, or the like; and

[0204] q.sub.i, q.sub.j, q.sub.k and q.sub.l are each independently H, halogen, C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.1-C.sub.12 alkoxyl, substituted C.sub.1-C.sub.12 alkoxyl, OJ.sub.j, SJ.sub.j, SOJ.sub.j, SO.sub.2J.sub.j, NJ.sub.jJ.sub.k, N.sub.3, CN, C(═O)OJ.sub.j, C(═O)NJ.sub.jJ.sub.k, C(═O)J.sub.j, O—C(═O)NJ.sub.jJ.sub.k, N(H)C(═NH)NJ.sub.jJ.sub.k, N(H)C(═O)NJ.sub.jJ.sub.k or N(H)C(═S)NJ.sub.jJ.sub.k;

[0205] q.sub.i and q.sub.j or q.sub.l and q.sub.k are both ═C(q.sub.g)(q.sub.h), wherein q.sub.g and q.sub.h are each independently H, halogen, C.sub.1-C.sub.12 alkyl or substituted C.sub.1-C.sub.12 alkyl.

[0206] One carbocyclic bicyclic nucleoside having a 4′-(CH.sub.2).sub.3-2′ crosslink and the alkenyl analog crosslink 4′-CH═CH—CH.sub.2-2′ have been described (Frier et al., Nucleic Acids Research, 1997, 25 (22), 4429-4443, and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740). Synthesis and preparation of carbocyclic bicyclic nucleosides is also described, along with their oligomerization and biochemical studies (Srivastava et al., J. Am. Chem. Soc. 2007, 129 (26), 8362-8379).

[0207] In certain embodiments, examples of nucleosides having a bicyclic sugar include, but are not limited to, compounds such as those shown below.

##STR00006## ##STR00007##

[0208] wherein Bx is a base moiety; and R is independently a protecting group, C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkoxy.

[0209] In certain embodiments (LNA), examples of a nucleoside having a bicyclic sugar include nucleosides represented by the following formula:

##STR00008##

[0210] [wherein

[0211] B is a nucleobase; and

[0212] X and Y are each independently a hydrogen atom, a protecting group of a hydroxyl group, a phosphate group that may be substituted, a phosphorus moiety, a covalent attachment to a support, or the like] (see WO 98/39352]. A typical example is a nucleotide represented by the following formula:

##STR00009##

[0213] In certain embodiments (GuNA), a nucleoside containing a bicyclic is a nucleoside represented by the following general formula:

##STR00010##

[0214] [wherein B is a nucleobase; R.sub.3, R.sub.4, R.sub.5, and R.sub.6 are each independently a hydrogen atom or a C.sub.1-6 alkyl group that may be substituted with one or more substituents;

[0215] R.sub.7 and R.sub.8 are each independently a hydrogen atom, a protecting group of a hydroxyl group, a phosphate group that may be substituted, a phosphorus moiety, a covalent attachment to a support, or the like; and

[0216] R.sub.9, R.sub.10, and R.sub.11 are each independently a hydrogen atom, a C.sub.1-6 alkyl group that may be substituted with one or more substituents, or a protecting group of an amino group] (see, for example, WO 2014/046212, and WO 2017/047816).

[0217] In certain embodiments (ALNA [mU]), a nucleoside containing a bicyclic sugar is a nucleoside represented by the following general formula (I):

##STR00011##

[0218] [wherein

[0219] B is a nucleobase;

[0220] R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are each independently a hydrogen atom or a C.sub.1-6 alkyl group that may be substituted with one or more substituents;

[0221] R.sub.5 and R.sub.6 are each independently a hydrogen atom, a protecting group of a hydroxyl group, a phosphate group that may be substituted, a phosphorus moiety, a covalent attachment to a support, or the like;

[0222] m is 1 or 2; and

[0223] X is a group represented by the following formula (II-1):

##STR00012##

[0224] the symbol

##STR00013##

[0225] described in the formula (II-1) represents a point of attachment to a 2′-amino group; and

[0226] one of R.sub.7 and R.sub.8 is a hydrogen atom and the other is a methyl group that may be substituted with one or more substituents] (see, for example, International Application PCT/JP2019/044182 (WO 2020/100826)). A typical specific example is a nucleoside in which one of R.sub.7 and R.sub.8 is a hydrogen atom and the other is an unsubstituted methyl group.

[0227] In certain embodiments (ALNA[ipU]), a nucleoside containing a bicyclic sugar is a nucleoside having the general formula (I) as defined in ALNA[mU] above, wherein

[0228] X is a group represented by the following formula (II-1):

##STR00014##

[0229] one of R.sub.7 and R.sub.8 is a hydrogen atom and the other is an isopropyl group that may be substituted with one or more substituents (see, for example, International Application PCT/JP2019/044182 (WO 2020/100826)). A typical specific example is a nucleoside in which one of R.sub.7 and R.sub.8 is a hydrogen atom and the other is an unsubstituted isopropyl group.

[0230] In certain embodiments (ALNA[Trz]), a nucleoside containing a bicyclic is a nucleoside having the above general formula (I), wherein X is a group represented by the following formula (II-2):

##STR00015##

[0231] wherein A is a triazolyl group that may be substituted with one or more substituents (see, for example, International Application PCT/JP2019/044182 (WO 2020/100826)). A typical example of ALNA[Trz] is a nucleoside in which A is a triazolyl group that may have one or more methyl groups, more specifically, a 1,5-dimethyl-1,2,4-triazol-3-yl group.

[0232] In certain embodiments (ALNA[Oxz]), it is a nucleoside having the general formula (I) defined in ALNA[mU] above, wherein

[0233] X is a group represented by the following formula (II-2):

##STR00016##

[0234] and A is an oxadiazolyl group that may be substituted with one or more substituents (see, for example, International Application PCT/JP2019/044182 (WO 2020/100826)). A typical specific example is a nucleoside or nucleotide in which A is an oxadiazolyl group that may have one or more methyl groups, more specifically, a 5-methyl-1,2,4-oxadiazol-3-yl group.

[0235] In certain embodiments (ALNA[Ms]), a nucleoside containing a bicyclic is a nucleoside having the above general formula (I), wherein X is a group represented by the following general formula (II-3):

##STR00017##

[0236] and M is a sulfonyl group substituted with a methyl group that may be substituted with one or more substituents (see, for example, International Application PCT/JP2019/044182 (WO 2020/100826)). A typical specific example of ALNA[Ms] is a nucleoside in which M is a sulfonyl group substituted with an unsubstituted methyl group.

[0237] In certain embodiments, a nucleoside is modified by replacement of a ribosyl ring with a sugar surrogate. Examples of such modifications include, but are not limited to, replacements of a ribosyl ring with a substitute ring system (sometimes referred to as a DNA analog), for example, a morpholino ring, a cyclohexenyl ring, a cyclohexyl ring or a tetrahydropyranyl ring, and, for example, those having one of the following formulas:

##STR00018##

[0238] In certain embodiments, a sugar surrogate having the following formula is selected:

##STR00019##

[0239] wherein

[0240] Bx is a heterocyclic base moiety;

[0241] T.sub.3 and T.sub.4 are each independently an internucleoside linking group linking a tetrahydropyran nucleoside analog to an oligomeric compound; or one of T.sub.3 and T.sub.4 is an internucleoside linking group linking a tetrahydropyran nucleoside analog to an oligomeric compound or an oligonucleotide, and the other of T.sub.3 and T.sub.4 is H, a hydroxyl protecting group, a linking conjugate group or a 5′ or 3′-terminal group;

[0242] q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7 are each independently H, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted C.sub.2-C.sub.6 alkynyl;

[0243] one of R.sub.1 and R.sub.2 is hydrogen, and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, OC(═X)J.sub.1, OC(═X)NJ.sub.1J.sub.2, NJ.sub.3C(═X)NJ.sub.1J.sub.2 and CN (wherein X is O, S or NJ.sub.1; and J.sub.1, J.sub.2 and J.sub.3 are each independently H or C.sub.1-C.sub.6 alkyl).

[0244] In certain embodiments, q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6, and q.sub.7 are each H. In certain embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6, and q.sub.7 is other than H. In certain embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6, and q.sub.7 is methyl. In certain embodiments, a THP nucleoside is provided in which one of R.sub.1 and R.sub.2 is F. In certain embodiments, R.sub.1 is fluoro and R.sub.2 is H; R.sub.1 is methoxy and R.sub.2 is H; and R.sub.1 is methoxyethoxy and R.sub.2 is H.

[0245] Examples of such sugar substitutes include, but are not limited to, those known in the art as hexitol nucleic acid (HNA), altitol nucleic acid (ANA) and mannitol nucleic acid (MNA) (see Leumann, C. J., Bioorg. & Med. Chem., 2002, 10, 841-854).

[0246] In certain embodiments, a sugar surrogate includes a ring having more than 5 atoms and more than one heteroatom. For example, a nucleoside containing a morpholino sugar moiety and use thereof in an oligomeric compound have been reported (see, for example, Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506).

[0247] As used herein, the term “morpholino” means a sugar surrogate having the following structure:

##STR00020##

[0248] In certain embodiments, a morpholino can be modified, for example, by adding or changing various substituents from the above morpholino structure. Such a sugar surrogate is referred to herein as a “modified morpholino.”

