Antisense nucleic acid for regulating expression and / or function of ATXN7 gene

Abstract

The present invention provides a single-stranded antisense oligonucleotide or pharmaceutically acceptable salt thereof for regulating the expression and / or function of the ATXN7 gene. In the single-stranded antisense oligonucleotide, each nucleotide is bonded by a phosphate group and / or modified phosphate group. The single-stranded antisense oligonucleotide includes a gap region, a 3' wing region bonded to the 3' terminal of the gap region, and a 5' wing region bonded to the 5' terminal of the gap region. The gap region is a deoxyribose-constituted nucleic acid in which a nucleic acid modified by a sugar moiety may be included. The 3' wing region and the 5' wing region are modified nucleic acids. The sugar modification constituting the single-stranded antisense oligonucleotide is a modified nucleic acid represented by formula (A1). The single-stranded antisense oligonucleotide has a base length of 12-30 mer. The base sequence of the antisense oligonucleotide is: a base sequence having a sequence identity of 90-100% with a base sequence that is complementary to at least one target region constituted at the same base length as the antisense oligonucleotide in the base sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; a base sequence that is complementary to the base sequence obtained by deleting, substituting, inserting, or adding one or more bases in the target region; or a base sequence that, under stringent conditions, hybridizes with an oligonucleotide having the target region.

Description

Antisense nucleic acid that regulates the expression and / or function of the ATXN7 gene

[0001] The present invention relates to an antisense nucleic acid that regulates the expression and / or function of the ATXN7 gene. The present invention also relates to an antisense nucleic acid (antisense oligonucleotide) that regulates the expression and / or function of the human ATXN7 gene, and a pharmaceutical composition containing the oligonucleotide.

[0002] The ATXN7 gene encodes a protein called ataxin-7. Ataxin-7 is a component of the Spt-Ada-Gcn5 acetyltransferase complex (hereinafter sometimes referred to as the "SAGA complex"). Ataxin-7 controls gene expression by regulating the histone acetyltransferase activity and histone deubiquitination activity of the SAGA complex. Abnormal expansion of the CAG repeat in the ATXN7 gene causes a genetic disease called spinocerebellar ataxia type 7 (hereinafter sometimes referred to as "spinocerebellar ataxia type 7" or "SCA7"). SCA7 is primarily caused by degeneration in the cerebellum, brainstem, eyes, etc., resulting in symptoms such as decreased motor function and decreased vision (Non-Patent Documents 1, 2, and 3).

[0003] Abnormal expansion of the CAG repeat in exon 3 (also referred to as "Exon 3") of the ATXN7 gene leads to the expression of mutant Ataxin-7, which contains polyglutamine (hereinafter sometimes referred to as "PolyQ") encoded by the CAG repeat. Mutant Ataxin-7 inhibits the histone acetyltransferase activity and histone deubiquitination activity of the SAGA complex, causing abnormal expression of downstream genes and contributing to the onset and progression of SCA7 (Figure 1) (Non-Patent Document 4).

[0004] Studies using ATXN7 genetically engineered mice (hereinafter sometimes referred to as "SCA7 mice") have reported that induction of mutant Ataxin-7 expression or abnormal expansion of CAG repeats in the ATXN7 gene causes motor dysfunction or visual impairment, which are major symptoms of SCA7 (Non-Patent Documents 5 and 6). It has also been shown that suppressing ATXN7 gene expression improves histone modification abnormalities, motor dysfunction, and visual impairment in SCA7 mice (Non-Patent Documents 7 and 8).

[0005] International Publication No. WO 1991 / 009033 International Publication No. WO 2009 / 064471 International Publication No. WO 99 / 14226 International Publication No. WO 2017 / 015555

[0006] Nat. Genet. 1997 Sep;17(1):65-70. Neurogenetics. 2015 Jan; 16(1): 11-21. Eur. J. Neurol. 2020 Nov;27(11):2267-2276. Neurotherapeutics. 2019 Oct; 16(4):1074-1096. Neuron. 2003 Feb 6;37(3):383-401. J. Neurosci. 2011 Nov 9;31(45):16269-78. Hum. Mol. Genet. 2013 Mar 1;22(5):890-903. Sci. Transl. Med. 2018 Oct 31;10(465):eaap8677. J. Med. Chem. 2016 Nov 10;59(21):9645-9667. J. Am. Chem. Soc. 2016 Dec 7;138(48):15663-15672. Nature. 2015 Feb 19;518(7539):409-12. J. Am. Chem. Soc. 2003 Jul 9;125(27):8307-17. Nucleic Acids Res. 2014 Dec 16;42(22):13456-68.

[0007] Spinocerebellar ataxia type 7 is an intractable disease that threatens the life and quality of life of patients. However, effective treatments for spinocerebellar ataxia type 7 are currently limited, and the development of new therapeutic agents is urgently needed.

[0008] In recent years, nucleic acid drugs have been put into practical use as new therapeutic agents. Nucleic acid drugs are pharmaceuticals manufactured by chemical synthesis, consisting of oligonucleotides in which nucleic acids or modified nucleic acids are linked together by a dozen or so bases. Nucleic acid drugs are known to act directly on messenger RNA or non-translated RNA involved in protein translation, or to act on target proteins like antibody drugs. There are several types of nucleic acid drugs, including (1) gapmer antisense oligonucleotides (hereinafter sometimes referred to as "gapmers"), which are designed to suppress the expression of a target gene; (2) small interfering RNA (siRNA); (3) splicing-controlling antisense oligonucleotides (hereinafter sometimes referred to as "SSO"), which affect splicing from pre-mRNA (hereinafter sometimes referred to as "pre-mRNA") and control the skipping or insertion of a target exon; and (4) aptamers, which are synthetic RNA / RNA that specifically bind to a target protein like an antibody.

[0009] By hybridizing to a target mRNA, gapmers can induce the RNA-cleaving enzyme RNase H and cleave the target mRNA (knockdown). For example, by using gapmers to knock down mRNA that causes disease, it is possible to suppress the expression of abnormal proteins, thereby improving the disease pathology. By hybridizing to a target pre-mRNA, SSOs can inhibit the function of splicing factors, thereby switching exon splicing (splice-out). For example, by splicing out a specific exon of an RNA that causes disease, it is possible to express a functional protein, even if its amino acid sequence is shorter than that of the native protein, thereby improving the disease pathology.

[0010] As described above, in spinocerebellar ataxia type 7, abnormal expansion of CAG repeat sequences has been observed within exon 3 of the human ATXN7 gene. The abnormally expanded CAG repeats cause abnormal structuring of ATXN7 mRNA or the insertion of abnormal polyarginine chains translated from the mRNA, resulting in cell death. Non-Patent Document 8 reports that screening of antisense oligonucleotides targeting the mouse atxn7 gene was performed, and that intravitreal administration of the obtained antisense oligonucleotides to model mice (SCA7 266Q knock-in mice) resulted in suppression of atxn7 gene expression, significant suppression of PolyQ-containing Ataxin-7 aggregation, and associated improvement in visual acuity loss. These results suggest that controlling the expression of the human ATXN7 gene may be able to treat human spinocerebellar ataxia type 7. However, Non-Patent Document 8 targets only the mouse atxn7 gene, and does not disclose specific antisense oligonucleotides for the human ATXN7 gene or their effects, nor does it examine the toxicity of the oligonucleotides, so further detailed studies are required for their therapeutic application. Given the above-mentioned background, there is a strong demand for pharmaceuticals that suppress the expression of the human ATXN7 gene, and the present inventors have conducted studies to create antisense oligonucleotides that suppress the expression of a mutant human ATXN7 gene containing an abnormally expanded CAG repeat (hereinafter sometimes simply referred to as the "human ATXN7 gene," "human ATXN7 mRNA," "mutant human ATXN7 gene," or "mutant human ATXN7 mRNA") or induce skipping of its third exon.

[0011] The present invention has been made in consideration of the above circumstances, and the problem that the present invention aims to solve is to provide a single-stranded antisense oligonucleotide that regulates the expression and / or function of human ATXN7, and an agent for regulating the expression and / or function of the human ATXN7 gene that contains the same.

[0012] As a result of intensive research to solve the above-mentioned problems, the present inventors have discovered a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof (hereinafter, sometimes referred to as the "antisense oligonucleotide of the present invention") that binds to and functions with human ATXN7 mRNA or its precursor, pre-mRNA, thereby effectively regulating the expression and / or function of human ATXN7 mRNA, and have completed the present invention. That is, the present invention is as follows.

[0013] [1] The antisense oligonucleotide of the present invention is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that regulates the expression and / or function of human ATXN7 gene, wherein each nucleotide in the single-stranded antisense oligonucleotide is linked via a phosphate group and / or a modified phosphate group, the single-stranded antisense oligonucleotide comprises a gap region, a 3' wing region linked to the 3' end of the gap region, and a 5' wing region linked to the 5' end of the gap region, the gap region is a nucleic acid composed of deoxyribose that may contain a nucleic acid whose sugar moiety is modified, the 3' wing region and the 5' wing region are modified nucleic acids, the sugar modification constituting the single-stranded antisense oligonucleotide is a modified nucleic acid represented by the following formula (A1), the base length of the single-stranded antisense oligonucleotide is 12 to 30 mer, and the base sequence of the single-stranded antisense oligonucleotide is The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof is a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to at least one target region of the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, which is the same base length as the single-stranded antisense oligonucleotide; a base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted, or added in the target region; or a base sequence which hybridizes under stringent conditions to an oligonucleotide having the target region. (wherein, base is a nucleic acid base, each independently being a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine, or uracil; R １ and R ２ are each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, or R １ and R ２ and X together form a ring, each X is independently a hydrogen atom, a hydroxyl group, a fluorine atom, an alkoxy group having 1 to 6 carbon atoms which may be substituted, or an amino group which may be substituted, and each Y is independently a hydrogen atom or an alkyl group having 1 to 8 carbon atoms which may have a carbon-oxygen double bond, or some carbon atoms of the alkyl group and X together form a ring.

[0014] The antisense oligonucleotide of the present invention is based on an oligonucleotide having a nucleic acid composed of deoxyribose, whose sugar moiety may contain a modified nucleic acid, in the gap region, and the above-mentioned specific modified nucleic acid is arranged in the 5' wing region and the 3' wing region. The sugar modification constituting the antisense oligonucleotide is the modified nucleic acid represented by the above formula (A1). The antisense oligonucleotide of the present invention containing the modified nucleic acid represented by formula (A1) is expected to have high binding affinity to human ATXN7 mRNA or its precursor mRNA. Furthermore, since the antisense oligonucleotide of the present invention is a so-called gapmer, it functions as a catalyst in the degradation reaction of the human ATXN7 gene by RNase, as described below. Therefore, it is believed that the desired effect can be sustained even with a small amount of administration.

[0015] [2] In the above [1], the base sequence of the single-stranded antisense oligonucleotide (gapmer) is preferably a base sequence having a sequence identity of 95% to 100% based on a base sequence complementary to at least one target region of the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, which has the same base length as the single-stranded antisense oligonucleotide (gapmer).

[0016] [3] In the above [1], the base sequence of the single-stranded antisense oligonucleotide (gapmer) is preferably a base sequence complementary to at least one target region of the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, the target region having the same base length as the single-stranded antisense oligonucleotide (gapmer).

[0017] [4] In any of the above [1] to [3], it is preferable that the number of bases in the gap region is 5 to 20 mer, the 3' wing region is composed of a 2 to 5 mer modified nucleic acid, and the 5' wing region is composed of a 2 to 5 mer modified nucleic acid.

[0018] [5] In any of the above [1] to [4], the base length of the single-stranded antisense oligonucleotide (gapmer) is preferably 15 to 22 mer.

[0019] [6] In any of the above items [1] to [5], it is preferred that the modified nucleic acid in the 3' wing region is selected from the group consisting of 2'-O-alkylated nucleic acids and cross-linked modified nucleic acids, and the modified nucleic acid in the 5' wing region is selected from the group consisting of 2'-O-alkylated nucleic acids and cross-linked modified nucleic acids.

[0020] [7] In any of the above [1] to [6], it is preferable that the number of bases in the gap region is 8 to 12 mer, the 3' wing region is composed of a 3 to 5 mer modified nucleic acid, and the 5' wing region is composed of a 3 to 5 mer modified nucleic acid.

[0021] [8] In any of the above [1] to [7], it is preferable that the 3'-wing region is composed of a 2'-MOE nucleic acid or a 2',4'-bridged modified nucleic acid, the 5'-wing region is composed of a 2'-MOE nucleic acid or a 2',4'-bridged modified nucleic acid, and at least one internucleotide bond of the single-stranded antisense oligonucleotide (gapmer) is a phosphorothioate bond.