[0249] In certain embodiments, an oligonucleotide includes one or more modified cyclohexenyl nucleosides, which are nucleosides having a 6-membered cyclohexenyl in place of a pentofuranosyl residue of a naturally occurring nucleoside. Examples of modified cyclohexenyl nucleosides include, but are not limited to, those described in the art (see, for example, according to sharing, WO 2010/036696 published on Apr. 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., Oligonucleotides, 2003, 13 (6), 479-489; Wang et al., J. Org. Chem., 2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001, 29 (24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wang et al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20 (4-7), 785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; WO 06/047842; and WO 01/049687; text of each of these is incorporated herein by reference in its entirety).

[0250] Certain modified cyclohexenyl nucleosides have the following formula:

##STR00021##

[0251] wherein

[0252] Bx is a heterocyclic base moiety;

[0253] T.sub.3 and T.sub.4 are each independently an internucleoside linking group linking a cyclohexenyl nucleoside analog to an oligonucleotide compound; or one of T.sub.3 and T.sub.4 is an internucleoside linking group linking a tetrahydropyran nucleoside analog to an oligonucleotide compound, and the other of T.sub.3 and T.sub.4 is H, a hydroxyl protecting group, a linking conjugate group or a 5′- or 3′-terminal group; and

[0254] q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6, q.sub.7, q.sub.8 and q.sub.9 are each independently H, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, substituted C.sub.2-C.sub.6 alkynyl or other sugar substituent.

[0255] Many other bicyclic and tricyclic sugar substitute ring systems are known in the art that can be used to modify a nucleoside for incorporation into an oligonucleotide (see, for example, a review: Leumann, Christian J., Bioorg. & Med. Chem., 2002, 10, 841-854). Such ring systems can undergo various additional substitutions for activity enhancement.

[0256] A method for preparing a modified sugar is well known to a person skilled in the art. Some representative U.S. patents that teach preparation of such modified sugars include, but are not limited to: U.S. Pat. Nos. 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; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032, as well as WO 2005/121371, and each of these is incorporated herein by reference in its entirety.

[0257] In a nucleotide having a modified sugar moiety, a nucleobase moiety (natural, modified or a combination thereof) is maintained during hybridization with an appropriate nucleic acid target.

[0258] Preferred modified sugars are sugar moieties of ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], or ALNA[Trz], and among these, the sugar moiety of ALNA[Ms] is more preferable. These modified sugars are novel, and a modified oligonucleotide for ATXN3 containing these modified sugars may hybridize to any region of ATXN3.

[0259] That is, in certain embodiments, the modified oligonucleotide of the present embodiment is a modified oligonucleotide having an activity of inhibiting ATXN3 expression and consisting of 12-24 residues, preferably 16, 17 or 18 bases, wherein a nucleobase sequence of the modified oligonucleotide has a complementarity of at least 85%, at least 90%, or at least 95% to an equal length portion of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing, and at least one of nucleosides forming the oligonucleotide has a modified sugar selected from sugar moieties of ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], or ALNA[Trz].

[0260] (Modified Nucleobases)

[0261] Modification or substitution of a nucleobase (or base) is structurally distinguishable from a naturally occurring or synthetic unmodified nucleobase and is further functionally interchangeable with such an unmodified nucleobase. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such a nucleobase modification can impart nuclease stability, binding affinity or some other beneficial biological properties to an oligonucleotide compound. As the modified oligonucleotide of the present invention, a modified oligonucleotide in which at least one nucleoside forming the modified oligonucleotide contains a modified nucleobase is preferably used. An example of the modified nucleobase is a 5-methylcytosine (5-me-C). The 5-methylcytosine means a cytosine modified with a methyl group attached to a 5-position. Certain nucleobase substitutions, including a 5-methylcytosine substitution, are particularly useful for increasing the binding affinity of an oligonucleotide. For example, 5-methylcytosine substitution has been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T., and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

[0262] Additional 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—CH.sub.3) 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, and 3-deazaguanine and 3-deazaadenine.

[0263] Heterocyclic base moieties can also include those in which a purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Examples of nucleobases that are particularly useful for increasing the binding affinity of a modified oligonucleotide include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines (including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine).

[0264] (Modified Internucleoside Linkage)

[0265] A naturally occurring internucleoside linkage of RNA and DNA is a 3′-5′ phosphodiester linkage. An oligonucleotide having one or more modified, that is, non-naturally occurring, internucleoside linkages is often preferred over an oligonucleotide having a naturally occurring internucleoside linkage because of properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid, and increased stability in the presence of nucleases.

[0266] An oligonucleotide having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom and internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, one of more of phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods for preparing phosphorous-containing and non-phosphorous-containing linkages are well known.

[0267] An internucleoside linkage of the modified oligonucleotide of the present invention may be any internucleoside linkage as long as the modified oligonucleotide has an activity of inhibiting ATXN3 expression. However, those in which at least one internucleoside linkage contains a phosphorothioate internucleoside linkage are preferably used. For example, all internucleoside linkages may be phosphorothioate internucleoside linkages.

[0268] (Modified Oligonucleotide Motif)

[0269] In certain embodiments, the modified oligonucleotide of the present invention can have a gapmer motif in order to obtain an increased resistance to degradation by nuclease, an increased cellular uptake, an increased binding affinity for a target nucleic acid, and/or an increased ATXN3 expression inhibitory activity.

[0270] A “gapmer” means a modified oligonucleotide in which an internal region having multiple nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides. An internal region can be referred to as a “gap segment” and an external region can be referred to as a “wing segment.” A wing segment existing on a 5′ side of a gap segment can be referred to as a “5′ wing segment,” and a wing segment existing in a 3′ side of a gap segment can be referred to as a “3′ wing segment.” In a gapmer, sugar moieties of nucleosides of wings that are close to a gap (a nucleoside of a 5′ wing on a most 3′-side and a nucleoside of a 3′ wing on a most 5′-side) are different from sugar moieties of adjacent gap nucleosides. For example, the sugar moieties of the nucleoside of the 5′ wing on the most 3′-side and the nucleoside of the 3′ wing on the most 5′-side are modified sugars, and the sugar moieties of the adjacent gap nucleosides are natural DNA sugar moieties.

[0271] A wing-gap-wing motif can be described as “X-Y-Z,” where “X” represents a sequence length of a 5′ wing region, “Y” represents a sequence length of a gap region, and “Z” represents a sequence length of a 3′ wing region.

[0272] In certain embodiments, the modified oligonucleotide of the present invention includes: (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 nucleosides of the 5′ wing segment and the 3′ wing segment each contain a modified sugar. Sugar moieties of nucleosides of a gap segment may be natural DNA sugar moieties only, or may include one or more modified sugars. The modified sugars are preferably selected from a group consisting of bicyclic sugars, 2′-MOE-modified sugars, and 2′-OMe-modified sugars. Examples of the bicyclic sugars include, for example, at least one sugar moiety of LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz] and ALNA[Trz].

[0273] The number of nucleosides contained in each of a gap segment, a 5′ wing segment, and a 3′ wing segment may be any number as long as it has an ATXN3 expression inhibitory activity. However, for example, it is 12 for the gap segment, 3 for the 5′ wing segment, and 3 for the 3′ wing segment, or it is 10 for the gap segment, 3 for the 5′ wing segment, and 3 for the 3′ wing segment. More preferably, it is 12 for the gap segment, 3 for the 5′ wing segment, and 3 for the 3′ wing segment.

[0274] In certain embodiments, a cap structure is included at one end or each of both ends of a modified oligonucleotide to enhance properties such as nuclease stability. Preferred cap structures include 4′,5′-methylene nucleotide, 1-(β-D-erythrofuranosyl) nucleotide, 4′-thionucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotide, α-nucleotide, modified base nucleotide, phosphorodithioate bond, threo-pentoflanosyl nucleotide, acyclic 3′,4′-seconucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety, 3′-3′-inverted debasement part, 3′-2′-inverted nucleotide moiety, 3′-2′-inverted debasement moiety, 1,4-butanediol phosphate, 3′-phosphoruamidate, hexyl phosphate, aminohexyl phosphate, 3′-phosphate, 3′-phospholothioate, phosphorodithioate, crosslinked methylphosphonate and non-crosslinked methylphosphonate 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 moiety, 5′-5′-inverted debasement moiety, 5′-phosphorumidate, 5′-phosphorothioate, 5′-amino, cross-linked and/or non-cross-linked 5′-phospholumidate, phosphorothioate, and 5′-mercapto moiety.

[0275] (Method for Evaluating the Compound of the Present Invention)

[0276] A method for evaluating the compound of the present invention may be any method as long as the method can very that the compound of the present invention inhibits an intracellular expression level of ATXN3. However, specifically, for example, the in vitro and in vivo ATXN3 expression measurement methods to be described below are used.

[0277] An in vitro ATXN3 expression measurement method for evaluating inhibition of intracellular ATXN3 expression by the compound of the present invention can use any cells in which ATXN3 is expressed (hereinafter, such cells may be referred to as “ATXN3-expressing cells”). However, for example, SH-SY5Y cells (human neuroblastoma cells, for example, ATCC CRL-2266) can be used.

[0278] A method for causing the compound of the present invention to be in contact with ATXN3-expressing cells is also not particularly limited. However, an example thereof is a method that is generally used for introducing a nucleic acid into cells. Specific examples thereof include a lipofection method, an electroporation method, a Gymnosis method, and the like. In the Gymnosis method, the compounds of the present invention can be treated, for example, at a final concentration of 0.3, 1, 3, 10 or 30 μM.

[0279] An intracellular mRNA level of ATXN3 can be assayed using various methods known in the art. Specific examples include Northern blot analysis, competitive polymerase chain reaction (PCR) or quantitative real-time PCR, and the like.