[0022] [9] In any one of [1] to [8] above, the base sequence of the single-stranded antisense oligonucleotide (gapmer) is, in the base sequence of SEQ ID NO: 1, at positions 384 to 385, 457 to 460, 831 to 833, 1118 to 1129, 1904 to 1907, 2170 to 2176, 3109, 3654 to 3667, 4088, 4271 to 4275, 4528 to 4532, 4732, 4850, 4872 to 4876, 4877 to 4878, 4879 to 4880, 4879 to 4881, 4879 to 4882, 4879 to 4883, 4879 to 4884, 4879 to 4885, 4879 to 4886, 4879 to 4888, 4879 to 4881, 4879 to 4882, 4879 to 4883, 4879 to 4884, 4879 to 4885, 4879 to 4886, 4879 to 4889 ... The base sequence is preferably a base sequence having 90% to 100% sequence identity based on a base sequence complementary to a target region consisting of a continuous 15-22mer starting from bases located at positions 78, 5462 to 5463, 5710 to 5712, 5783, 5814 to 5819, 5950, 6402 to 6404, 6805 to 6806, or 6851; a base sequence complementary to a base sequence in the above target region in which one or several bases have been deleted, substituted, inserted, or added; or a base sequence that hybridizes under stringent conditions to an oligonucleotide having the above target region.

[0023]

[10] In any of the above [1] to [9], the base sequence of the single-stranded antisense oligonucleotide (gapmer) is a base sequence that has 90% to 100% sequence identity with respect to a base sequence complementary to a target region consisting of a continuous 15-22mer starting from bases located at positions 384 to 385, 457 to 460, 831 to 833, 1118 to 1126, 2174 to 2176, 3654, 4088, 4271 to 4275, 4528, 4850, 4873, 5462, 5710 to 5712, 5816 to 5819, 6402 to 6404, 6805 to 6806, or 6851 in the base sequence set forth in SEQ ID NO: 1, counting from the 5' end; Preferably, the 3' wing region is a 2-5 mer, and the 5' wing region is a 2-5 mer.

[0024]

[11] In any of the above items [1] to

[10] , the base sequence of the single-stranded antisense oligonucleotide (gapmer) is preferably a base sequence that has 90% to 100% sequence identity with respect to a base sequence complementary to a target region consisting of a continuous 15-22mer starting from the base at positions 1122, 2174, 4272, 4528, 5462, 5712, or 5818, counting from the 5' end, in the base sequence set forth in SEQ ID NO: 1.

[0025]

[12] In any of the above [1] to

[11] , the base sequence of the single-stranded antisense oligonucleotide (gapmer) is preferably one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 10 to 16, 18, 20, 21, 23 to 25, 27, 28, 31 to 35, 37, 41, 42, 45, 47 to 49, 52, 54 to 60, 63 to 70, 73 to 83, and 85 to 197.

[0026]

[13] In any of the above [1] to

[12] , the base sequence of the single-stranded antisense oligonucleotide (gapmer) is preferably one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 10 to 13, 15, 18, 21, 24, 27, 33 to 35, 37, 41, 45, 54 to 60, 65, 66, 68 to 70, 74 to 81, and 83.

[0027]

[14] The antisense oligonucleotide of the present invention is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that induces skipping of exon 3 in the human ATXN7 gene, wherein each nucleotide in the single-stranded antisense oligonucleotide is linked via a phosphate group and / or a modified phosphate group, the single-stranded antisense oligonucleotide comprises a modified nucleic acid having at least one modified sugar, and the sugar modification constituting the single-stranded antisense oligonucleotide is a modified nucleic acid represented by the following formula (A2) and / or formula (A3), the base length of the single-stranded antisense oligonucleotide is 15 to 30 mer, and the base sequence of the single-stranded antisense oligonucleotide is a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to at least one target region having the same base length as the single-stranded antisense oligonucleotide in the base sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7, a base sequence complementary to a base sequence in the target region in which one or several bases have been deleted, substituted, inserted or added, or The antisense oligonucleotide is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, which has a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region. (wherein, base is a nucleic acid base, each independently being a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine, or uracil; R １ and R ２ are each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, or R １ and R ２ and together form a ring, and R ３ are each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, and R ４ and R ５ are each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, or R ４ and R ５and are taken together to form a carbon-oxygen double bond or ring, each Z is independently an oxygen atom or an optionally substituted nitrogen atom, and n is an integer of 0 to 2.

[0028] The sugar moiety of the antisense oligonucleotide of the present invention may contain a modified nucleic acid. The antisense oligonucleotide of the present invention containing the modified nucleic acid represented by formula (A2) and / or formula (A3) is expected to have high binding affinity to human ATXN7 pre-mRNA. Furthermore, since the antisense oligonucleotide of the present invention is a so-called splicing control antisense oligonucleotide (SSO), it inhibits the function of a splicing control factor by hybridizing to the human ATXN7 pre-mRNA described below, thereby inducing skipping of the desired exon 3.

[0029]

[15] In the above

[14] , the base sequence of the single-stranded antisense oligonucleotide (SSO) is preferably a base sequence complementary to at least one target region in the base sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7, the target region having the same base length as the single-stranded antisense oligonucleotide (SSO).

[0030]

[16] In the above

[14] or

[15] , the single-stranded antisense oligonucleotide (SSO) preferably has a base length of 18 to 25 mer.

[0031]

[17] In any one of the above

[14] to

[16] , the sugar modification constituting the single-stranded antisense oligonucleotide (SSO) is represented by the above formula (A2), wherein R ３ are preferably each independently a methyl group, a methoxyethyl group, or an N-methylpropanamide group.

[0032]

[18] In any of the above

[14] to

[17] , the base sequence of the single-stranded antisense oligonucleotide (SSO) is a base sequence having 90% to 100% sequence identity based on a base sequence complementary to a target region consisting of 18 to 25 consecutive bases from the bases located at positions 6, 8 to 15, 26, 282, 287, 292, 297, 302, or 317, counting from the 5' end, in the base sequence of SEQ ID NO: 6, or a target region consisting of 18 to 25 consecutive bases from the bases located at positions 49450, 49452 to 49459, 49470, 49726, 49731, 49736, 49741, 49746, 49761, or 49781, counting from the 5' end, in the base sequence of SEQ ID NO: 7; It is preferable that the base sequence is complementary to a base sequence in which one or several bases have been deleted, substituted, inserted or added in the target region, or that the base sequence hybridizes under stringent conditions to an oligonucleotide having the target region.

[0033]

[19] In any of the above items

[14] to

[18] , the base sequence of the single-stranded antisense oligonucleotide (SSO) is preferably a base sequence having a sequence identity of 90% to 100% based on a base sequence complementary to a target region consisting of 18 to 25 consecutive bases from bases located at positions 8 to 11, 282, 287, or 302, counting from the 5' end, in the base sequence of SEQ ID NO: 6, or a target region consisting of 18 to 25 consecutive bases from bases located at positions 49452 to 49455, 49726, 49731, or 49746, counting from the 5' end, in the base sequence of SEQ ID NO: 7.

[0034]

[20] In any of the above

[14] to

[19] , the base sequence of the single-stranded antisense oligonucleotide (SSO) is preferably one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 236, 260 to 263, 265 to 267, 269, 271, 292, and 293.

[0035]

[21] The double-stranded antisense oligonucleotide according to the present invention is a double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, comprising: a single-stranded antisense oligonucleotide (gapmer or SSO) according to any one of [1] to

[20] above; and a second-strand oligonucleotide hybridized to the single-stranded antisense oligonucleotide, wherein the base sequence of the second-strand oligonucleotide is complementary to the base sequence of the single-stranded antisense oligonucleotide and has a sequence identity of 90% to 100% based on the base sequence of the complementary strand.

[0036]

[22] In the above

[21] , the double-stranded antisense oligonucleotide is preferably at least one double-stranded antisense oligonucleotide selected from the group consisting of double-stranded oligonucleotide sequences SP-AX7-195, SP-AX7-205, SP-AX7-206, and SP-AX7-209.

[0037]

[23] The antisense oligonucleotide complex of the present invention is an oligonucleotide complex or a pharmaceutically acceptable salt thereof, comprising: a single-stranded antisense oligonucleotide of any one of [1] to

[20] above or a pharmaceutically acceptable salt thereof, or a double-stranded antisense oligonucleotide of

[21] or

[22] above or a pharmaceutically acceptable salt thereof; and an additional substance bound to the single-stranded antisense oligonucleotide or the second strand oligonucleotide directly or via a linker bond, wherein the linker bonds are each independently a phosphodiester bond or a phosphorothioate bond, and the additional substance is at least one selected from the group consisting of polyethylene glycol, a peptide, an alkyl chain, a ligand compound, an antibody, a protein, a nucleic acid, and a sugar chain.

[0038]

[24] A pharmaceutical product according to the present invention comprises, as an active ingredient, the single-stranded antisense oligonucleotide of any one of [1] to

[20] above or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide of

[21] or

[22] above or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex of

[23] above or a pharmaceutically acceptable salt thereof.

[0039]

[25] The agent for regulating the expression and / or function of the human ATXN7 gene according to the present invention comprises, as an active ingredient, any one of the single-stranded antisense oligonucleotides [1] to

[20] above or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide

[21] or

[22] above or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex

[23] above or a pharmaceutically acceptable salt thereof.

[0040]

[26] A therapeutic agent for spinocerebellar ataxia type 7 according to the present invention comprises, as an active ingredient, any one of the single-stranded antisense oligonucleotides [1] to

[20] above or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide

[21] or

[22] above or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex

[23] above or a pharmaceutically acceptable salt thereof.

[0041]

[27] A preventive agent for spinocerebellar ataxia type 7 according to the present invention comprises, as an active ingredient, any one of the single-stranded antisense oligonucleotides [1] to

[20] above or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide

[21] or

[22] above or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex

[23] above or a pharmaceutically acceptable salt thereof.

[0042]

[28] A method for treating or preventing spinocerebellar ataxia type 7 according to the present invention comprises the step of administering, as an active ingredient, to an individual suffering from spinocerebellar ataxia type 7, the single-stranded antisense oligonucleotide of any of [1] to

[20] above or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide of

[21] or

[22] above or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex of

[23] above or a pharmaceutically acceptable salt thereof.

[0043]

[29] The present invention provides a single-stranded antisense oligonucleotide according to any one of [1] to

[20] above or a pharmaceutically acceptable salt thereof, a double-stranded antisense oligonucleotide according to

[21] or

[22] above or a pharmaceutically acceptable salt thereof, or an antisense oligonucleotide conjugate according to

[23] above or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of spinocerebellar ataxia type 7.

[0044]

[30] The present invention provides a single-stranded antisense oligonucleotide according to any one of [1] to

[20] above or a pharmaceutically acceptable salt thereof, a double-stranded antisense oligonucleotide according to

[21] or

[22] above or a pharmaceutically acceptable salt thereof, or an antisense oligonucleotide conjugate according to

[23] above or a pharmaceutically acceptable salt thereof, for use in producing an agent for treating or preventing spinocerebellar ataxia type 7.

[0045] According to the present invention, it is possible to provide a single-stranded antisense oligonucleotide that regulates the expression and / or function of the human ATXN7 gene, and an agent for regulating the expression and / or function of the human ATXN7 gene comprising the same. Furthermore, according to the present invention, it is possible to provide a single-stranded antisense oligonucleotide that regulates the expression and / or function of the human ATXN7 gene and can be used for the treatment or prevention of spinocerebellar ataxia type 7.

[0046] FIG. 1 is a schematic diagram illustrating a hypothesis that spinocerebellar ataxia type 7 is caused by abnormal expansion of CAG repeats in the ATXN7 gene. FIG. 2 is a schematic diagram illustrating an example of the gapmer configuration in a single-stranded antisense oligonucleotide according to this embodiment. FIG. 3 is a schematic diagram illustrating the mechanism by which ATXN7 gene expression is suppressed when a gapmer in a single-stranded antisense oligonucleotide according to this embodiment is used. FIG. 4 is a schematic diagram illustrating the mechanism by which exon 3 of the ATXN7 gene is skipped and expression of the Ataxin-7 protein containing PolyQ is suppressed when an SSO in a single-stranded antisense oligonucleotide according to this embodiment is used. FIG. 5 is a schematic diagram illustrating the sequence design position of a single-stranded antisense oligonucleotide (SSO) according to this embodiment. FIG. 6 is the structural formula of an oligonucleotide with sequence name SP-AX7-180 to which tocopherol is bound as an additional substance. Figure 7 is a photograph of a non-denaturing acrylamide gel electrophoresis of a double-stranded antisense oligonucleotide in which a complementary strand is hybridized to a single-stranded antisense oligonucleotide (SSO) according to this embodiment. Figure 8 is an agarose gel electrophoresis image showing the exon skipping activity of the ATXN7 gene when the SSO in the single-stranded antisense oligonucleotide according to this embodiment is used. Figure 9 shows the results of sequence analysis confirming that the third exon of the ATXN7 gene is skipped when the SSO in the single-stranded antisense oligonucleotide according to this embodiment is used. Figure 10 is an agarose gel electrophoresis image showing the skipping of the third exon in spinocerebellar ataxia type 7 patient fibroblasts (SCA7 patient strain) when the SSO in the single-stranded antisense oligonucleotide according to this embodiment is used. Figure 11 shows the results of confirming the suppression of Ataxin-7 protein expression in spinocerebellar ataxia type 7 patient fibroblasts (SCA7 patient strain) when the SSO in the single-stranded antisense oligonucleotide according to this embodiment is used.

[0047] <Single-stranded antisense oligonucleotide that regulates expression and / or function of human ATXN7 gene> The single-stranded antisense oligonucleotide of this embodiment is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that regulates expression and / or function of human ATXN7 gene, wherein each nucleotide in the single-stranded antisense oligonucleotide is linked via a phosphate group and / or a modified phosphate group, wherein the single-stranded antisense oligonucleotide (gapmer) comprises a gap region, a 3' wing region linked to the 3' end of the gap region, and a 5' wing region linked to the 5' end of the gap region, wherein the gap region is a nucleic acid composed of deoxyribose that may contain a nucleic acid whose sugar moiety is modified, wherein the 3' wing region and the 5' wing region are modified nucleic acids, wherein the sugar modification constituting the single-stranded antisense oligonucleotide is a modified nucleic acid represented by the following formula (A1), wherein the base length of the single-stranded antisense oligonucleotide (gapmer) is 12 to 30 mer, and wherein the base sequence of the single-stranded antisense oligonucleotide (gapmer) is The single-stranded antisense oligonucleotide (gapmer) or a pharmaceutically acceptable salt thereof is a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to at least one target region of the same base length as the single-stranded antisense oligonucleotide (gapmer) in the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; a base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted, or added in the target region; or a base sequence which hybridizes under stringent conditions to an oligonucleotide having the target region. (wherein, base is a nucleic acid base, each independently being a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine, or uracil; R １ and R ２ are each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, or R１ and R ２ and X together form a ring, each X is independently a hydrogen atom, a hydroxyl group, a fluorine atom, an alkoxy group having 1 to 6 carbon atoms which may be substituted, or an amino group which may be substituted, and each Y is independently a hydrogen atom or an alkyl group having 1 to 8 carbon atoms which may have a carbon-oxygen double bond, or some carbon atoms of the alkyl group and X together form a ring.

[0048] The single-stranded antisense oligonucleotide of this embodiment is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that induces skipping of exon 3 in the human ATXN7 gene, wherein each nucleotide in the single-stranded antisense oligonucleotide (SSO) is linked via a phosphate group and / or a modified phosphate group, the single-stranded antisense oligonucleotide (SSO) comprises a modified nucleic acid having at least one modified sugar, and the sugar modification constituting the single-stranded antisense oligonucleotide is a modified nucleic acid represented by the following formula (A2) and / or formula (A3), the base length of the single-stranded antisense oligonucleotide (SSO) is 15 to 30 mer, and the base sequence of the single-stranded antisense oligonucleotide (SSO) is a base sequence having a sequence identity of 90% or more and 100% or less with respect to a base sequence complementary to at least one target region of the base sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7, which is composed of the same base length as the single-stranded antisense oligonucleotide (SSO), The single-stranded antisense oligonucleotide (SSO) or a pharmaceutically acceptable salt thereof is a base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted or added in the target region, or a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region. (wherein, base is a nucleic acid base, each independently being a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine, or uracil; R １ and R ２are each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, or R １ and R ２ and together form a ring, and R ３ are each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, and R ４ and R ５ are each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, or R ４ and R ５ and are taken together to form a carbon-oxygen double bond or ring, each Z is independently an oxygen atom or an optionally substituted nitrogen atom, and n is an integer of 0 to 2.

[0049] In one aspect of this embodiment, the single-stranded antisense oligonucleotide (SSO) can also be understood as a single-stranded antisense oligonucleotide (SSO) or a pharmaceutically acceptable salt thereof that hybridizes to exon 3 (an exon containing a CAG repeat) of human ATXN7 mRNA precursor or RNA in the vicinity thereof, thereby inducing skipping of the exon 3.

[0050] The single-stranded antisense oligonucleotide (SSO) may be a double-stranded antisense oligonucleotide composed of a second strand oligonucleotide hybridized to the single-stranded antisense oligonucleotide (SSO), but is preferably a single-stranded antisense oligonucleotide. In this embodiment, the term "modified nucleic acid having a modified sugar" includes morpholino nucleic acid, which will be described in detail below.

[0051] The antisense oligonucleotide or pharmaceutically acceptable salt thereof having the above-described configuration can regulate the expression and / or function of the human ATXN7 gene. In one aspect of this embodiment, the oligonucleotide or pharmaceutically acceptable salt thereof can also be understood as "an oligonucleotide or pharmaceutically acceptable salt thereof that regulates the expression and / or function of the human ATXN7 gene." The present invention suppresses the expression of a human ATXN7 gene containing an abnormally expanded CAG repeat or induces skipping of the third exon in which the CAG repeat is located, thereby improving various symptoms of spinocerebellar ataxia type 7, including motor dysfunction and visual impairment. This is described in detail below. For convenience, the oligonucleotide according to this embodiment may be referred to as an "antisense oligonucleotide," "single-stranded antisense oligonucleotide," "double-stranded antisense oligonucleotide," etc., but is not intended to be limited to these terms.

[0052] <Definitions of Terms, etc.> First, definitions of terms used in this specification will be explained below.

[0053] (ATXN7 gene) In this embodiment, the "ATXN7 gene" can be defined according to Nat. Genet. 1997 Sep; 17(1):65-70. (Non-Patent Document 1). Synonyms of "ATXN7" include Ataxin-7, SCA7, OPCA3, SGF73, ADCAII, etc.

[0054] (Single-stranded antisense oligonucleotide) In this embodiment, a "single-stranded antisense oligonucleotide" or "antisense oligonucleotide" (hereinafter sometimes referred to as "ASO") refers to an oligonucleotide or a pharmacologically acceptable salt thereof that is complementary to the mRNA, pre-mRNA, or non-coding RNA (ncRNA) of a target gene (hereinafter these three may be collectively referred to as "target RNA"). Antisense oligonucleotides are composed of DNA, RNA, and / or analogs thereof. Antisense oligonucleotides control / regulate the function or expression of the target mRNA, pre-mRNA, or ncRNA by forming a double strand (hereinafter sometimes referred to as "hybridizing") with the target mRNA, pre-mRNA, or ncRNA. Antisense oligonucleotides include those that have a base sequence that is completely complementary to the base sequence of the target mRNA, mRNA precursor, or ncRNA, those that have a base sequence in which one or more bases have been deleted, substituted, inserted, or added in the complementary base sequence, and those that contain bases that form wobble base pairs in their base sequence.

[0055] Furthermore, the antisense oligonucleotide of the present invention may further contain modified nucleotides known in the art other than "modified nucleic acids in which the sugar moiety is a modified sugar" (sugar-modified modified nucleotides) described below. Examples of modified nucleotides known in the art include sugar-modified modified nucleotides, as well as phosphate-modified nucleotides and nucleobase-modified nucleotides described below. The antisense oligonucleotide of this embodiment is not particularly limited in terms of the structure of both termini, and may be, for example, -OH or -OR (where R represents an alkyl chain, a phosphate ester, or an additional substance described below). The single-stranded antisense oligonucleotide of this embodiment may be in a single-stranded form, or may hybridize with a second-strand oligonucleotide described below to form a double-stranded form. A double-stranded oligonucleotide consisting of the single-stranded antisense oligonucleotide and a second-strand oligonucleotide hybridized to the single-stranded antisense oligonucleotide may be referred to as a "double-stranded antisense oligonucleotide."

[0056] (Oligonucleotide) In this embodiment, "oligonucleotide" refers to a polymer of nucleotides in which 2 to 30 identical or different nucleotides are linked together via phosphodiester bonds or other bonds. The oligonucleotide can also be understood as being composed of a nucleic acid base moiety, a phosphate moiety, and a sugar moiety, as shown in the following structural formula:

[0057]

[0058] The oligonucleotides are broadly classified into natural oligonucleotides and non-natural oligonucleotides. "Natural oligonucleotides" refer to oligonucleotides composed of naturally occurring nucleotides. "Non-natural oligonucleotides" refer to oligonucleotides containing at least one modified nucleotide as a constituent unit, as described below. "Non-natural oligonucleotides" preferably include modified sugar derivatives in which the sugar moiety is modified; phosphorothioate derivatives in which one non-bridging oxygen atom in the phosphodiester bond is replaced with a sulfur atom; phosphorodithioate derivatives in which two non-bridging oxygen atoms in the phosphodiester bond are replaced with sulfur atoms; ester derivatives in which the phosphodiester bond is triesterized; phosphoamide derivatives in which the phosphodiester bond is amidated; boranophosphate derivatives in which the phosphodiester bond is boronated; alkylphosphonate (e.g., methylphosphonate, methoxypropylphosphonate, etc.) derivatives in which the non-bridging oxygen atom in the phosphodiester bond is replaced with an alkyl group; amide derivatives in which the phosphodiester bond is replaced with an amide bond; and modified base derivatives in which the nucleobase is modified. More preferably, the unnatural oligonucleotide includes a modified sugar derivative in which the 2'-position of the sugar moiety is modified; a bridged modified sugar derivative in which the 2'- and 4'-positions of the sugar moiety are bridged; a phosphorothioate derivative in which one non-bridging oxygen atom of the phosphodiester bond is replaced with a sulfur atom; a phosphoamide derivative in which the phosphodiester bond is amidated; and a derivative in which the sugar moiety is modified with a modified sugar (e.g., a 2'-position modified sugar and / or a bridged sugar) described below and one non-bridging oxygen atom of the phosphodiester bond is replaced with a sulfur atom.

[0059] (Nucleoside) In this embodiment, "nucleoside" refers to a compound in which a purine base or a pyrimidine base is bound to a sugar. A naturally occurring nucleoside may be referred to as a "natural nucleoside." A modified nucleoside that does not occur in nature may be referred to as a "modified nucleoside." A modified nucleoside in which the sugar moiety is modified may be referred to as a "modified sugar nucleoside." A modified sugar nucleoside having a substituent at the 2'-position of the sugar moiety is referred to as a "2'-substituted nucleoside," and "2'-substituted" means that the 2'-position of the sugar moiety is substituted with at least one substituent other than H (hydrogen atom) or OH (hydroxyl group).

[0060] (Nucleotide) In this embodiment, "nucleotide" refers to a compound in which a phosphate group is bound to the sugar of the above-mentioned nucleoside. A naturally occurring nucleotide may be referred to as a "natural nucleotide." A non-naturally occurring modified nucleotide may be referred to as a "modified nucleotide" or "modified nucleic acid." Examples of "modified nucleotide" or "modified nucleic acid" include a compound in which a phosphate group is bound to the sugar moiety of the above-mentioned modified nucleoside, a compound in which a modified phosphate group (described below) is bound to the sugar moiety of the above-mentioned modified nucleoside, a compound in which a modified phosphate group (described below) is bound to the sugar moiety of the above-mentioned modified nucleoside, and a compound in which a modified phosphate group (described below) is bound to the sugar moiety of a natural nucleoside.

[0061] (Sugar modification, modified sugar) In this embodiment, "sugar modification" means that the sugar moiety of the nucleotide is modified. The modified sugar moiety may be specifically referred to as a "modified sugar". Modified nucleotides that have been sugar-modified can be used as modified nucleic acids, and examples thereof include 2'-O-alkylated nucleic acids (e.g., 2'-O-methyl nucleic acid, 2'-O-methoxyethyl nucleic acid (2'-MOE nucleic acid)), 2'-F-modified nucleic acids (2'-fluoro-nucleic acids), 5'-alkylated nucleic acids (e.g., 5'-methyl nucleic acid, 5'-dimethyl nucleic acid), 5'-cycloalkylated nucleic acids (e.g., 5'-cyclopropyl nucleic acid), 2',4'-BNA (Bridged Nucleic Acid, hereinafter sometimes referred to as "LNA"), AmNA (Amido-bridged artificial nucleic acid), GuNA (Guanidino-bridged artificial nucleic acid), and the like. acid), scpBNA (2'-O,4'-C-Spirocycloalkylene bridged nucleic acid), ENA (2'-O,4'-C-Ethylene-Bridged Nucleic Acid), S-cEt (2',4'-constrained Ethyl Nucleic Acid), BANA (1'-C,3'-O-Propylene-Bridged Altriol Nucleic Acid), and the like. Examples of 2'-O-alkylated nucleic acids include those containing structures represented by the symbols "A(M)", "A(m)", "C(M)", "5(m)", "G(M)", "G(m)", "U(M)", and "T(m)" as described below. Examples of LNAs include those containing structures represented by the symbols "A(L)", "5(L)", "G(L)", and "T(L)" as described below.