[0280] An intracellular protein level of ATXN3 can be assayed using various methods known in the art. Specific examples thereof include immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assay, protein activity assay (for example, caspase activity assay), immunohistochemistry, immunocytochemistry or fluorescence activated cell sorting (FACS), and the like.

[0281] An example of an in vivo ATXN3 expression measurement method for evaluating inhibition of intracellular ATXN3 expression by the compound of the present invention is a method in which the compound of the present invention is administered to an animal expressing ATXN3 and the above-described ATXN3 expression level analysis is performed with respect to the cells.

[0282] The compound of the present invention can be synthesized using a phosphoramidite method using commercially available amidites (including also LNA) for DNA and RNA synthesis. Artificial nucleic acids ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz] and ALNA[Trz] can synthesize an oligomer using a method described in International Application PCT/JP 2019/044182 (WO 2020/100826) (see a reference example to be described below).

[0283] (Treatment of ATXN3-Related Diseases with the Compound of the Present Invention)

[0284] The compound of the present invention can treat ATXN3-related diseases by inhibiting ATXN3-expression. An ATXN3-related disease is not particularly limited as long as it is a disease caused by an ATXN3 abnormality. However, an example thereof is a neurodegenerative disease.

[0285] Further, the compound of the present invention can ameliorate one or more symptoms of a neurodegenerative disease. Examples of the symptoms include ataxia, neuropathy and aggregate formation. An example of a neurodegenerative disease is spinocerebellar ataxia type 3.

[0286] In spinocerebellar ataxia type 3, occurrence of CAG repeat expansion in ATXN3 causes mitochondrial disorders, transcriptional abnormalities, calcium homeostasis abnormalities, autophagy abnormalities, axonal transport abnormalities and the like due to RNA toxicity or its translation product, polyglutamine, and motor dysfunction occurs following cerebellar Purkinje cell dysfunction and shedding. It is known that motor dysfunction is suppressed by administration of antisense oligonucleotide of ATXN3, which causes inhibition of ATXN3 mRNA expression, to spinocerebellar ataxia type 3 model animals that express CAG repeat expansion of ATXN3. Therefore, the modified oligonucleotide of the present invention that strongly inhibits ATXN3 expression can treat, prevent, or delay progression of spinocerebellar ataxia type 3.

[0287] Therefore, the present invention provides: a modified oligonucleotide for use in treating, preventing or delaying progression of ATXN3-related diseases; a pharmaceutical composition for use in treating, preventing or delaying progression of ATXN3-related diseases; use of a modified oligonucleotide for treating, preventing or delaying progression of ATXN3-related diseases; use of a modified oligonucleotide in producing a medicament for treating, preventing or delaying progression of ATXN3-related diseases; a modified oligonucleotide for use in producing a medicament for treating, preventing or delaying progression of ATXN3-related diseases; and a method for treating, preventing or delaying progression of ATXN3-related diseases, including administering an effective amount of a modified oligonucleotide to a subject in need thereof.

[0288] (2) Pharmacological Composition Containing a Modified Oligonucleotide

[0289] The compound of the present invention, or a pharmaceutically acceptable salt thereof, and the compound of the present invention containing a pharmaceutically acceptable carrier can be used as pharmaceutical compositions. For preparation of a pharmaceutical composition, the modified oligonucleotide can be mixed with one or more pharmaceutically acceptable active or inactive substances. A composition and a method for formulating a pharmaceutical composition can be selected according to several criteria including a route of administration, a severity of a disease, or a dose to be administered.

[0290] For example, as a composition for parenteral administration, for example, an injection is used. Such an injection is prepared according to a method commonly known per se, for example, by dissolving, suspending or emulsifying the modified oligonucleotide in a sterile aqueous or oily solution usually used for injections. As an aqueous solution for injection, for example, phosphate buffered saline, physiological saline, an isotonic solution containing glucose or other adjuvants, and the like are used, and may be used in combination with a suitable solubilizing agent, for example, alcohol (for example, ethanol), polyalcohol (for example, propylene glycol, polyethylene glycol), nonionic surfactant [for example, poly solvate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], and the like. Further, a buffering agent, a pH adjusting agent, an isotonizing agent, a soothing agent, a preservative, a stabilizer, and the like can be contained. Such compositions are produced using commonly known methods.

[0291] Examples of parenteral administration include, but are not limited to, subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, intracranial administration, intrathecal administration, and intracerebroventricular administration. Administration may be continuous or long-term, or may be short-term or intermittent.

[0292] Examples of 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, and the like. Such compositions are produced using methods commonly known and contain carriers, diluents or excipients commonly used in the pharmaceutical field. As carriers and excipients for tablets, for example, lactose, starch, sucrose, magnesium stearate, and the like are used, and as a diluent, for example, physiological saline is used.

[0293] Further, the pharmacological composition of the present invention can include a nucleic acid introduction reagent. As the nucleic acid introduction reagent, liposome, lipofectin, lipofectamine, DOGS (transfectum), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, or cationic lipids such as poly (ethyleneimine) (PEI), or the like can be used.

[0294] Further, the modified oligonucleotide contained in the pharmaceutical composition of the present invention is preferably conjugated with conjugate groups of protein, biotin, phenazine, vitamin, peptide, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin and pigments, which have affinity for in vivo molecules such as fatty acids, cholesterol, carbohydrates, phospholipids, and antibodies, at one or more sites. The conjugated modified oligonucleotide can be produced using a commonly known method, and can be selected to enhance its activity, tissue distribution, cell distribution or cell uptake.

[0295] The conjugate group is directly attached to the modified oligonucleotide, or the conjugate group is attached to the modified oligonucleotide by a linking moiety selected from amino, hydroxy, carboxylic acid, thiol, unsaturated moiety (for example, a double or triple bond), 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidemethyl) cyclohexane-1-carboxylate (SMCC), 6-aminocaproic acid (AHEX or AHA), azide, substituted C.sub.1-C.sub.10 alkyl, substituted or unsubstituted C.sub.2-C.sub.10 alkenyl, and substituted or unsubstituted C.sub.2-C.sub.10 alkynyl. Here, a substituent is selected from amino, alkoxy, carboxy, zazide, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

[0296] An administration form of the pharmaceutical composition of the present invention may be systemic administration such as oral administration, intravenous administration, or intraarterial administration, or may be local administration such as intracranial administration, intrathecal administration, or intracerebroventricular administration, but is not limited to these. Dosage of the pharmaceutical composition of the present invention can be changed as appropriate depending on a purpose of use, severity of the disease, age, body weight, gender, and the like of a patient. However, an amount of the modified oligonucleotide can usually be selected from a range of 0.1 ng-100 mg/kg/day, preferably 1 ng-10 mg/kg/day.

NON-LIMITING DISCLOSURE AND INCORPORATION BY REFERENCE

[0297] While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references described in this application is incorporated herein by reference in its entirety.

Example 1

[0298] Synthesis and Purification of Modified Oligonucleotide Compound for In Vitro Evaluation

[0299] Using ALNA[Ms] amidites (synthesized using a method described in International Application PCT/JP 2019/044182 (WO 2020/100826)), a modified oligonucleotide compound was synthesized using a DNA/RNA oligonucleotide automatic synthesizer nS-8II (manufactured by GeneDesign, Inc.) on a 0.5 μmol scale using a CPG or a polystyrene carrier. All the amidites were prepared in a 0.1 M acetonitrile solution; a coupling time in unnatural nucleosides was 10 minutes; and other steps were performed under standard conditions of nS-8II. Activator 42 (Sigma-Aldrich) was used as an activator, Sulfurizing Reagent II (Gren Research Corporation) was used for thiolation, and Oxidizer (Sigma-Aldrich) was used for oxidation. A synthesized oligonucleotide was excised from the carrier and a base moiety was deprotected by adding a 28% aqueous ammonia solution and causing the mixture to react at 85° C. for 8 hours. After ammonia was concentrated and distilled off, simple purification with NAP10 was performed, and then reverse phase HPLC purification was performed.

[0300] The synthesized modified oligonucleotide compounds are shown in Table 1 (18-residue modified oligonucleotides) and Table 2 (16-residue modified oligonucleotides). In the notation of the compounds, each nucleotide is represented by three letters. However, a 3′-end nucleotide is represented by two letters since there is no internucleoside linkage.

[0301] 1) The first letter is capitalized and indicates the following nucleobases:

[0302] A=adenine, T=thymine, G=guanine, C=cytosine, U=uracil, M=5-methylcytosine;

[0303] 2) The second letter indicates the following sugar moieties:

[0304] m=ALNA[Ms], and d=2′-deoxyribose; and

[0305] 3) The third letter indicates the following internucleoside linkage:

[0306] s=phosphorothioate, p=phosphodiester.

[0307] A target starting position indicates an ATXN3 pre-mRNA 5′ target site of a modified oligonucleotide (a position of SEQ ID NO: 1 in the sequence listing corresponding to a 3′ end of the modified oligonucleotide), and a target ending position indicates an ATXN3 pre-mRNA 3′ target site of a modified oligonucleotide (a position of SEQ ID NO: 1 in the sequence listing corresponding to a 5′ end of the modified oligonucleotide).

Example 2

[0308] Purification and Purity Confirmation of Modified Oligonucleotide Compound

[0309] Purification and purity confirmation of a synthesized modified oligonucleotide compound were performed by reverse phase HPLC under the following conditions.