[0062] In one aspect of this embodiment, the modification of the sugar constituting the oligonucleotide is preferably a group represented by the following formula (A1): In another aspect of this embodiment, the modified sugar constituting the oligonucleotide can also be understood to be represented by the following formula (A1):

[0063]

[0064] In the above formula (A1), base is a nucleic acid base, and each base is independently a group represented by adenine, guanine, cytosine, 5-methylcytosine (meaning a cytosine modified with a methyl group attached to the 5-position), thymine, or uracil; R １ and R ２ are each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, or R １ and R ２ are taken together to form a ring (preferably an optionally substituted 3- to 6-membered cycloalkyl ring or saturated heterocyclic ring), each X is independently a hydrogen atom, a hydroxyl group, a fluorine atom, an optionally substituted alkoxy group of 1 to 6 carbon atoms, or an optionally substituted amino group, each Y is independently a hydrogen atom or an alkyl group of 1 to 8 carbon atoms which may have a carbon-oxygen double bond, or some carbon atoms of the alkyl group and X are taken together to form a ring.

[0065] Examples of the "optionally substituted alkyl group" include an unsubstituted alkyl group, a hydroxyl group, a fluoro group, an alkoxy group (-OR ６ a carbamoyl group, and an amino group (-NR ７ R ８ and alkyl groups substituted with one or more substituents selected from the group consisting of groups represented by the formula: ６ R each independently represents a linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group having 3 to 6 carbon atoms, or a cycloalkyl group. ７ and R ８ are each independently a hydrogen atom, a linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group or a cycloalkyl group having 3 to 6 carbon atoms, or R ７ and R ８ In the "optionally substituted alkyl group", the "optionally substituted alkyl group having 1 to 6 carbon atoms" refers to a linear, branched or cyclic alkyl group having 1 to 6 carbon atoms which may have the above-mentioned substituent.

[0066] Examples of the "optionally substituted alkoxy group" include an unsubstituted alkoxy group, as well as a hydroxyl group, a fluoro group, an alkoxy group (-OR ６ ), amino group (—NR ７ R ８ ), and alkylamide groups (—(CO)—NR ９ R １０ and an alkoxy group substituted with one or more substituents selected from the group consisting of groups represented by the following formula: ６ R each independently represents a linear alkyl group having 1 to 6 carbon atoms, or a branched alkyl group or cycloalkyl group having 3 to 6 carbon atoms. ７ and R ８ are each independently a hydrogen atom, a linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group or a cycloalkyl group having 3 to 6 carbon atoms, or R ７ and R ８ R represents a 4- to 6-membered heterocyclic ring formed by combining with ９ and R １０ are each independently a hydrogen atom, a linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group or a cycloalkyl group having 3 to 6 carbon atoms, or R ９ and R １０ In the "optionally substituted alkoxy group", the "optionally substituted alkoxy group having 1 to 6 carbon atoms" refers to a linear, branched or cyclic alkoxy group having 1 to 6 carbon atoms which may have the above-mentioned substituent.

[0067] Examples of the "optionally substituted amino group" include an unsubstituted amino group, a linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group having 3 to 6 carbon atoms, or a cyclic alkyl group (each of which is a linear alkoxy group having 1 to 6 carbon atoms, a branched alkoxy group having 3 to 6 carbon atoms, or a -(CO)-NR ９ R １０and an amino group substituted with one or more substituents selected from the group consisting of an acyl group having 1 to 6 carbon atoms and an amidine group which may be substituted with a linear alkyl group having 1 to 6 carbon atoms or a branched alkyl group having 3 to 6 carbon atoms at 1 to 3 positions. ９ and R １０ are each independently a hydrogen atom, a linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group or a cycloalkyl group having 3 to 6 carbon atoms, or R ９ and R １０ and , taken together, form a 4- to 6-membered heterocyclic ring.

[0068] Examples of "an alkyl group which may have a carbon-oxygen double bond" include an unsubstituted alkyl group, an unsubstituted cycloalkyl group (e.g., a cyclopropyl group), an alkyl group having a carbon-oxygen double bond, and a cycloalkyl group having a carbon-oxygen double bond. In the "an alkyl group which may have a carbon-oxygen double bond," an "alkyl group having 1 to 8 carbon atoms which may have a carbon-oxygen double bond" refers to a linear alkyl group having 1 to 8 carbon atoms, or a branched or cyclic alkyl group having 3 to 8 carbon atoms, which may have a carbon-oxygen double bond. The number of carbon atoms in the "alkyl group having 1 to 8 carbon atoms which has a carbon-oxygen double bond" may include the carbon constituting the carbon-oxygen double bond. Therefore, in this embodiment, the "alkyl group having 1 to 8 carbon atoms which has a carbon-oxygen double bond" may include an aldehyde group. When Y is an aldehyde group and X is an amino group which may be substituted, the ring formed by X and Y together can be understood to have a structure containing an amide bond.

[0069] "R １ and R ２ Examples of the "optionally substituted cycloalkyl ring or saturated heterocyclic ring formed by combining " and " include unsubstituted cycloalkyl rings or saturated heterocyclic rings, as well as hydroxyl groups, fluoro groups, alkoxy groups (-OR ６ ) and amino groups (—NR ７ R ８) substituted with one or more substituents selected from the group consisting of cycloalkyl rings or saturated heterocyclic rings. ６ R each independently represents a linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group having 3 to 6 carbon atoms, or a cycloalkyl group. ７ and R ８ are each independently a hydrogen atom, a linear alkyl group having 1 to 6 carbon atoms, a branched alkyl group or a cycloalkyl group having 3 to 6 carbon atoms, or R ７ and R ８ represents a 4- to 6-membered heterocyclic ring formed by combining with "R １ and R ２ and "R １ and R ２ The term "an optionally substituted 3- to 6-membered cycloalkyl ring or saturated heterocyclic ring formed by taking these together" refers to a 3- to 6-membered cycloalkyl ring or saturated heterocyclic ring which may have the above-mentioned substituents.

[0070] In one aspect of this embodiment, the sugar modification constituting the oligonucleotide is preferably a group represented by the following formula (A2) and / or formula (A3): In another aspect of this embodiment, the modified sugar constituting the oligonucleotide can also be understood to be represented by the following formula (A2) and / or formula (A3):

[0071]

[0072] In formula (A2) and / or formula (A3), base is a nucleic acid base, each independently being a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine, or uracil; R １ and R ２ are each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, or R １ and R ２ are taken together to form a ring (preferably an optionally substituted 3- to 6-membered cycloalkyl ring or saturated heterocyclic ring), and R ３ are each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, and R４ and R ５ are each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, or R ４ and R ５ and are taken together to form a carbon-oxygen double bond or ring, each Z is independently an oxygen atom or an optionally substituted nitrogen atom, and n is an integer of 0 to 2.

[0073] In another aspect of this embodiment, the modification of the sugar constituting the oligonucleotide is a group represented by formula (A2) above, and the R ３ are each independently a methyl group, a methoxyethyl group, a hydroxyethyl group, or an N-methylpropanamide group (—CH ２ CH ２ -CONH-CH ３ In another aspect of this embodiment, the sugar modification constituting the single-stranded antisense oligonucleotide (SSO) is represented by the formula (A2), and in formula (A2), R ３ are preferably each independently a methyl group, a methoxyethyl group, or an N-methylpropanamide group.

[0074] In another aspect of this embodiment, the modification of the sugar constituting the oligonucleotide is a group represented by formula (A3), and the R １ and R ２ are each independently a hydrogen atom or a methyl group, or R １ and R ２ and R are cyclopropane rings formed together. ４ and R ５ are each independently a hydrogen atom or a methyl group, or R ４ and R ５and n are each independently an oxygen atom, a nitrogen atom substituted with an alkyl group having 1 to 3 carbon atoms, or a nitrogen atom substituted with an amidine group which may be substituted at 1 to 3 positions with a linear alkyl group having 1 to 4 carbon atoms or a branched alkyl group having 3 to 4 carbon atoms, and n is preferably 0 or 1.

[0075] (Nucleotide Modifications Known in the Art Other Than Sugar Modifications) Nucleotide modifications known in the art other than the sugar modifications described above can be used as modified nucleic acids for producing single-stranded antisense oligonucleotides of the present invention. Known nucleotide modifications include phosphate group modifications and nucleobase modifications, which will be described later. Examples of such nucleotide modifications include the nucleotide modifications described in W. Brad Wan et. al. J. Med. Chem. (2016) 59:9645-9667. (Non-Patent Document 9) and the like. These nucleotide modifications can be carried out based on methods known in the art described in the literature cited in the above literature.

[0076] (Phosphate Group) In this embodiment, the term "phosphate group" refers to a nucleotide in which the phosphate moiety is bound in the form of a naturally occurring phosphodiester bond.

[0077] (Phosphate Group Modification, Modified Phosphate Group) In this embodiment, "phosphate group modification" means that the phosphate moiety of the nucleotide has been modified. The modified phosphate moiety may be specifically referred to as a "modified phosphate group." Examples of linkages containing the modified phosphate group include phosphorothioate linkages (linkages indicated by the symbol "∧" described below), phosphorodithioate linkages, phosphoramidate linkages, boranophosphate linkages, alkylphosphonate linkages, phosphorodiamidate linkages, phosphorodiamidiothioate linkages, and phosphorodiamidiodithioate linkages. Modified internucleoside linkages can be used to alter, typically increase, the nuclease resistance of oligonucleotides compared to natural phosphate linkages. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture or as separate enantiomers. Methods for preparing phosphorus-containing and non-phosphorus-containing internucleoside linkages are well known to those skilled in the art.

[0078] Representative internucleoside linkages having a chiral center include, but are not limited to, phosphorothioate and alkylphosphonate. Modified oligonucleotides containing internucleoside linkages having a chiral center can be prepared as modified oligonucleotides containing stereorandom internucleoside linkages or as modified oligonucleotides containing phosphorothioate linkages in a specific stereochemical configuration. In certain embodiments, the modified oligonucleotide contains phosphorothioate internucleoside linkages, and all phosphorothioate internucleoside linkages are stereorandom. In certain embodiments, the modified oligonucleotide is a modified oligonucleotide containing one or more specific phosphorothioate internucleoside linkages in a specific, independently selected stereochemical configuration. In certain embodiments, a specific arrangement of phosphorothioate linkages is present in at least 50% of the oligonucleotides. In certain embodiments, a specific arrangement of phosphorothioate linkages is present in at least 75% of the oligonucleotides. In certain embodiments, a specific arrangement of phosphorothioate linkages is present in at least 90% of the oligonucleotides. These can be produced using synthetic methods known in the art, for example, the methods described in J. Am. Chem. Soc. 2003 Jul 9;125(27):8307-17. (Non-Patent Document 12), Nucleic Acids Res. 2014 Dec 16;42(22):13456-68. (Non-Patent Document 13), and WO 2017 / 015555. Unless otherwise specified, the chiral internucleoside linkages of the modified oligonucleotides described herein can be stereorandom or can be in a specific stereochemical configuration.

[0079] (Nucleobase modification, modified nucleobase) In this embodiment, "nucleobase modification" means that the nucleobase portion of the nucleotide has been modified. The modified nucleobase portion may be particularly referred to as a "modified nucleobase". Examples of modified nucleobases include 5-methylcytosine, 5-hydroxymethylcytosine (meaning cytosine modified with a hydroxymethyl group attached to the 5-position), and 5-propynylcytosine (meaning cytosine modified with a propynyl group attached to the 5-position).