[0310] Reversed phase HPLC (purification)

[0311] Mobile phase:

[0312] Solution A: 400 mM hexafluoroisopropanol, 15 mM triethylamine

[0313] Solution B: methanol

[0314] Gradient: A:B=83:17.fwdarw.70:30 (10 min) Columns used:

[0315] Preparative isolation Waters XBridge @ Oligonucleotide BEH C18 OBDTM Prep Column, 130 Å, 2.5 μm, 10 mm*50 mm

[0316] Flow rate:

[0317] Preparative isolation 5 mL/min

[0318] Column temperature: 60° C.

[0319] Detection: UV (260 nm)

[0320] Reversed phase HPLC (purity confirmation)

[0321] Mobile phase:

[0322] Solution A: 400 mM hexafluoroisopropanol, 15 mM triethylamine aqueous solution

[0323] Solution B: methanol

[0324] Gradient: A:B=82:18.fwdarw.73.5:26.5 (5 min)

[0325] Columns used:

[0326] Waters XBridge@ Oligonucleotide BEH C18 OBDTM Prep Column, 130 Å, 2.5 μm, 4.6 mm*50 mm

[0327] Flow rate: 1 mL/min

[0328] Column temperature: 60° C.

[0329] Detection: UV (260 nm)

Example 3

[0330] In Vitro ATXN3 Expression Ratio Test (Gymnosis Method)

[0331] At the same time as seeding 3×10.sup.3 SH-SY5Y cells per well, a synthesized modified oligonucleotide was added at a final concentration of 3 or 10 μmol/L and incubated in a CO.sub.2 incubator for 3 days. After RNA was extracted from the cells, a reverse transcription reaction was performed to obtain cDNA. Using the obtained cDNA, quantitative real-time PCR with primers and probes for ATXN3 or GAPDH was performed. An ATXN3 mRNA level was obtained by calculating an amount ratio with respect to GAPDH. An ATXN3 expression ratio was calculated as a percentage by comparing an ATXN3 mRNA level in cells to which the modified oligonucleotide was added to an ATXN3 mRNA level in cells to which the modified oligonucleotide was not added. Tables 1 and 3 show the results of the ATXN3 expression ratios when 18-residue modified oligonucleotides were added at a final concentration of 3 μmol/L, and Table 2 shows the results of the ATXN3 expression ratios when 16-residue modified oligonucleotides were added at a final concentration of 10 μmol/L.

Example 4

[0332] In Vitro ATXN3 Expression Ratio Test (Gymnosis Method)

[0333] 650528, which is a representative modified oligonucleotide described in WO 2018/089805, 1100673, which is a representative modified oligonucleotide described in WO 2019/217708, or 1287095, which is a representative modified oligonucleotide described in WO 2020/172559, was synthesized. Target starting positions and target ending positions of these modified oligonucleotides are as shown in Table 4. Synthesis was performed using 2′-MOE (2′-O-methoxyethyl) amidite in the same way as in Examples 1 and 2. At the same time as seeding 3×10.sup.3 SH-SY5Y cells per well, a synthesized modified oligonucleotide was added at a final concentration of 3 or 10 μmol/L and incubated in a CO.sub.2 incubator for 3 days. After RNA was extracted from the cells, a reverse transcription reaction was performed to obtain cDNA. Using the obtained cDNA, quantitative real-time PCR with primers and probes for ATXN3 or GAPDH was performed. An ATXN3 mRNA level was obtained by calculating an amount ratio with respect to GAPDH. An ATXN3 expression ratio was calculated as a percentage by comparing an ATXN3 mRNA level in cells to which the modified oligonucleotide was added to an ATXN3 mRNA level in cells to which the modified oligonucleotide was not added. The results are shown in Table 4.

TABLE-US-00002 TABLE 1-1 ATXN3 Target Target expression Compound SEQ starting ending ratio ID ID position position Sequence (3 μM)   1   3   256   273 GmsMmsGmsTdsAdsGdsGdsTasGdsAdsTdsGdsMdsTdsMdsMmsMmsMm 80.0   2   4   404   421 AmsGmsGmsAdsAdsTdsMdsMdsTdsMdsMdsGdsAdsAdsAdsAmsGmsMm 75.3   3   5   405   422 AmsAmsGmsGdsAdsAdsTdsMdsMdsTdsMdsMdsGdsAdsAdsAmsAmsGm 86.9   4   6   407   424 TmsGmsAmsAdsGdsGdsAdsAdsTdsMdsMdsTdsMcsMdsGdsAmsAmsAm 90.2   5   7   408   425 GmsTmsGmsAdsAdsGdsGdsAdsAdsTdsMdsMdsTdsMdsMdsGmsAmsAm 74.5   6   8   409   426 GmsGmsTmsGdsAdsAdsGdsGdsAdsAdsTdsMdsMdsTdsMdsMmsGmsAm 84.8   7   9   410   428 GmsGmsGmsTdsGdsAdsAdsGdsGdsAdsAdsTdsMdsMdsTdsMmsMmsGm 82.2   8  10   541   558 GmsAmsMmsTdsMdsMdsAdsAdsTdsAdsTdsAdsGdsAdsAdsTmsTmsGm 76.1   9  11   647   664 GmsTmsMmsAdsGdsGdsMdsMdsMdsTdsAdsMdsAdsGdsTdsGmsGmsGm 73.1  10  12   648   665 TmsGmsTmsMdsAdsGdsGdsMdsMdsMdsTdsAdsMdsAdsGdsTmsGmsGm 79.6  11  13   697   714 MmsGmsAmsMdsTdsTdsMdsMdsMdsTdsMdsTdsTdsTdsAdsGmsGmsGm 79.2  12  14   698   715 AmsMmsGmsAdsMdsTdsTdsMdsMdsMdsTdsGdsTdsTdsTdsAmsGmsGm 68.8  13  15   699   716 GmsAmsMmsGdsAdsMdsTdsTdsMdsMdsMdsTdsGdsTdsTdsTmsAmsGm 64.4  14  16   767   784 GmsMmsMmsTdsGdsMdsTdsMdsMdsGdsGdsAdsAdsAdsGdsAmsTmsGm 78.3  15  17  2043  2060 AmsGmsTmsAdsGdsTdsGdsTdsGdsGdsAdsAdsMdsAdsTdsGmsTmsTm 65.7  16  18  2045  2062 GmsAmsAmsGdsTdsAdsGdsTdsGdsTdsGdsGdsAdsAdsMdsAmsTmsGm 75.4  17  19  2046  2063 GmsGmsAmsAdsGdsTdsAdsGdsTdsGdsTdsGdsGdsAdsAdsMmsAmsTm 67.5  18  20  2047  2064 AmsGmsGmsAdsAdsGdsTdsAdsGdsTdsGdsTdsGdsGdsAdsAmsMmsAm 69.0  19  21  2048  2065 MmsAmsGmsGdsAdsAdsGdsTdsAdsGdsTdsGdsTdsGdsGdsAmsAmsMm 71.2  20  22  2092  2109 GmsTmsMmsMdsMdsAdsAdsMdsTdsAdsGdsGdsTdsAdsAdsMmsAmsAm 68.8  21  23  2159  2176 TmsMmsGmsGdsGdsTdsAdsAdsGdsTdsAdsGdsAdsTdsTdsTmsTmsMm 51.2  22  24  2502  2519 AmsGdsGmsMdsMdsTdsAdsTdsAdsMdsAdsMdsAdsTdsGdsMmsTmsGm 79.9  23  25  2513  2530 GmsAmsAmsGdsTdsAdsTdsMdsTdsGdsTdsAdsGdsGdsMdsMmsTmsAm 57.9  24  26  2646  2663 GmsGmsAmsMdsTdsGdsTdsAdsTdsAdsGdsGdsAdsGdsAdsTmsTmsAm 55.0  25  27  2650  2667 GmsAmsAmsTdsGdsGdsAdsMdsTdsGdsTdsAdsTdsAdsGdsGmsAmsGm 63.2  26  28  4011  4028 MmsMmsTmsMdsTdsTdsAdsGdsTdsGdsMdsTdsMdsTdsMdsMmsGmsAm 75.2  27  29  4012  4029 TmsMmsMmsTdsMdsTdsTdsAdsGdsTdsGdsMdsTdsMdsTdsMmsMmsGm 73.3  28  30  4041  4058 AmsAmsGmsMdsTdsGdsTdsMdsAdsAdsTdsMdsTdsAdsGdsMmsAmsGm 65.2  29  31  4046  4063 GmsGmsGmsAdsMdsAdsAdsGdsMdsTdsGdsTdsMdsAdsAdsTmsMmsTm 80.1  30  32  4267  4284 TmsAmsGmsGdsMdsMdsMdsAdsGdsAdsMdsTdsTdsMdsAdsTmsTmsGm 77.5  31  33  4268  4285 TmsTmsAmsGdsGdsMdsMdsMdsAdsGdsAdsMdsTdsTdsMdsAmsTmsTm 76.0  32  34  4269  4286 TmsTmsTdsAdsGdsGdsMdsMdsMdsAdsGdsAdsMdsTdsTdsMmsAmsTm 72.0  33  35  5179  5196 MmsAmsAmsTdsGdsAdsMdsAdsAdsTdsGdsAdsGdsMdsMdsMmsAmsMm 78.7  34  36  4863  5860 MmsTmsTmsAdsTdsAdsGdsGdsAdsTdsGdsMdsAdsGdsGdsTmsAmsAm 63.9  35  37  5844  5861 GmsGmsTmsTdsAdsTdsAdsGdsGdsAdsTdsgddsMdsAdsGdsGmsmsAm 54.2  36  38  5845  5862 AmsGmsGmsTdsTdsAdsTdsAdsGdsGdsAdsTdsGdsMdsAdsGmsGmsTm 45.8  37  39  5853  5870 AmsTmsAmsAdsTdsGdsGdsGdsAdsGdsGdsTdsTdsAdsTdsAmsGmsGm 70.3  38  40  7203  7220 GmsAmsAmsTdsAdsAdsGdsTdsAdsGdsMdsTdsTdsGdsGdsAmsGmsMm 75.9  39  41  7208  7225 GmsTmsAmsAdsGdsGdsAdsAdsTdsAdsAdsGdsTdsAdsGdsAmsTmsTm 75.2  40  42  7535  7552 GmsAmsMmsAdsMdsMdsAdsGdsGdsTdsTdsTdsGdsMdsMdsMmsMmsTm 70.9  41  43  7536  7553 AmsGmsAmsMdsAdsMdsMdsAdsGdsGdsTdsTdsTdsGdsMdsMmsMmsMm 73.3  42  44  7741  7758 AmsTmsGmsGdsGdsAdsGdsGdsTdsAdsGdsGdsTdsAdsMdsAmsAmsMm 72.6  43  45  7788  7805 GmsGmsAmsMdsTdsGdsMdsAdsMdsMdsMdsAdsAdsGdsMdsTmsMmsAm 72.2  44  46  7792  7809 AmsGmsAmsGdsGdsGdsAdsMdsTdsGdsMdsAdsMdsMdsMdsAmsAmsGm 68.4  45  47  7793  7810 AmsAmsGmsAdsGdsGdsGdsAdsMdsTdsGdsMdsAdsMdsMdsMmsAmsAm 70.3  46  48 10997 11014 MmsMmsMmsMdsAdsMdsTdsMdsTdsAdsAdsTdsAdsTdsGdsAmsAmsGm 70.4  47  49 11642 11659 GmsTmsMmsGdsMdsTdsMdsAdsGdsGdsAdsGdsAdsGdsGdsTmsTmsMm 62.2  48  50 11643 11650 GmsGmsTmsMdsGdsMdsTdsMdsAdsGdsGdsAdsGdsAdsGdsGmsTmsTm 68.3  49  51 11644 11651 AmsGmsGmsTdsMdsGdsMdsTdsMdsAdsGdsGdsAdsGdsAdsGmsGmsTm 63.0  50  52 11645 11662 TmsAmsGmsGdsTdsMdsGdsMdsTdsMdsAdsGdsGdsAdsGdsAmsGmsGm 66.3  51  53 11646 11663 AmsTmsAmsGdsGdsTdsMdsGdsMdsTdsMdsAdsGdsGdsAdsGmsAmsGm 74.9  52  54 11651 11668 MmsAmsGmsAdsGdsAdsTdsAdsGdsGdsTdsMdsGdsMdsTdsMmsAmsGm 72.9  53  55 11658 11672 TmsMmsTmsGdsMdsAdsGdsAdsGdsAdsTdsAdsGdsGdsTdsMmsGmsMm 62.2  54  56 11734 11751 MmsGmsGmsTdsAdsAdsAdsGdsMdsAdsMdsTdsAdsGdsAdsTmsGmsGm 62.9  55  57 11735 11752 AmsMmsGmsGdsTdsAdsAdsAdsGdsMdsAdsMdsTdsAdsGdsAmsTmsGm 58.8  56  58 11741 11758 AmsGmsTmsGdsGdsMdsAdsMdsGdsGdsTdsAdsAdsAdsGdsMmsAmsMm 73.4  57  59 11799 11816 AmsGmsTmsGdsMdsTdsGdsGdsGdsMdsMdsTdsAdsMdsTdsMmsTmsGm 81.2  58  60 11800 11817 AmsAmsGmsTdsGdsMdsTdsGdsGdsGdsMdsMdsTdsAdsMdsTmsMmsTm 86.4  59  61 12710 12727 GmsMmsTmsAdsTdsGdsTdsTdsAdsGdsTdsTdsGdsTdsGdsTmsAmsMm 59.0  60  62 13128 13153 AmsAmsMmsGdsTdsMdsTdsMdsTdsGdsMdsAdsGdsMdsAdsGmsMmsTm 87.0  61  63 15115 15132 GmsAmsAmsGdsMdsTdsAdsAdsGdsTdsAdsGdsGdsTdsGdsAmsMmsTm 48.7  62  64 15116 15133 TmsGmsAmsAdsGdsMdsTdsAdsAdsGdsTdsAdsGdsGdsTdsGmsAmsMm 58.9  63  65 15703 15720 AmsGmsAmsGdsGdsTdsGdsAdsTdsTdsMdsMdsMdsAdsMdsTmsMmsGm 78.7  64  66 15704 15721 TmsAmsGmsAdsGdsGdsTdsGdsAdsTdsTdsMdsMdsMdsAdsMmsTmsMm 56.8  65  67 15705 15722 MmsTmsAmsGdsAdsGdsGdsTdsGdsAdsTdsTdsMdsMdsMdsAmsMmsTm 75.9  66  68 15706 15723 MmsMmsTmsAdsGdsAdsGdsGdsTdsGdsAdsTdsTdsMdsMdsMmsAmsMm 67.4  67  69 18749 18766 GmsGmsTmsAdsAdsTdsAdsGdsMdsTdsAdsAdsGdsMdsAdsAmsGmsAm 86.4  68  70 19061 19078 GmsMmsTmsAdsMdsTdsMdsTdsGdsGdsTdsAdsMdsAdsGdsGmsAmsAm 71.4  69  71 19163 19180 MmsMmsTmsAdsGdsTdsMdsAdsMdsTdsTdsTdsGdsAdsTdsAmsGmsAm 64.3  70  72 19355 19372 GmsMmsAmsMdsTdsAdsAdsAdsGdsGdsMdsTdsMdsAdsTdsMmsTmsGm 90.2  71  73 19738 19753 GmsAmsAmsMdsAdsTdsMdsTdsTdsGdsAdsGdsTdsAdsGdsGmsTmsGm 95.8  72  74 19737 19754 GmsGmsAmsAdsMdsAdsTdsMdsTdsTdsGdsAdsGdsTdsAdsGmsGmsTm 63.1  73  75 19738 19755 AmsGmsGmsAdsAdsMdsAdsTdsMdsTdsTdsGdsAdsGdsTdsAmsGmsGm 76.5  74  76 19739 19756 MmsAmsGmsGdsAdsAdsMdsAdsTdsMdsTdsTdsGdsAdsGdsTmsAmsGm 83.0  75  77 20833 20850 TmsGmsTmsTdsMdsAdsGdsGdsGdsTdsAdsGdsAdsTdsGdsTmsMmsAm 79.8  76  78 20835 20852 GmsGmsTmsGdsTdsTdsMdsAdsGdsGdsGdsTdsAdsGdsAdsTmsGmsTm 58.5  77  79 20836 20853 AmsGmsGmsTdsGdsTdsTdsMdsAdsGdsGdsGdsTdsAdsGdsAmsTmsGm 80.6  78  80 21059 21078 GmsGmsGmsTdsGdsGdsGdsAdsMdsAdsTdsAdsAdsMdsAdsGmsGmsMm 91.8  79  81 21060 21077 AmsGmsGmsGdsTdsGdsGdsGdsAdsMdsAdsTdsAdsAdsMdsAmsGmsGm 97.5  80  82 21482 21499 GmsGmsAmsTdsAdsMdsTdsMdsTdsGdsMdsMdsMdsTdsGdsTmsTmsMm 54.4  81  83 21483 21500 TmsGmsGmsAdsTdsAdsMdsTdsMdsTdsGdsMdsMdsMdsTdsGmsTmsGm 65.1  82  84 22109 22126 AmsAmsGmsMdsTdsAdsGdsAdsTdsGdsMdsTdsAdsMdsAdsMmsAmsMm 92.0  83  85 22195 22212 MmsAmsAmsAdsMdsGdsTdsGdsTdsGdsGdsTdsTdsTdsGdsMmsAmsTm 70.1  84  86 22198 22213 TmsMmsAmsAdsAdsMdsGdsTdsGdsTdsGdsGdsTdsTdsTdsGmsMmsAm 95.4  85  87 22197 22214 GmsTmsMmsAdsAdsAdsMdsGdsTdsGdsTdsGdsGdsTdsTdsTmsGmsMm 82.2  86  88 22196 22215 TmsGmsTmsMdsAdsAdsAdsMdsGdsTdsGdsTdsGdsGdsTdsTmsTmsGm 85.4  87  89 22200 22217 GmsGmsTmsGdsTdsMdsAdsAdsAdsMdsGdsTdsGdsTdsGdsGmsTmsTm 55.3  88  90 22913 22930 GmsTmsAmsMdsMdsTdsTdsMdsMdsTdsGdsTdsGdsAdsTdsGmsGmsTm 70.1  89  91 23048 23063 MmsAmsGmsGdsMdsTdsAdsMdsTdsMdsAdsMdsTdsTdsAdsMmsTmsGm 76.0  90  92 23047 23064 GmsMmsAmsGdsGdsMdsTdsAdsMdsTdsMdsAdsMdsTdsTdsAmsMmsTm 90.4  91  93 23048 23065 AmsGmsMmsAdsGdsGdsMdsTdsAdsMdsTdsMdsAdsMdsTdsTmsAmsMm 70.9  92  94 23049 23065 GmsAmsGmsMdsAdsGdsGdsMdsTdsAdsMdsTdsMdsAdsMdsTmsTmsAm 88.9  93  95 23050 23067 TmsGmsAmsGdsMdsAdsGdsGdsMdsTdsAdsMdsTdsMdsAdsMmsTmsTm 84.8  94  96 23055 23072 TmsTmsGmsAdsMdsTdsGdsAdsGdsMdsAdsGdsGdsMdsTdsAmsMmsTm 70.5  95  97 23058 23073 MmsTmsTmsGdsAdsMdsTdsGdsAdsGdsMdsAdsGdsGdsMdsTmsAmsMm 85.6  96  98 23057 23074 AmsMmsTmsTdsGdsAdsMdsTdsGdsAdsGdsMdsAdsGdsGdsMmsTmsAm 74.4  97  99 24651 24868 MmsAmsGmsGdsMdsMdsMdsTdsTdsMdsMdsAdsTdsAdsGdsTmsGmsTm 72.4  98 100 24652 24869 MmsMmsAmsGdsGdsMdsMdsMdsTdsTdsMdsMdsAdsTdsAdsGmsTmsGm 80.1  99 101 24653 24870 TmsMmsMmsAdsGdsGdsMdsMdsMdsTdsTdsMdsMdsAdsTdsAmsGmsTm 77.4 100 102 27368 27405 MmsGmsTmsGdsTdsGdsMdsTdsAdsGdsTdsAdsTdsTdsTdsGmsTmsTm 56.2 101 103 27359 27406 MmsMmsGmsTdsGdsTdsGdsMdsTdsAdsGdsTdsAdsTdsTdsTmsGmsTm 80.0 102 104 27391 27408 GmsGmsMmsMdsGdsTdsGdsTdsGdsMdsTdsAdsGdsTdsAdsTmsTmsTm 83.6 103 105 27392 27409 TmsGmsGmsMdsMdsGdsTdsGdsTdsGdsMdsTdsAdsGdsTdsAmsTmsTm 67.8 104 106 27393 27410 TmsTmsGmsGdsMdsMdsGdsTdsGdsTdsGdsMdsTdsAdsGdsTmsAmsTm 61.9 105 107 27394 27411 AmsTmsTmsGdsGdsMdsMdsGdsTdsGdsTdsGdsMdsTdsAdsGmsTmsAm 66.8 106 108 27395 27412 GmsAmsTmsTdsGdsGdsMdsMdsGdsTdsGdsTdsGdsMdsTdsAmsGmsTm 83.2 107 109 30559 30576 AmsAmsGmsGdsTdsTdsGdsMdsMdsTdsGdsAdsMdsMdsMdsTmsGmsTm 89.8 108 110 30560 30577 MmsAmsAmsGdsGdsTdsTdsGdsMdsMdsTdsGdsAdsMdsMdsMmsTmsGm 84.5 109 111 30561 30578 AmsMmsAmsAdsGdsGdsTdsTdsGdsMdsMdsTdsGdsAdsMdsMmsMmsTm 75.1 110 112 30564 30581 GmsGmsTmsAdsMdsAdsAdsGdsGdsTdsTdsGdsMdsMdsTdsGmsAmsMm 64.3 111 113 30565 30582 GmsGmsGmsTdsAdsMdsAdsAdsGdsGdsTdsTdsGdsMdsMdsTmsGmsAm 73.8 112 114 31570 31587 TmsAmsGmsTdsAdsGdsAdsGdsTdsTdsTdsTdsGdsMdsTdsTmsGmsGm 54.3 113 115 31574 31592 GmsAmsTmsGdsTdsAdsGdsTdsAdsGdsAdsGdsTdsTdsTdsTmsGmsMm 57.3 114 116 31575 31592 TmsGmsAmsTdsGdsTdsAdsGdsTdsAdsGdsAdsGdsTdsTdsTmsTmsGm 49.3 115 117 31576 31593 MmsTmsGmsAdsTdsGdsTdsAdsGdsTdsAdsGdsAdsGdsTdsTmsTmsTm 44.1 116 118 31841 31858 GmsTmsGmsGdsTdsGdsGdsMdsTdsMdsAdsAdsGdsTdsAdsTmsTmsAm 58.2 117 119 32007 32024 MmsAmsAmsGdsTdsTdsGdsGdsTdsTdsTdsGdsTdsGdsGdsTmsAmsMm 65.1 118 120 32008 32025 GmsMmsAmsAdsGdsTdsTdsGdsGdsTdsTdsTdsGdsTdsGdsGmsTmsAm 56.4 119 121 32009 32026 TmsGmsMmsAdsAdsGdsTdsTdsGdsGdsTdsTdsTdsGdsTdsGmsGmsTm 56.1 120 122 32010 32027 MmsTmsGmsMdsAdsAdsGdsTdsTdsGdsGdsTdsTdsTdsGdsTmsGmsGm 72.1 121 123 32011 32028 MmsMmsTmsGdsMdsAdsAdsGdsTdsTdsGdsGdsTdsTdsTdsGmsTmsGm 62.0 122 124 32012 32029 TmsMmsMmsTdsGdsMdsAdsAdsGdsTdsTdsGdsGdsTdsTdstsmGmsTm 76.6 123 125 32127 32144 TmsMmsTmsAdsGdsGdsMdsAdsAdsTdsTdsGdsTdsGdsGdsTmsGmsGm 60.5 124 126 33630 33647 GmsMmsAmsTdsGdsGdsMdsTdsAdsAdsTdsGdsAdsTdsGdsMmsAmsMm 83.5 125 127 35711 35728 TmsGmsTmsGdsAdsAdsGdsGdsTdsAdsGdsMdsGdsAdsAdsMmsAmsTm 65.3 126 128 35712 35729 GmsTmsGmsTdsGdsAdsAdsGdsGdsTdsAdsGdsMdsGdsAdsAmsMmsAm 68.9 127 129 36411 36428 GmsTmsAmsAdsMdsTdsMdsTdsGdsMdsAdsMdsTdsTdsMdsMmsMmsAm 64.9 128 130 36413 36430 AmsAmsGmsTdsAdsAdsMdsTdsMdsTdsGdsMdsAdsMdsTdsTmsMmsMm 74.7 129 131 36607 36624 GmsTmsMmsAdsTdsMdsMdsMdsTdsAdsTdsGdsTdsMdsTdsTmsAmsTm 47.4 130 132 38559 38576 GmsMmsAmsAdsMdsAdsGdsTdsMdsTdsGdsGdsAdsTdsTdsMmsTmsMm 52.8 131 133 38557 38604 GmsAmsGmsGdsTdsGdsTdsMdsMdsTdsTdsGdsTdsTdsAdsMmsTmsGm 65.6 132 134 38588 38605 GmsGmsAmsGdsGdsTdsGdsTmsMdsMdsTdsTdsGdsTdsTdsAmsMmsTm 58.0 133 135 38589 28605 TmsGmsGmsAdsGdsGdsTdsGdsTdsMdsMdsTdsTdsGdsTdsTmsAmsMm 61.6 134 136 38590 28607 GmsTmsGmsGdsAdsGdsGdsTdsGdsTdsMdsMdsTdsTdsGdsTmsTmsAm 63.6 135 137 38593 38610 AmsTmsTmsGdsTdsGdsGdsAdsGdsGdsTdsGdsTdsMdsMdsTmsTmsGm 62.1 136 138 38594 28611 MmsAmsTmsTdsGdsTdsGdsGdsAdsGdsGdsTdsGdsTdsMdsMmsTmsTm 64.2 137 139 38596 38613 GmsAmsMmsAdsTdsTdsGdsTdsGdsGdsAdsGdsGdsTdsGdsTmsMmsMm 82.5 138 140 38597 38614 GmsGmsAmsMdsAdsTdsTdsGdsTdsGdsGdsAdsGdsGdsTdsGmsTmsMm 86.2 139 141 38598 38615 MmsGmsGmsAdsMdsAdsTdsTdsGdsTdsGdsGdsAdsGdsGdsTmsGmsTm 85.8 140 142 38599 38616 AmsMmsGmsGdsAdsMdsAdsTdsTdsGdsTdsGdsGdsAdsGdsGmsTmsGm 93.2 141 143 38600 38617 MmsAmsMmsGdsGdsAdsMdsAdsTdsTdsGdsTdsGdsGdsAdsGmsGmsTm 87.0 142 144 38601 38618 MmsMmsAmsMdsGdsGdsAdsMdsAdsTdsTdsGdsTdsGdsGdsAmsGmsGm 73.1 143 145 38604 38621 MmsMmsTmsMdsMdsAdsMdsGdsGdsAdsMdsAdsTdsTdsGdsTmsGmsGm 61.6 144 146 38606 38623 GmsGmsMmsMdsTdsMdsMdsAdsMdsGdsGdsAdsMdsAdsTdsTmsGmsTm 72.0 145 147 38607 38624 TmsGmsGmsMdsMdsTdsMdsMdsAdsMdsGdsGdsAdsMdsAdsTmsTmsGm 68.9 146 148 40453 40470 GmsTmsMmsAdsTdsAdsTdsGdsGdsTdsMdsAdsGdsGdsGdsTmsAmsTm 21.6 147 149 40454 40471 TmsGmsTmsMdsAdsTdsAdsTdsGdsGdsTdsMdsAdsGdsGdsGmsTmsAm 51.7 148 150 40455 40472 AmsTmsGmsTdsMdsAdsTdsAdsTdsGdsGdsTdsMdsAdsGdsGmsGmsTm 46.9 149 151 40456 40473 TmsAmsTmsGdsTdsMdsAdsTdsAdsTdsGdsGdsTdsMdsAdsGmsGmsGm 51.3 150 152 40475 40492 TmsGmsMmsMdsMdsAdsGdsGdsAdsGdsTdsAdsTdsAdsGdsAmsTmsGm 69.5 151 153 40539 40556 TmsAmsMmsAdsGdsMdsAdsMdsTdsTdsAdsMdsGdsGdsAdsGmsTmsGm 71.3 152 154 40540 40557 TmsTmsAmsMdsAdsGdsMdsAdsMdsTdsTdsAdsMdsGdsGdsAmsGmsTm 82.7 153 155 41281 41298 GmsMmsTmsAdsTdsGdsGdsAdsTdsGdsTdsAdsMdsTdsAdsMmsAmsGm 56.9 154 156 41392 41409 AmsAmsGmsAdsMdsMdsMdsTdsTdsTdsGdsAdsGdsGdsMdsTmsTmsTm 63.6 155 157 41393 41410 TmsAmsAmsGdsAdsMdsMdsMdsTdsTdsTdsGdsAdsGdsGdsMmsTmsTm 58.3 156 158 45892 45909 GmsTmsAmsGdsMdsTdsMdsTdsTdsGdsGdsMdsAdsMdsTdsAmsGmsMm 57.0