[0080] (DNA or RNA Analogs) The term "DNA or RNA analogs" refers to molecules having a structure similar to that of DNA or RNA. Examples include peptide nucleic acids (pNA) and morpholino nucleic acids (PMO). The nucleic acids used in the present invention are not limited to those modified in the sugar moiety of the nucleic acid, and morpholino nucleic acids or peptide nucleic acids may also be used. That is, in another embodiment of the single-stranded antisense oligonucleotide of the present invention, the single-stranded antisense oligonucleotide may be composed of a DNA or RNA analog in the sequence listed in the "Antisense Nucleic Acid_Base Sequence (5'-3')" column of Tables 1-1 to 1-3 or Tables 2-1 to 2-3 described below. The DNA or RNA analogs include at least peptide nucleic acids or morpholino nucleic acids. The synthesis of the antisense oligonucleotides of the present invention containing peptide nucleic acids or morpholino nucleic acids is carried out according to standard methods. Morpholino oligonucleotides can be prepared using the methods described, for example, in WO 1991 / 009033 (Patent Document 1), WO 2009 / 064471 (Patent Document 2), J. Am. Chem. Soc. Am. Chem. Soc. 2016 Dec 7; 138(48): 15663-15672. (Non-Patent Document 10).

[0081] (ncRNA) In this embodiment, "ncRNA" refers to a general term for RNA that is not involved in protein translation. Examples of the ncRNA include ribosomal RNA, transfer RNA, miRNA (micro RNA), and Natural Antisense Transcript (NAT).

[0082] (Nucleobase Moiety of Oligonucleotide) Examples of the nucleobase moiety of the oligonucleotide include thyminyl, cytosinyl, adeninyl, guaninyl, 5-methylcytosinyl, uracilyl, 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl, 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl, and 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl groups. Preferred examples of the nucleobase moiety include thyminyl, cytosinyl, adeninyl, guaninyl, 5-methylcytosinyl, and uracilyl groups. Among these nucleobases, uracil (U) and thymine (T) are interchangeable. Both uracil (U) and thymine (T) can form base pairs with adenine (A) in a complementary strand, as well as in the nucleobase portion of antisense oligonucleotides.

[0083] (Target RNA) In this embodiment, the term "target RNA" refers to RNA whose function and / or expression is inhibited by binding of the single-stranded antisense oligonucleotide. In other words, in this embodiment, the target RNA refers to ATXN7 mRNA (hereinafter, sometimes referred to as "hATXN7") and human ATXN7 pre-mRNA (hereinafter, sometimes referred to as "human ATXN7 pre-mRNA"). Examples of the target RNA include human ATXN7 mRNA having the nucleotide sequence set forth in SEQ ID NO: 1, exon 3 of human ATXN7 having the nucleotide sequence set forth in SEQ ID NO: 6, and human ATXN7 pre-mRNA having the nucleotide sequence set forth in SEQ ID NO: 7.

[0084] (Binding to target RNA) In this embodiment, "binding to target RNA" means that the nucleic acid bases of the single-stranded antisense oligonucleotide form a double strand with the nucleic acid bases of the target RNA due to complementarity with the target RNA. The double strand may be formed in at least a portion of the target RNA. The strength of binding to the target RNA can be measured, for example, by an index of thermal stability. An example of an index of thermal stability is the melting temperature (Tm value) of the double strand. The Tm value is preferably 40 to 90°C, more preferably 50 to 70°C. "Complementarity" as used herein means that the nucleic acid base sequence of the oligonucleotide and another nucleic acid sequence arranged in the opposite direction to each other can hydrogen bond with each other. "Complementary nucleobase pair" means nucleobases that can form hydrogen bonds with each other, and examples of complementary nucleobase pairs include adenine (A) and thymine (T) or uracil (U), and cytosine (C) or 5-methylcytosine (5-mC) and guanine (G). Note that complementary oligonucleotides and / or oligonucleic acids do not necessarily need to have completely complementary nucleobases at each nucleoside, and some deletions, substitutions, insertions, or additions may be permitted.

[0085] (Target region) The target region refers to a region in human ATXN7 mRNA and pre-mRNA (hereinafter, collectively referred to as "human ATXN7 RNA") to which the single-stranded antisense oligonucleotide binds. The target region includes the target region consisting of the indicated base sequence, human ATXN7 mRNA, and a region on ATXN7 pre-mRNA.

[0086] (Pre-mRNA) The pre-mRNA (pre-mRNA) refers to the primary transcript of RNA transcribed from DNA. That is, the pre-mRNA is RNA containing exon regions, intron regions, and untranslated regions (UTRs). The pre-mRNA can also be understood as RNA before splicing after transcription. When the pre-mRNA is spliced, it becomes mRNA.

[0087] (Binding to target region) The binding to target region means that the single-stranded antisense oligonucleotide of the present invention forms a double strand with target region.However, the single-stranded antisense oligonucleotide of the present invention does not necessarily need to form a double strand with the entire target region, but can form a double strand with a part of the target region.That is, the single-stranded antisense oligonucleotide of the present invention is preferably one that has complete complementarity with target region, but as long as it binds to the target RNA of human ATXN7, it can be complementary to at least a part of the target region.

[0088] (Part of the target region) The part of the target region means a region of the target region having a length of 10 to 15 nucleotide bases.

[0089] (Complementary to at least a portion of the target region) "Complementary to at least a portion of the target region" means complementary to the bases of at least a portion of the target region on the target RNA, which also includes complementary to the bases of a region on the mRNA or pre-mRNA corresponding to at least a portion of the region.

[0090] The present inventors focused on antisense oligonucleotides that regulate the expression and / or function of human ATXN7 RNA, and investigated the creation of gapmers that suppress the expression of human ATXN7 RNA or SSOs that induce skipping of exon 3 of human ATXN7 RNA.

[0091] <Single-stranded antisense oligonucleotide: gapmer sequence design> Single-stranded antisense oligonucleotide gapmers are compounds for inhibiting the expression of human ATXN7 RNA. Therefore, the gapmer structure is designed to bind (hybridize) to the target region of human ATXN7 RNA. In this case, the gapmer contains a DNA strand of four or more bases as a gap region that can be recognized by RNase H, which specifically cleaves RNA in an RNA / DNA duplex after hybridization. In addition, modified nucleic acids are arranged at both ends to maintain resistance to degradation by other nucleases, etc. Furthermore, the gapmer has appropriately arranged bonds containing modified phosphate groups, as necessary.

[0092] <Base Sequence of Single-Stranded Antisense Oligonucleotide Functioning as a Gapmer> The base sequence of the single-stranded antisense oligonucleotide (gapmer) according to this embodiment is: (A) in the base sequence of SEQ ID NO: 1, counting from the 5' end, at positions 384 to 385, 457 to 460, 831 to 833, 1118 to 1129, 1904 to 1907, 2170 to 2176, 3109, 3654 to 3667, 4088, 4271 to 4275, 4528 to 4532, 4732, 4850, 4872 to 4878, 5462 (B) a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to a target region consisting of a consecutive 12-30 mer (preferably 15-22 mer) starting from bases at positions 5463 to 5463, 5710 to 5712, 5783, 5814 to 5819, 5950, 6402 to 6404, 6805 to 6806, or 6851, or (C) a base sequence complementary to a base sequence in the target region in which one or several bases have been deleted, substituted, inserted, or added, or (D) a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region. In this embodiment, each base sequence shown in the sequence listing is used to indicate only the sequence information of the nucleic acid base portion. The structural information of the oligonucleotide, including the sugar moiety and the phosphate moiety in addition to the nucleic acid base moiety, is shown in the format shown in Tables 3-1 to 3-10 below.

[0093] In one aspect of the single-stranded antisense oligonucleotide (gapmer) according to this embodiment, the target region is, counting from the 5' end, at positions 384 to 385, 457 to 460, 831 to 833, 1118 to 1129, 1904 to 1907, 2170 to 2176, 3109, 3654 to 3667, 4088, 4271 to 4275, 4528 to 4532, 4732, It is more preferable that the base sequence is a 12- to 30-mer or 15- to 22-mer contiguous base sequence starting from bases located at positions 4850, 4872 to 4878, 5462 to 5463, 5710 to 5712, 5783, 5814 to 5819, 5950, 6402 to 6404, 6805 to 6806, or 6851, and that the 3' wing region is a 3- to 5-mer and the 5' wing region is a 3- to 5-mer.

[0094] In another aspect of the single-stranded antisense oligonucleotide (gapmer) according to this embodiment, the target region is, counting from the 5' end, at positions 384 to 385, 457 to 460, 831 to 833, 1118 to 1126, 2174 to 2176, 3654, 4088, 4271 to 4275, 4528, 4850, 4871, 4872, 4873, 4874, 4875, 4876, 4877, 4878, 4879 ...9, 4871, 4876, 4878, 4879, 4879, 4879, 4871, 4872, 4874, 4875, 4876, 4877, 4878, 4879, 4879, 4879, 4879, 4871, 4876, 4879, 4879, 4879, 4879, 4879, 4871, 4879, 4871, 4872, 4874, 4875, 4876, 4877, 4879, 4879, 4879, 4879, 4879, 4879, 4879, 4979, 5079, 5079, 50 It is more preferred that the base sequence is a 12-30 mer, 15-22 mer, or 18-22 mer consisting of consecutive bases starting from bases at positions 3, 5462, 5710 to 5712, 5816 to 5819, 6402 to 6404, 6805 to 6806, or 6851, and that the 3' wing region is a 3-5 mer and the 5' wing region is a 3-5 mer.

[0095] In another aspect of the single-stranded antisense oligonucleotide (gapmer) according to this embodiment, the base sequence of the single-stranded antisense oligonucleotide (gapmer) is preferably a base sequence that has 90% to 100% sequence identity with respect to a base sequence complementary to a target region consisting of a continuous 15-22mer starting from the base at positions 1122, 2174, 4272, 4528, 5462, 5712, or 5818, counting from the 5' end, in the base sequence set forth in SEQ ID NO: 1.

[0096] In one aspect of the single-stranded antisense oligonucleotide (gapmer) according to this embodiment, the base sequence of the single-stranded antisense oligonucleotide is preferably one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 10-16, 18, 20, 21, 23-25, 27, 28, 31-35, 37, 41, 42, 45, 47-49, 52, 54-60, 63-70, 73-83, and 85-197.

[0097] In one aspect of the single-stranded antisense oligonucleotide (gapmer) according to this embodiment, the base sequence of the single-stranded antisense oligonucleotide is more preferably one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 10 to 13, 15, 18, 21, 24, 27, 33 to 35, 37, 41, 45, 54 to 60, 65, 66, 68 to 70, 74 to 81, and 83.

[0098] One embodiment of the single-stranded antisense oligonucleotide (gapmer) of the present invention is a single-stranded antisense oligonucleotide (gapmer) that regulates expression of human ATXN7 RNA, and has any of the nucleotide sequences listed in Tables 1-1 to 1-3. The single-stranded antisense oligonucleotide (gapmer) is complementary to a target region in human ATXN7 mRNA listed in Tables 1-1 to 1-3. Note that the single-stranded antisense oligonucleotide (gapmer) may extend by 1 to 5 nucleotides on the 3' and / or 5' ends, as long as it contains a nucleotide sequence listed in Tables 1-1 to 1-3. The target region can be said to be a region in human ATXN7 mRNA that is particularly involved in regulating expression of human ATXN7 mRNA (e.g., a region having a secondary structure of mRNA that is easily bound by antisense nucleotides). For example, if the 5'-end position in Table 1-1 is "1122" and the 3'-end position is "1137," the base sequence from 1122 to 1137 counting from the 5'-end in the base sequence set forth in SEQ ID NO: 1 is the target region in the mRNA of human ATXN7 targeted by the corresponding single-stranded antisense oligonucleotide (gapmer).

[0099]

[0100]

[0101]

[0102] In the above Tables 1-1 to 1-3 and the later-described Tables 2-1 to 2-3, the symbol "A ＊ ", symbol "C ＊ ", symbol "G ＊ " and the symbol "T ＊ " are each selected from natural nucleosides (a, c, 5(x), g, and t, as described below) or modified nucleosides (including modified sugar nucleosides). ＊ " is selected from A(M), A(m), or A(L) as described below, and the symbol "C ＊ " is selected from C(M), 5(m), or 5(L) as described below, and the symbol "G ＊" is selected from G(M), G(m), or G(L) as described below, and the symbol "T ＊ " is selected from U(M), T(m), or T(L) as described below. In this case, the symbol "C" contained in the single-stranded antisense oligonucleotide ＊ " may be U(M).

[0103] <Single-stranded antisense oligonucleotide: SSO sequence design> The single-stranded antisense oligonucleotide SSO is a compound for skipping exon 3 of the human ATXN7 gene, which contains a CAG repeat region. Therefore, the structure of the SSO is designed so that it can bind to the target region of human ATXN7 RNA (exon 3 or its vicinity) (Figure 5). In this case, sugar-modified nucleic acids are arranged on the single-stranded antisense oligonucleotide to prevent it from being recognized by nucleases, which are nucleic acid-degrading enzymes.

[0104] <Base sequence of single-stranded antisense oligonucleotide functioning as SSO> The base sequence of the single-stranded antisense oligonucleotide (SSO) according to this embodiment is: (A) A base sequence having 90% or more and 100% sequence identity to a base sequence complementary to a target region consisting of a contiguous 18-25 mer (preferably 20-25 mer) from the base located at position 6, 8 to 15, 26, 282, 287, 292, 297, 302, or 317 in the base sequence of SEQ ID NO: 6, counting from the 5' end; or a base sequence having 90% or more and 100% sequence identity to a base sequence complementary to a target region consisting of a contiguous 15-30 mer (preferably 18-25 mer) from the base located at position 49450, 49452 to 49459, 49470, 49726, 49731, 49736, 49741, 49746, 49761, or 49781 in the base sequence of SEQ ID NO: 7, counting from the 5' end; or (B) a base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted, or added in the target region, or (C) a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region. In this embodiment, each base sequence shown in the sequence listing is used to indicate only the sequence information of the nucleobase portion. The structural information of the antisense oligonucleotide (SSO), including the nucleobase portion, sugar portion, and phosphate portion, is shown in the format shown in Tables 4-1 to 4-9 and Tables 6-1 to 6-3, which will be described later.

[0105] In another aspect of the single-stranded antisense oligonucleotide (SSO) according to this embodiment, the target region is preferably a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to a target region consisting of a contiguous 18-25 mer starting from bases located at positions 8 to 11, 282, 287, or 302, counting from the 5' end, or a target region consisting of a contiguous 18-25 mer starting from bases located at positions 49452 to 49455, 49726, 49731, or 49746, counting from the 5' end, in the base sequence set forth in SEQ ID NO: 7.

[0106] In one aspect of the single-stranded antisense oligonucleotide (SSO) according to this embodiment, the base sequence of the single-stranded antisense oligonucleotide is preferably one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 236, 260 to 263, 265 to 267, 269, 271, 292, and 293. In another aspect of this embodiment, when the SSO is a double-stranded antisense oligonucleotide, the double-stranded antisense oligonucleotide is preferably at least one double-stranded antisense oligonucleotide selected from the group consisting of the double-stranded oligonucleotide sequence names SP-AX7-195, SP-AX7-205, SP-AX7-206, and SP-AX7-209 in Tables 6-1 to 6-3 described below.

[0107] One embodiment of the single-stranded antisense oligonucleotide (SSO) of the present invention is a single-stranded antisense oligonucleotide capable of skipping exon 3 of human ATXN7 mRNA, which has any of the nucleotide sequences listed in Tables 2-1 to 2-3, and which is complementary to a target region in the pre-mRNA of the human ATXN7 gene. Furthermore, as long as the single-stranded antisense oligonucleotide contains a nucleotide sequence listed in Tables 2-1 to 2-3, it may extend by 1 to 5 bases on the 3' and / or 5' side. The target region can be said to be a region in the human ATXN7 pre-mRNA that is particularly involved in the regulation of expression of the human ATXN7 gene (e.g., a region having a secondary structure in the pre-mRNA that is easily bound by antisense nucleotides). For example, in Table 2-1, if the 5'-terminal position of SEQ ID NO:6 (corresponding to exon 3 of SEQ ID NO:7) is "5" and the 3'-terminal position is "24," the contiguous 20-mer nucleotide sequence from the 5th to the 24th nucleotide counting from the 5'-terminal in the nucleotide sequence of SEQ ID NO:6 is the target region in human ATXN7 RNA targeted by the corresponding single-stranded antisense oligonucleotide (SSO). Also, in Table 2-1, if the 5'-terminal position of SEQ ID NO:6 is "*" and the 3'-terminal position is "15," this means that the 5'-terminal position of the target RNA in the nucleotide sequence of SEQ ID NO:6 is not present, but the 5'-terminal position of the target RNA is located in the second intron linked to exon 3 in the human ATXN7 precursor, and the 3'-terminal position of the target RNA is the 15th base counting from the 5'-terminal of SEQ ID NO:6. In this case, the continuous base sequence from the 5'-terminal position to the 3'-terminal position set forth in SEQ ID NO: 7 becomes the target region in the human ATXN7 pre-mRNA targeted by the corresponding single-stranded antisense oligonucleotide (SSO).Furthermore, in Table 2-2, when the 5'-end position of SEQ ID NO: 6 is "327" and the 3'-end position is "*", this means that the 5'-end position of the target RNA in the base sequence of SEQ ID NO: 6 is the 327th base counting from the 5'-end position of SEQ ID NO: 6, the 3'-end position of the target RNA is not in SEQ ID NO: 6, and the 3'-end position of the target RNA is in the third intron linked to the third exon in the human ATXN7 pre-mRNA. In this case, the consecutive base sequence from the 5'-end position to the 3'-end position of SEQ ID NO: 7 is the target region in human ATXN7 RNA targeted by the corresponding single-stranded antisense oligonucleotide (SSO).

[0108]

[0109]

[0110]

[0111] The single-stranded antisense oligonucleotide (gapmer or SSO) can bind to a target region of human ATXN7 RNA. As used herein, the term "binding to a target region of human ATXN7" of the single-stranded antisense oligonucleotide of the present invention encompasses direct binding of the single-stranded antisense oligonucleotide of the present invention to human ATXN7 mRNA and direct binding to human ATXN7 pre-mRNA.

[0112] The base sequence of the single-stranded antisense oligonucleotide (gapmer or SSO) according to this embodiment is a base sequence complementary to the above-mentioned predetermined target region in the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and preferably has a sequence identity of 95% or more and 100% or less, more preferably 98% or more and 100% or less, and even more preferably 100% sequence identity.

[0113] In this embodiment, "sequence identity" refers to the percentage (%) of identical bases in the total overlapping base sequence in the optimal alignment when two base sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm can take into account the introduction of gaps into one or both of the sequences for optimal alignment). The "sequence identity" of a base sequence can be easily confirmed by those skilled in the art. For example, NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) can be used.

[0114] In this embodiment, examples of "a base sequence in which one or several bases have been deleted, substituted, inserted or added" include a base sequence that has, due to the deletion, substitution, insertion or addition, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more sequence identity to the base sequence before the deletion, substitution, insertion or addition. The specific number of "one or several bases" may be one, two, three, four, or five of the above-mentioned deletions, substitutions, insertions or additions, each independently, or a combination of multiple deletions, substitutions, insertions or additions.

[0115] In this embodiment, "stringent conditions" refers to conditions in which the sample is incubated for 12 hours at room temperature in a solution containing 6xSSC (1xSSC has the composition: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5x Denhardt's solution, 100 μg / mL denatured salmon sperm DNA, and 50% (v / v) formamide, followed by washing in 0.5xSSC at a temperature of 50°C or higher. More stringent conditions are also encompassed, such as incubation at 45°C or 60°C for 12 hours, washing in 0.2xSSC or 0.1xSSC, or washing at a temperature of 60°C or 65°C or higher.

[0116] <Pharmacologically Acceptable Salts> The single-stranded antisense oligonucleotide (gapmer or SSO) according to this embodiment may be in the form of a pharmacologically acceptable salt. Here, "pharmacologically acceptable salts" refers to salts of the single-stranded antisense oligonucleotide of the present invention, which are physiologically acceptable salts of the single-stranded antisense oligonucleotide of the present invention, i.e., salts that retain the desired biological activity of the single-stranded antisense oligonucleotide and do not retain undesired toxicological effects. The same applies to the double-stranded antisense oligonucleotides and antisense oligonucleotide complexes described below.

[0117] Pharmaceutically Acceptable Salts In one aspect of this embodiment, the single-stranded antisense oligonucleotide (gapmer or SSO) may be in the form of a pharmaceutically acceptable salt. Here, "pharmaceutically acceptable salts" refers to the pharmacologically acceptable salts described above that are acid addition salts or base addition salts. Examples of acid addition salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate, as well as organic acid salts such as citrate, oxalate, phthalate, fumarate, maleate, succinate, malate, acetate, formate, propionate, benzoate, trifluoroacetate, methanesulfonate, benzenesulfonate, para-toluenesulfonate, and camphorsulfonate. Examples of base addition salts include inorganic base salts such as sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, and aluminum salts, as well as organic base salts such as trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, tromethamine [tris(hydroxymethyl)methylamine], tert-butylamine, cyclohexylamine, dicyclohexylamine, and N,N-dibenzylethylamine. Further examples include salts with basic or acidic amino acids such as arginine, lysine, ornithine, aspartic acid, and glutamic acid (amino acid salts). The same applies to the double-stranded antisense oligonucleotides and antisense oligonucleotide complexes described below.

[0118] <Gapmer Structure in Single-Stranded Antisense Oligonucleotide> The single-stranded antisense oligonucleotide (gapmer) according to this embodiment comprises a gap region, a 3' wing region attached to the 3' end of the gap region, and a 5' wing region attached to the 5' end of the gap region (see, for example, FIG. 2). The gapmer is preferably in a single-stranded form. In one aspect of this embodiment, the gapmer may hybridize with a second-strand oligonucleotide described below to form a double-stranded form (double-stranded antisense oligonucleotide). The base sequence of the second-strand oligonucleotide is complementary to the base sequence of the gapmer and has a sequence identity of 90% to 100% based on the base sequence of the complementary strand.

[0119] The single-stranded antisense oligonucleotide is a so-called gapmer-type single-stranded antisense oligonucleotide. Gapmer-type single-stranded antisense oligonucleotides inhibit the function of or cleave target RNA through the following mechanism: First, the gapmer binds to the target region of the target RNA (top to center of Figure 3). Next, RNase H, an RNase, recognizes and binds to the complex of the gapmer and target RNA (center of Figure 3). The target RNA is then cleaved and degraded by an enzymatic degradation reaction catalyzed by RNase H. At this time, the gapmer is not affected by enzymatic degradation by RNase H (bottom of Figure 3). Therefore, the gapmer can bind to another target RNA and cleave and degrade that RNA. In this way, gapmer-type single-stranded antisense oligonucleotides function as a catalyst in the enzymatic degradation reaction catalyzed by RNase H, and are therefore thought to have a sustained effect even when administered in small amounts.

[0120] Furthermore, in this embodiment, the single-stranded antisense oligonucleotide (gapmer) can be suitably used to regulate the expression of the human ATXN7 gene (including mutant human ATXN7 genes) through the mechanism described above (including when it acts via regulating the maturation of human ATXN7 pre-mRNA). Furthermore, according to this embodiment, the effect of regulating the expression of the human ATXN7 gene by the gapmer can be exerted even through intrathecal administration, which is an administration route commonly used in clinical applications. Here, "regulating the expression of the human ATXN7 gene" means at least suppressing the expression of the human ATXN7 gene.

[0121] (Gap Region) The gap region in the single-stranded antisense oligonucleotide (gapmer) is preferably a 5-20 mer nucleic acid composed of deoxyribose, which may contain a modified nucleic acid in the sugar moiety. In other words, the gap region can also be understood as a 5-20 mer nucleic acid containing deoxyribose, which may contain a modified deoxyribose in the sugar moiety. The gap region can also be understood as a 5-20 mer nucleic acid composed of natural nucleotides, non-natural nucleotides, or both, whose sugar moiety is deoxyribose. By using deoxyribose or modified deoxyribose in the sugar moiety, the gap region can form a complex (double-stranded) recognizable by RNase H with the target RNA, such as human ATXN7 mRNA. Here, an example of a nucleic acid containing modified deoxyribose is 5'-CP nucleic acid (5'-cyclopropyl nucleic acid).

[0122] The number of bases in the gap region of the single-stranded antisense oligonucleotide (gapmer) is preferably 5 to 20 mer, more preferably 6 to 17 mer, even more preferably 8 to 13 mer, even more preferably 8 to 12 mer, and particularly preferably 7 to 11 mer.

[0123] Examples of natural nucleotides in which the sugar moiety in the single-stranded antisense oligonucleotide (gapmer) is deoxyribose include deoxyadenosine monophosphate, deoxyguanosine monophosphate, thymidine monophosphate, deoxycytidine monophosphate, deoxy-5-methylcytidine monophosphate (also referred to as "5-methyldeoxycytidine"), etc. In other words, natural nucleotides constituting the gap region include those containing structural formulas represented by the symbols a, g, t, c, and 5(x), which will be described later.

[0124] Examples of non-natural nucleotides in which the sugar moiety in the single-stranded antisense oligonucleotide (gapmer) is deoxyribose or modified deoxyribose include 5'-CP nucleic acid, 2-thio-thymidine monophosphate, 2-aminoadenosine monophosphate, and 7-deazaguanosine monophosphate.