TABLE-US-00003 TABLE 2-1 ATKN3 Target Target expression Compound SEQ starting ending ratio ID ID position position Sequence (10 μM) 157 159  1512  1527 TmsMmsMmsGdsAdsGdsAdsTdsGdsTdsTdsTdsTdsMmsTmsAm 47.8 158 160  1521  1536 TmsMmsAmsAdsAdsAdsGdsTdsTdsTdsCdsCdsGdsAmsGmsAm 56.4 159 161  1568  1583 MmsAmsAmsCdsTdsAdsTdsAdsCdsGdsAdsCdsTdsGmsAmsGm 74.6 160 162  2051  2056 TmsMmsAmsGdsGdsAdsAdsGdsTdsAdsGdsTdsGdsTmsGmsGm 62.1 161 163  2162  2177 TmsTmsMmsGdsGdsGdsTdsAdsAdsGdsTdsAdsGdsAmsTmsTm 49.4 162 164  3931  3946 GmsMmsAmsCdsTdsAdsTdsCdsTdsGdsCdsCdsAdsGmsGmsGm 63.6 163 165  4038  4053 GmsTmsMmsAdsAdsTdsCdsTdsAdsGdsCdsAdsGdsMmsAmsTm 65.3 164 166  4259  4274 MmsTmsTmsCdsAdsTdsTdsGdsTdsGdsTdsTdsGdsGmsGmsMm 83.8 165 167  4277  4292 TmsAmsTmsGdsAdsTdsTdsTdsTdsAdsGdsGdsCdsMmsMmsAm 86.9 166 168  5668  8653 TmsMmsAmsGdsTdsTdsTdsGdsGdsAdsGdsTdsTdsTmsAmamm 34.2 167 169  5762  5777 GmsAmsTmsTdsTdsGdsTdsGdsTdsGdsCdsTdsCdsMmsTmsMm 35.7 168 170  7602  7617 GmsMmsTmsTdsTdsCdsCdsCdsAdsTdsGdsCdsTdsAmsMmsMm 81.8 169 171  7817  7832 GmsMmsTmsCdsAdsCdsTdsCdsCdsTdsGdsGdsAdsMmsAmsAm 85.0 170 172  9928  9940 AmsMmsGmsTdsGdsCdsGdsAdsTdsAdsAdsTdsCdsTmsTmsMm 85.1 171 173 10273 10288 AmsGmsAmsAdsAdsTdsGdsTdsAdsAdsGdsCdsCdsGmsGmsGm 68.3 172 174 10489 10424 GmsTmsMmsAdsTdsTdsAdsGdsCdsGdsTdsGdsCdsAmsTmsAm 80.1 173 175 10535 10571 TmsGmsGmsGdsAdsTdsGdsTdsAdsAdsGdsCdsAdsAmsMmsAm 85.5 174 176 10584 10599 GmsAmsMmsTdsAdsTdsTdsGdsAdsAdsAdsGdsGdsMmsTmsGm 56.6 175 177 11002 11017 AmsTmsGmsCdsCdsCdsCdsAdsCdsTdsCdsTdsAdsAmsTmsAm 52.9 176 178 11617 11632 TmsMmsTmsGdsGdsAdsTdsAdsCdsAdsAdsCdsTdsMmsAmsMm 64.2 177 179 11650 11665 AmsGmsAmsTdsAdsGdsGdsTdsCdsGdsCdsTdsCdsAmsGmsGm 29.3 178 180 11654 11669 GmsMmsAmsGdsAdsGdsAdsTdsAdsGdsGdsTdsCdsGmsMmsTm 64.9 179 181 11738 11753 MmsAmsMmsGdsGdsTdsAdsAdsAdsGdsCdsAdsCdsTmsAmsGm 57.9 180 182 11744 11759 MmsAmsGmsTdsGdsGdsCdsAdsCdsGdsGdsTdsAdsAmsAmsGm 55.8 181 183 11810 11825 GmsAmsAmsAdsCdsGdsCdsCdsAdsAdsGdsTdsGdsMmsTmsGm 80.0 182 184 12026 12091 TmsAmsAmsGdsTdsTdsCdsTdsCdsCdsCdsTdsGdsGmsGmsMm 26.2 183 185 13141 13156 AmsTmsGmsAdsAdsCdsGdsTdsCdsTdsCdsTdsGdsMmsAmsGm 67.8 184 186 14074 14089 TmsTmsGmsAdsCdsAdsCdsTdsAdsTdsTdsTdsCdsAmsGmsGm 40.8 185 187 14213 14228 AmsTmsAmsGdsCdsTdsTdsGdsCdsTdsCdsAdsAdsGmsAmsMm 30.4 186 188 14325 14340 GmsMmsTmsGdsCdsAdsTdsGdsTdsAdsTdsCdsAdsMmsTmsAm 58.2 187 189 15114 15129 GmsMmsTmsAdsAdsGdsTdsAdsGdsGdsTdsGdsAdsMmsTmsTm 57.9 188 190 15124 15139 TmsTmsAmsGdsGdsAdsTdsGdsAdsAdsGdsCdsTdsAmsAmsGm 72.5 189 191 16211 16228 MmsTmsGmsCdsAdsCdsTdsCdsGdsGdsCdsCdsCdsTmsAmsAm 57.0 190 192 16740 16758 AmsGmsAmsTdsTdsCdsCdsAdsAdsTdsTdsGdsTdsGmsAmsGm 29.4 191 193 18015 18030 AmsGmsMmsCdsTdsAdsTdsCdsAdsCdsCdsAdsCdsGmsTmsMm 42.5 192 194 18033 18048 MmsAmsGmsCdsGdsTdsCdsAdsCdsCdsCdsAdsAdsAmsTmsMm 30.2 193 195 18108 18123 TmsAmsMmsTdsTdsAdsGdsAdsAdsGdsGdsGdsCdsTmsGmsMm 79.8 194 196 19166 19181 GmsMmsMmsTdsAdsGdsTdsCdsAdsCdsTdsTdsTdsGmsAmsTm 56.7 195 197 20832 20847 TmsMmsAmsGdsGdsGdsTdsAdsGdsAdsTdsGdsTdsMmsAmsAm 38.1 196 198 20839 20854 TmsAmsGmsGdsTdsGdsTdsTdsCdsAdsGdsGdsGdsTmsAmsGm 37.0 197 199 20848 20863 AmsGmsTmsCdsAdsAdsAdsGdsCdsTdsAdsGdsGdsTmsGmsTm 48.2 198 200 21487 21502 AmsAmaTmsGdsGdsAdsTdsAdsCdsTdsCdsTdsGdsMmsMmsMm 55.6 199 201 22116 22131 TmsMmsMmsCdsCdsAdsAdsGdsCdsTdsAdsGdsAdsTmsGmsMm 66.0 200 202 22194 22209 AmsMmsGmsTdsGdsTdsGdsGdsTdsTdsTdsGdsCdsAmsTmsMm 39.3 201 203 22199 22214 GmsTmsMmsAdsAdsAdsCdsGdsTdsGdsTdsGdsGdsTmsTmsTm 22.6 202 204 22203 22218 AmsGmsGmsTdsGdsTdsCdsAdsAdsAdsCdsGdsTdsGmsTmsGm 22.1 203 205 22210 22225 MmsTmsAmsTdsTdsTdsAdsAdsGdsGdsTdsGdsTdsMmsAmsAm 70.6 204 206 22998 23013 AmsTmsMmsAdsGdsAdsCdsTdsGdsCdsCdsTdsGdsMmsAmsTm 66.8 205 207 26470 26485 GmsMmsAmsCdsTdsTdsAdsCdsAdsAdsGdsGdsCdsTmsGmsGm 59.3 206 208 27062 27077 GmsGmsTmsGdsCdsTdsGdsCdsTdsAdsAdsTdsAdsTmsAmsMm 27.6 207 209 27271 27285 GmsMmsAmsCdsTdsAdsAdsTdsAdsAdsGdsCdsAdsMmsTmsAm 48.9 208 210 29393 29408 TmsMmsAmsTdsGdsCdsAdsGdsCdsCdsTdsGdsTdsTmsAmsMm 42.4 209 211 30535 30550 AmsAmsGmsCdsAdsCdsTdsAdsGdsTdsTdsAdsTdsTmsGmsMm 62.8 210 212 30556 30571 TmsGmsMmsCdsTdsGdsAdsCdsCdsCdsTdsGdsTdsTmsTmsAm 45.4 211 213 30930 30945 AmsGmsMmsTdsTdsGdsAdsTdsAdsAdsCdsCdsTdsTmsAmsGm 50.0 212 214 31576 31591 GmsAmsTmsGdsTdsAdsGdsTdsAdsGdsAdsGdsTdsTmsTmsTm 13.7 213 215 31580 31595 AmsGmsMmsTdsGdsAdsTdsGdsTdsAdsGdsTdsAdsGmsAmsGm 51.7 214 216 32006 32021 GmsTmsTmsGdsGdsTdsTdsTdsGdsTdsGdsGdsTdsAmsMmsTm 17.1 215 217 33237 33252 GmsTmsAmsGdsGdsAdsTdsTdsCdsTdsGdsGdsTdsTmsAmsMm 43.9 216 218 34900 34915 AmsTmsAmsAdsGdsTdsAdsCdsCdsTdsTdsTdsGdsAmsTmsMm 57.7 217 219 35710 35725 GmsAmsAmsGdsGdsTdsAdsGdsCdsGdsAdsAdsCdsAmstsmgm 41.7 218 220 35841 35856 GmsGmsTmsAdsAdsCdsTdsGdsCdsTdsCdsCdsTdsTmsAmsAm 21.2 219 221 36413 36428 GmsTmsAmsAdsCdsTdsCdsTdsGdsCdsAdsCdsTdsTmsMmsMm 36.7 220 222 36417 36432 GmsAmsAmsAdsGdsTdsAdsAdsCdsTdsCdsTdsGdsMmsAmsMm 49.2 221 223 36426 36441 MmsGmsAmsGdsAdsCdsCdsTdsGdsGdsadaAdsAdsGmsTmsAm 51.8 222 224 36606 36624 GmsTmsMmsAdsTdsCdsCdsCdsTdsAdsTdsGdsTdsMmsTmsTm 18.7 223 225 37690 37705 GmsTmsGmsTdsTdsTdsTdsGdsGdsCdsTdsGdsTdsAmsMmsTm 33.0 224 226 38556 38571 AmsGmsTmsCdsTdsGdsGdsAdsTdsTdsCdsTdsCdsTmsAmsMm 47.5 225 227 40450 40467 AmsTmsAmsTdsGdsGdsTdsCdsAdsGdsGdsGdsTdsAmsTmsGm 48.0 226 228 40470 40465 GmsAmsGmsTdsAdsTdsAdsGdsAdsTdsGdsCdsTdsAmsTmsGm 33.6 227 229 41285 41380 AmsTmsGmsCdsTdsAdsTdsGdsGdsAdsTdsGdsTdsAmsMmsTm 75.8 228 230 41294 41309 GmsAmsGmsTdsAdsTdsTdsAdsCdsAdsTdsGdsCdsTmsAmsTm 22.0 229 231 42110 42125 TmsTmsTmsCdsTdsAdsTdsTdsTdsGdsAdsGdsCdsAmsMmsGm 38.2 230 232 42805 42820 GmsTmsAmsTdsAdsCdsAdsGdsTdsTdsGdsAdsAdsGmsGmsGm 69.2 231 233 43091 43106 GmsMmsTmsGdsTdsAdsAdsGdsGdsTdsTdsTdsTdsGmsAmsTm 49.4 232 234 43182 43197 GmsGmsAmsGdsAdsCdsTdsTdsGdsCdsCdsTdsGdsMmsAmsTm 38.7 233 235 43209 43224 GmsTmsAmsCdsGdsTdsAdsTdsGdsTdsTdsTdsAdsGmsTmsMm 66.5 234 236 43218 43233 TmsMmsAmsTdsGdsGdsTdsGdsGdsGdsTdsAdsCdsGmstdmam 51.5 235 237 43933 43948 GmsTmsGmsTdsAdsAdsGdsGdsGdsAdsAdsAdsCdsTmsTmsMm 69.5 236 238 44050 44065 MmsTmsTmsGdsAdsGdsGdsGdsAdsGdsTdsAdsGdsTmsAmsMm 59.2