[0125] In addition, the gap region in the single-stranded antisense oligonucleotide (gapmer) may be a nucleic acid in which some of the sugar moieties of natural nucleotides whose sugar moieties are deoxyribose are modified sugars, as long as the effects of the present invention are achieved. That is, in one aspect of this embodiment, the gap region may be a nucleic acid in which some of the sugar moieties are deoxyribose and other sugar moieties are modified sugars (e.g., modified deoxyribose).

[0126] (3' Wing Region) The 3' wing region in the single-stranded antisense oligonucleotide (gapmer) is a modified nucleic acid. In other words, the 3' wing region can be understood to be composed of modified nucleotides, and "modified nucleotides" include 2'-position modified nucleic acids (e.g., 2'-O-alkylated nucleic acids) and bridged modified nucleic acids. The modified nucleic acid in the 3' wing region is preferably selected from the group consisting of 2'-O-alkylated nucleic acids or bridged modified nucleic acids. Furthermore, the modified nucleic acid in the 3' wing region is preferably 2'-O-methyl nucleic acid and 2'-MOE nucleic acid as 2'-position modified nucleic acids, and LNA, AmNA, GuNA, and scpBNA as bridged modified nucleic acids, and more preferably includes at least one selected from the group of modified nucleic acids consisting of these. By having the 3' wing region and the 5' wing region described below composed of the specified modified nucleotides, high binding affinity to the target RNA can be expected, and ultimately the function of the target RNA can be effectively inhibited. In one aspect of this embodiment, the 3' wing region may be a modified nucleic acid in which the sugar moiety is a modified sugar. Examples of modified nucleic acids in which the sugar moiety is a modified sugar include those listed above (Sugar modification, Modified sugar). In one aspect of this embodiment, the modified nucleic acid in the 3' wing region may be composed solely of 2'-MOE nucleic acid. In this case, cytotoxicity tends to be suppressed. Note that multiple types of modified nucleic acids in the 3' wing region may be contained in a single gapmer.

[0127] The number of bases in the 3' wing region is preferably 2 to 5 mer, more preferably 3 to 5 mer.

[0128] (5' Wing Region) The 5' wing region of the single-stranded antisense oligonucleotide (gapmer) is a modified nucleic acid. In other words, the 5' wing region can be understood to be composed of modified nucleotides, and "modified nucleotides" include 2'-position modified nucleic acids (e.g., 2'-O-alkylated nucleic acids) and bridged modified nucleic acids. The modified nucleic acid in the 5' wing region is preferably selected from the group consisting of 2'-O-alkylated nucleic acids and bridged modified nucleic acids. Furthermore, the modified nucleic acid in the 5' wing region preferably includes at least one selected from the group of modified nucleic acids, such as 2'-O-methyl nucleic acids and 2'-MOE nucleic acids as 2'-position modified nucleic acids and LNA, AmNA, GuNA, and scpBNA as bridged modified nucleic acids. In one aspect of this embodiment, the 5' wing region may be a modified nucleic acid in which the sugar moiety is a modified sugar. Examples of modified nucleic acids in which the sugar moiety is a modified sugar include those listed above (Sugar modification, modified sugar). In one aspect of this embodiment, the modified nucleic acid in the 5' wing region may be composed solely of 2'-MOE nucleic acid. In this case, cytotoxicity tends to be suppressed. Note that multiple types of modified nucleic acid in the 5' wing region may be contained in one gapmer. In another aspect of this embodiment, the modified nucleic acid in the 3' wing region and the 5' wing region may be composed solely of 2'-MOE nucleic acid.

[0129] The number of bases in the 5' wing region is preferably 2 to 5 mer, more preferably 3 to 5 mer.

[0130] In one aspect of the single-stranded antisense oligonucleotide (gapmer) of this embodiment, it is preferred that the number of bases in the gap region is 6 to 17 mer, the number of bases in the 3' wing region is 2 to 5 mer, and the number of bases in the 5' wing region is 2 to 5 mer.

[0131] In one aspect of the single-stranded antisense oligonucleotide (gapmer) according to this embodiment, it is more preferred that the number of bases in the gap region is 7 to 13 mer, the number of bases in the 3' wing region is 3 to 5 mer, and the number of bases in the 5' wing region is 3 to 5 mer.

[0132] In one aspect of the single-stranded antisense oligonucleotide (gapmer) according to this embodiment, it is more preferred that the number of bases in the gap region is 8 to 12 mer, the number of bases in the 3' wing region is 3 to 5 mer, and the number of bases in the 5' wing region is 3 to 5 mer.

[0133] In one aspect of the single-stranded antisense oligonucleotide (gapmer) according to this embodiment, it is more preferred that the number of bases in the gap region is 7 to 11 mer, the number of bases in the 3' wing region is 3 to 5 mer, and the number of bases in the 5' wing region is 3 to 5 mer.

[0134] The base length of the single-stranded antisense oligonucleotide (gapmer) of the present invention is 12 to 30 mer, preferably 12 to 22 mer, more preferably 15 to 22 mer, even more preferably 18 to 22 mer, and particularly preferably 18 to 20 mer. When the base length of the gapmer of the present invention is 15 to 22 mer, 18 to 22 mer, or 18 to 20 mer, binding to human ATXN7 mRNA or human ATXN7 pre-mRNA is particularly strong, and regulation of human AXTN7 gene expression can be more effectively achieved. In one aspect of this embodiment, when the base length of the gapmer is within the above-mentioned range, binding to off-target genes is suppressed, thereby avoiding undesired suppression or regulation of off-target gene expression, and the antisense oligonucleotide can be expected to be safer.

[0135] In this embodiment, the gapmer has each nucleoside linked via a phosphate group and / or a modified phosphate group, and is preferably linked via a phosphodiester bond or a phosphorothioate bond.

[0136] One embodiment of the single-stranded antisense oligonucleotide (gapmer) of the present invention is a single-stranded antisense oligonucleotide having a gap region consisting of 5 to 20 mers, a 5' wing region consisting of 2 to 5 mers, and a 3' wing region consisting of 2 to 5 mers. The gap region is located between the 5' wing region and the 3' wing region. Preferably, the 5' wing region and the 3' wing region each contain at least one 2'-O-alkylated nucleic acid or 2',4'-bridged modified nucleic acid. In one aspect of this embodiment, the 3' wing region may be composed of a 2'-MOE nucleic acid or a 2',4'-bridged modified nucleic acid, and the 5' wing region may be composed of a 2'-MOE nucleic acid or a 2',4'-bridged modified nucleic acid. The 2'-O-alkylated nucleic acid may be a 2'-O-alkylated nucleic acid of D-ribofuranose (e.g., a 2'-O-methylated nucleic acid, a 2'-MOE nucleic acid, etc.). The gapmer may also form a double strand by hybridizing with a second strand oligonucleotide.

[0137] (Method of notating gapmer structure) When notating the structure of the single-stranded antisense oligonucleotide (gapmer) of the present invention, the notation "X(5')-X(gap)-X(3')" may be used. In the above notation, "X(5')" indicates the number of bases in the 5' wing region, "X(3')" indicates the number of bases in the 3' wing region, and "X(gap)" indicates the number of bases in the gap region.

[0138] Examples of "X(5')-X(gap)-X(3')" include 2-8-4, 2-8-3, 2-8-5, 2-9-2, 2-9-3, 2-9-4, 2-9-5, 2-10-3, 2-10-4, 2-10-5, 2-11-3, 2-11-4, 2-11-5, 2-12-3, 2-12-4, 2-12-5, 3-8-2, 3-8-3, 3-8-4, 3-8-5, 3-9-3, 3-9-4, 3-9-5, 3-10-3, 3-10-4, 3-10-5, 3-11-3, 3-11-4, 3-11-5, 3-12-3, 3-12-4, 3-12-5, 3-13-3, 4 -8-2, 4-8-3, 4-8-4, 4-8-5, 4-9-3, 4-9-4, 4-9-5, 4-10-3, 4-10-4, 4-10-5, 4-11-2, 4-11-3, 4-11-4, 4-11-5, 4-12-3, 4-12-4, 4-12-5, 5-8-2, 5-8-3, 5-8-4, 5-8-5, 5-9-2, 5-9-3, 5-9-4, 5-9-5, 5-10-2, 5-10-3, 5-10-4, 5-10-5, 5-11-2, 5-11-3, 5-11-4, 5-11-5, 5-12-3, 5-12-4, 5-12-5, etc. For example, "2-8-4" means that the 5' wing region is a 2-mer oligonucleotide, the 3' wing region is a 4-mer oligonucleotide, and the gap region is an 8-mer oligonucleotide.

[0139] <Structure of SSO of Single-Stranded Antisense Oligonucleotide> The SSO of the single-stranded antisense oligonucleotide according to this embodiment is composed of a natural oligonucleotide and / or a non-natural oligonucleotide. The SSO is preferably in a single-stranded form. In one aspect of this embodiment, the SSO may hybridize with a second-strand oligonucleotide described below to form a double-stranded form (double-stranded antisense oligonucleotide). The base sequence of the second-strand oligonucleotide preferably has a sequence identity of 90% to 100% based on a base sequence complementary to the base sequence of the SSO.

[0140] <Mechanism of Exon Skipping by SSO> The single-stranded antisense oligonucleotide (SSO) skips an exon in a target RNA and regulates the expression and / or function of the target RNA through the following mechanism (see Figure 4). An exon is included in mRNA only when both splice sites are recognized by the spliceosome complex. Targeting a splice site with an antisense oligonucleotide (SSO) inhibits splicing and induces exon skipping. Exon skipping can be induced by binding to an exon with a single-stranded antisense oligonucleotide (SSO) that targets either or both of the 5' splice site and 3' splice site of an exon, or the interior of an exon. That is, the SSO binds to the target region of the target RNA and inhibits splicing of the exon, thereby allowing the target exon to be skipped, thereby regulating the expression and / or function of the target RNA.

[0141] Furthermore, in this embodiment, the single-stranded antisense oligonucleotide (SSO) can be suitably used to induce modified human ATXN7 mRNA in which exon 3 of human ATXN7 mRNA has been skipped by the mechanism described above. More specifically, the SSO binds to exon 3, which is the target region of the target RNA (human ATXN7 pre-mRNA), or a region adjacent thereto (upper panel of Figure 4), and inhibits splicing of exon 3. As a result, exon 3 containing the abnormally expanded CAG found in patients with spinocerebellar ataxia type 7 is skipped, thereby regulating its expression and / or function. Furthermore, according to this embodiment, the effect of SSO on regulating the expression of modified human ATXN7 mRNA in which exon 3 has been skipped can be exerted even through administration routes commonly used in clinical applications, such as intravenous administration, intrathecal administration, and intracerebroventricular administration. Here, "the third exon of human ATXN7 mRNA is skipped" at least means that the third exon of human ATXN7 mRNA is skipped, resulting in the induction of expression of modified ATXN7 mRNA.

[0142] Examples of natural nucleotides in which the sugar moiety in the single-stranded antisense oligonucleotide (SSO) is deoxyribose include deoxyadenosine monophosphate, deoxyguanosine monophosphate, thymidine monophosphate, deoxycytidine monophosphate, deoxy-5-methylcytidine monophosphate, etc. In other words, natural nucleotides constituting the SSO include those containing structural formulas represented by the symbols a, g, t, c, and 5(x), which will be described later.

[0143] Examples of the non-natural nucleotide in which the sugar moiety in the single-stranded antisense oligonucleotide (SSO) is deoxyribose include 2-thio-thymidine monophosphate, 2-aminoadenosine monophosphate, and 7-deazaguanosine monophosphate.

[0144] The single-stranded antisense oligonucleotide (SSO) of the present invention has a base length of 10 to 30 mer, preferably 15 to 25 mer, more preferably 18 to 25 mer, and even more preferably 20 to 25 mer. When the base length of the SSO of the present invention is 15 to 25 mer, 18 to 25 mer, or 20 to 25 mer, it exhibits particularly strong binding to human ATXN7 pre-mRNA and can more effectively induce highly efficient skipping of exon 3 of human ATXN7 mRNA.

[0145] In this embodiment, the single-stranded antisense oligonucleotide (SSO) has each nucleotide linked via a phosphate group and / or a modified phosphate group, and is preferably linked via a phosphodiester bond or a phosphorothioate bond.

[0146] <Double-Stranded Antisense Oligonucleotide> The double-stranded antisense oligonucleotide of this embodiment is a double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, comprising the single-stranded antisense oligonucleotide (gapmer or SSO) and a second-strand oligonucleotide hybridized to the single-stranded antisense oligonucleotide. The base sequence of the second-strand oligonucleotide is complementary to the base sequence of the single-stranded antisense oligonucleotide and has a sequence identity of 90% to 100% with respect to the base sequence of the complementary strand.