TABLE-US-00004 TABLE 3 ATXN3 Target Target expression Compound SEQ starting ending ratio ID ID position position Sequence (3 μM) 237 272 11648 11665 AmsGmsAmsTdsAdsGdsGdsTdsMdsGdsMdsTdsMdsAdsGdsGmsAmsGm 73.3 238 273 11649 11666 GmsAmsGmsAdsTdsAdsGdsGdsTdsMosGdsMdsTdsMdsAdsGmsGmsAm 56.1 239 274 11650 11667 AmsGmsAmsGdsAdsTdsAdsGdsGdsTdsMdsGdsMdsTdsMdsAmsGmsGm 64.6 240 275 20837 20854 TmsAmsGmsGdsTdsGdsTdsTdsMdsAdsGdsGdsGdsTdsAdsGmsAmsTm 62.1 241 276 20838 20855 MmsTmsAmsGdsGdsTdsGdsTdsTdsMdsAdsGdsGdsGdsTdsAmsGmsAm 77.3 242 277 22192 22209 AmsMmsEmsTdsGdsTdsGdsGdsTdsTdsTdsGdsMdsAdsTdsMmsMmsMm 57.7 243 278 22193 22210 AmsAmsMmsGdsTdsGdsTdsGdsGdsTdsTdsTdsGdsMdsAdsTmsMmsMm 74.5 244 279 22194 22211 AmsAmsAmsMdsGdsTdsGdsTdsGdsGdsTdsTdsTdsGdsMdsAmsTmsMm 70.0 245 280 22199 22216 GmsTmsGmsTdsMdsAdsAdsAdsMdsGdsTdsGdsTdsGdsGdsTmsTmsTm 54.2 246 281 22201 22218 AmsGmsGmsTdsGdsTdsMdsAdsAdsAdsMdsGdsTdsGdsTdsGmsCmsTm 48.9 247 282 22202 22219 AmsAmsGmsGdsTdsGdsTdsMdsAdsAdsAdsMdsGdsTdsGdsTmsGmsGm 54.6 248 283 22203 22220 TmsAmsAmsGdsGdsTdsGdsTdsMdsAdsAdsAdsMdsGdsTdsGmsTmsGm 74.8 249 284 32006 32023 AmsAmsGmsTdsTdsGdsGdsTdsTdsrdsGdsTdsGdsGdsTdsAmsMmsTm 65.9 250 285 36412 36429 AmsGmsTmsAdsAdsMdsTdsMdsTdsGdsMdsAdsMdsTdsTdsMmsMmsMm 65.5

TABLE-US-00005 TABLE 4 Target Target compound starting ending 3 10 ID position position μM μM WO2018/089805 650528 44828 44845 82.0 61.8 WO2019/217708 1100673 43010 43029 52.4 35.4 WO2020/172559 1287095 44221 44240 41.7 29.0

REFERENCE EXAMPLE

[0334] Scheme of methods for synthesizing ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, ALNA[ipU]-containing nucleosides, ALNA[Trz]-containing nucleosides, and ALNA[Oxz]-containing nucleosides are shown below. The starting compounds (1a, 1d, and 1g) can be synthesized using a method described in WO 2017/047816.

[0335] Synthesis of ALNA[Ms]-T

##STR00022##

[0336] Synthesis of ALNA[Ms]-mC

##STR00023##

[0337] Synthesis of ALNA[Ms]-G

##STR00024## ##STR00025##

[0338] Synthesis of ALNA[Ms]-A

##STR00026##

[0339] Synthesis of ALNA[mU]-T

##STR00027##

[0340] Synthesis of ALNA[mU]-mC

##STR00028##

[0341] Synthesis of ALNA[mU]-G

##STR00029## ##STR00030##

[0342] Synthesis of ALNA [mU]-A

##STR00031##

[0343] Synthesis of ALNA [ipU]-T

##STR00032##

[0344] Synthesis of ALNA[ipU]-mC

##STR00033##

[0345] Synthesis ALNA[ipU]-G

##STR00034##

[0346] Synthesis of ALNA[ipU]-A

##STR00035##

[0347] Synthesis of ALNA[Trz]-T

##STR00036## ##STR00037##

[0348] Synthesis of ALNA[Trz]-mC

##STR00038## ##STR00039##

[0349] Synthesis ALNA[Trz]-G

##STR00040## ##STR00041##

[0350] Synthesis of ALNA[Trz]-A

##STR00042## ##STR00043##

[0351] Synthesis of ALNA[Oxz]-T

##STR00044##

[0352] Synthesis of ALNA[Oxz]-mC

##STR00045## ##STR00046##

[0353] In the present specification, unless specifically stated otherwise, the use of a singular form includes a plural form. In the present specification, unless specifically stated otherwise, the use of “or” means “and/or.” Further, the use of the term “including” and its other forms such as “includes” and “included” are not limiting. Further, unless specifically stated otherwise, the term “element” or the like includes an element that includes one unit and an element that includes more than one subunit.

[0354] 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, reports, books and articles, are expressly incorporated herein by reference, with respect to the portions of the document discussed herein, and in their entirety.

[0355] Unless a specific definition is given, the nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medical chemistry and pharmaceutical chemistry, and their procedures and techniques described herein are those that are well known in the art and are commonly used. Standard techniques can be used for chemical synthesis and chemical analysis as used herein. Where permitted, all patents, patent applications, published patent applications and other publications referred to throughout the disclosure of this specification, and GenBank accession numbers and relevant sequence information as well as other data available through databases such as the National Center for Biotechnology Information (NCBI), are incorporated by reference with respect to portions of the documents discussed herein, and in its entirety.

[0356] Further, this specification is filed together with a sequence listing in electronic format. However, the information of the sequence listing described in the electronic format is incorporated herein by reference in its entirety.