[0147] The double-stranded antisense oligonucleotide can be dissociated in solution and separated into the single-stranded antisense oligonucleotide and the second-stranded oligonucleotide.The separated single-stranded antisense oligonucleotide can bind to the target RNA.The single-stranded antisense oligonucleotide can also be understood as a "first-stranded oligonucleotide" in relation to the second-stranded oligonucleotide.In addition, among the oligonucleotides constituting the double-stranded antisense oligonucleotide, the first-stranded oligonucleotide has an antisense strand to the target RNA, but for convenience, the double-stranded oligonucleotide consisting of the first-stranded oligonucleotide and the second-stranded oligonucleotide will be referred to as a "double-stranded antisense oligonucleotide".

[0148] <<Method for Producing Single-Stranded Antisense Oligonucleotides>> The single-stranded antisense oligonucleotides (gapmers or SSOs) of the present invention can be produced by solid-phase synthesis using the phosphoramidite method. For example, a single-stranded oligonucleotide having a predetermined base sequence is first synthesized on a solid support using a commercially available automated nucleic acid synthesizer. Next, the synthesized single-stranded oligonucleotide is cleaved from the solid support using a basic substance or the like, and deprotected to obtain a crude single-stranded oligonucleotide. The crude single-stranded oligonucleotide obtained is then purified using HPLC or the like. The single-stranded antisense oligonucleotides of the present invention can be produced by any method known to those skilled in the art, without being limited to the above-described production method, by appropriately modifying the base sequence, modification site, etc. of the nucleic acid. 2'-MOE nucleic acids can be produced using amidites that are commercially available as reagents. LNAs can be produced by the method described in WO 99 / 14226 (Patent Document 3). In addition, morpholino oligonucleotides (PMO) can be produced according to the methods described in International Publication No. 1991 / 009033 (Patent Document 1), International Publication No. 2009 / 064471 (Patent Document 2), or J. Am. Chem. Soc. 2016 Dec 7; 138(48): 15663-15672. (Non-Patent Document 10).

[0149] <<Method for Producing Double-Stranded Antisense Oligonucleotide>> The double-stranded antisense oligonucleotide of the present invention can be produced by first producing an oligonucleotide (second strand oligonucleotide) having a predetermined sequence identity based on a base sequence complementary to the single-stranded antisense oligonucleotide using a production method similar to that of the single-stranded antisense oligonucleotide, and then hybridizing the single-stranded antisense oligonucleotide and the second strand oligonucleotide.

[0150] <Antisense Oligonucleotide Complex> The antisense oligonucleotide complex of this embodiment comprises: the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof; or the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof; and an additional substance bound to the single-stranded antisense oligonucleotide or the second-stranded oligonucleotide directly or via a linker bond. The additional substance is selected from the group consisting of polyethylene glycol, peptide, alkyl chains that may contain unsaturated bonds (e.g., saturated aliphatic hydrocarbons, unsaturated aliphatic hydrocarbons, etc.), ligand compounds (e.g., compounds with organ-targeting function, compounds with cell-targeting function, etc.), antibodies, proteins, sugar chains (e.g., carbohydrates, polysaccharides, etc.), and oligomeric nucleic acids. The additional substance may be bound to the oligonucleotide via a linker substance. Examples of the linker substance include linkers composed of a phosphate group (including a modified phosphate group), an alkyl chain, a polyethylene glycol chain, a peptide chain, a disulfide group, an ester group, an amide group, a carbonyl group, etc., and / or a combination thereof. The method for binding the additional substance to the oligonucleotide includes, for example, a production method using solid phase synthesis by the phosphoramidite method.

[0151] In one aspect of this embodiment, the antisense oligonucleotide complex comprises the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, and an additional substance bound to the single-stranded antisense oligonucleotide or the second strand oligonucleotide directly or via a linker bond, wherein the additional substance is selected from the group consisting of polyethylene glycol, peptide, alkyl chains which may contain unsaturated bonds (e.g., saturated aliphatic hydrocarbons, unsaturated aliphatic hydrocarbons, etc.), ligand compounds (e.g., compounds having organ-targeting function, compounds having cell-targeting function, etc.), antibodies, proteins, sugar chains (e.g., carbohydrates, polysaccharides, etc.), and oligomeric nucleic acids.

[0152] In this embodiment, the term "additional substance" refers to a substance bound to the oligonucleotide and used to impart a predetermined effect. The additional substance may be bound to the 5' end, the 3' end, or both the 5' and 3' ends of the oligonucleotide. In one aspect of this embodiment, the additional substance is preferably bound to either the 5' end or the 3' end of the oligonucleotide. Alternatively, the additional substance may be directly covalently bound to the oligonucleotide. In one aspect of this embodiment, the additional substance may be attached to a single-stranded antisense oligonucleotide (corresponding to the first strand oligonucleotide in a double-stranded antisense oligonucleotide) or to the second strand oligonucleotide in a double-stranded antisense oligonucleotide.

[0153] Examples of peptides that can be used as the additional substance include, but are not limited to, CPPs (Cell Penetrating Peptides), nuclear transport peptides, TAT (Trans-Activator of Transcription Protein), polyarginine, glucagon-like peptide-1 analogue peptides, synthetic cyclic RGD peptides, and brain-translocating peptides.

[0154] Examples of the ligand compound used as the additional substance include, but are not limited to, N-acetylgalactosamine (GalNAc), sugars (glucose, mannose, etc.), lipids (cholesterol, palmitic acid, docosahexaenoic acid, etc.), vitamins (folic acid, vitamin A, vitamin E (tocopherol), etc.), amino acids, and monoamine receptor ligands (indatraline, etc.).

[0155] The antibody used as the additional substance is an immunoglobulin and / or a single domain antibody (also called a nanobody), and examples thereof include, but are not limited to, the following: an anti-insulin receptor antibody, an anti-transferrin receptor antibody, an anti-LDL receptor-related protein antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-HER2 antibody, and an anti-CD98 antibody.

[0156] Examples of proteins that can be used as the additional substance include, but are not limited to, the following: albumin

[0157] <<Expression Regulator (Expression and / or Function Regulator) of Human ATXN7 Gene>> The expression regulator (expression and / or function regulator) of the human ATXN7 gene according to this embodiment comprises, as an active ingredient, the single-stranded antisense oligonucleotide (gapmer or SSO), the double-stranded antisense oligonucleotide, or the antisense oligonucleotide conjugate of the present invention. In one aspect of this embodiment, the expression regulator can also be understood as an expression inhibitor for the human ATXN7 gene. The expression regulator includes a gapmer that induces degradation of human ATXN7 RNA via the RNase RNase H, and an SSO that induces degradation of modified human ATXN7 RNA (RNA in which the third exon of human ATXN7 mRNA has been skipped) by a quality control mechanism present in cells. In another aspect of this embodiment, the expression regulator can also be understood as an expression inhibitor for a mutant human ATXN7 gene containing an abnormally expanded CAG repeat. In another aspect of this embodiment, the expression regulator can also be understood as an inhibitor of the abnormal structure of a mutant human ATXN7 gene containing an abnormally expanded CAG repeat. In another aspect of this embodiment, the expression regulator can also be understood as an inhibitor of the expression of a human mutant ATXN7 protein with an abnormal polyarginine chain insertion translated from a mutant human ATXN7 gene containing an abnormally expanded CAG repeat. In another aspect of this embodiment, the expression regulator can also be understood as an inhibitor of cell death mediated by the abnormal structure of the mutant human ATXN7 gene and / or the human mutant ATXN7 protein with an abnormal polyarginine chain insertion. The single-stranded antisense oligonucleotide of the present invention binds to human mutant ATXN7 mRNA or human mutant ATXN7 pre-mRNA, thereby inhibiting the expression of the human mutant ATXN7 gene and suppressing its abnormal structure and / or cell death induction caused by its translation product. Any administration method and formulation known in the art can be used for the expression regulator of the human ATXN7 gene of the present invention.

[0158] <<Skipping Agent for Exon 3 of Human ATXN7 Gene>> The skipping agent for exon 3 of the human ATXN7 gene according to this embodiment comprises, as an active ingredient, the single-stranded antisense oligonucleotide (SSO), the double-stranded antisense oligonucleotide, or the antisense oligonucleotide conjugate of the present invention. The single-stranded antisense oligonucleotide of the present invention is capable of skipping exon 3 of the human ATXN7 gene by binding to human ATXN7 pre-mRNA. In one aspect of this embodiment, the skipping agent can be understood as a skipping agent for exon 3 containing an abnormally expanded CAG repeat in a mutant human ATXN7 gene. In another aspect of this embodiment, the skipping agent can be understood as an inhibitor of abnormal structuring of a mutant human ATXN7 gene by skipping exon 3 containing an abnormally expanded CAG repeat from the mutant human ATXN7 gene. In another aspect of this embodiment, the skipping agent can be understood as an agent for suppressing the expression of an abnormal polyarginine chain-inserted human mutant ATXN7 protein by skipping exon 3 containing an abnormally expanded CAG repeat from the mutant human ATXN7 gene. Any administration method and formulation known in the art can be used for the skipping agent.

[0159] <<Modified Human ATXN7 Gene Inducer>> The modified human ATXN7 gene inducer of this embodiment comprises the single-stranded antisense oligonucleotide (SSO), the double-stranded antisense oligonucleotide, or the antisense oligonucleotide conjugate of the present invention as an active ingredient. The single-stranded antisense oligonucleotide (SSO) of the present invention is a modified human ATXN7 inducer that skips exon 3 of the human ATXN7 gene and does not contain an abnormally expanded CAG repeat sequence. In one aspect of this embodiment, the inducer can also be understood as a modified human ATXN7 inducer in which exon 3 has been skipped (removed) from a mutant human ATXN7 gene containing an abnormally expanded CAG repeat. Any administration method and formulation known in the art can be used for the inducer.

[0160] <<Pharmaceutical Composition (Drug) Containing Antisense Oligonucleotide or the Like as an Active Ingredient>> The pharmaceutical composition (drug) according to this embodiment contains, as an active ingredient, the single-stranded antisense oligonucleotide of the present invention or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof. Any administration method and formulation known in the art can be used for the pharmaceutical composition of this embodiment. Hereinafter, the pharmaceutical composition may be referred to as a "pharmaceutical composition of antisense oligonucleotide or the like."

[0161] The pharmaceutical composition is used for treating or preventing a disease associated with the human ATXN7 gene, i.e., a disease that can be caused by the abnormal structure of the human mutant ATXN7 gene and / or its translation product by suppressing the expression of the human mutant ATXN7 gene or skipping the third exon containing the abnormally expanded CAG repeat. In other words, the pharmaceutical composition can be used for treating or preventing spinocerebellar ataxia type 7 by suppressing the expression of the mutant human ATXN7 gene.

[0162] <Therapeutic and Preventive Agents for Spinocerebellar Degeneration Type 7> The therapeutic agent for spinocerebellar degeneration type 7 according to this embodiment comprises, as an active ingredient, the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof. The preventive agent for spinocerebellar degeneration type 7 according to this embodiment comprises, as an active ingredient, the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide conjugate or a pharmaceutically acceptable salt thereof.

[0163] <Individual> In the present invention, an individual refers to a mammal. Preferably, it is a human, monkey, marmoset, dog, pig, rabbit, guinea pig, rat, or mouse. More preferably, it is a human.

[0164] When administering the antisense oligonucleotide of the present invention or a pharmaceutical composition thereof (including a therapeutic or preventive agent for spinocerebellar ataxia type 7), the administration method and dosage form are not particularly limited. That is, any administration method and formulation known in the art can be used as the administration method and formulation of the antisense oligonucleotide of the present invention. Examples of administration methods include oral administration and parenteral administration. Examples of parenteral administration include ophthalmic administration, intravitreal administration, intravaginal administration, intrarectal administration, intranasal administration, transdermal administration, intravenous injection, infusion, subcutaneous administration, intraperitoneal administration or intramuscular injection, pulmonary administration by aspiration or inhalation, intrathecal administration, and intraventricular administration.

[0165] Various pharmaceutical additives such as excipients, binders, wetting agents, disintegrants, lubricants, diluents, flavoring agents, fragrances, solubilizers, suspending agents, emulsifiers, stabilizers, preservatives, and isotonic agents may be mixed into the formulations of the antisense oligonucleotides of the present invention as needed.

[0166] When pharmaceutical compositions such as the antisense oligonucleotides of the present invention are administered locally, formulations such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders can be used.

[0167] When pharmaceutical compositions of the present invention, such as antisense oligonucleotides, are orally administered, formulations such as powders, granules, suspensions or solutions dissolved in water or non-aqueous media, capsules, powders, tablets, etc. can be used.

[0168] When pharmaceutical compositions such as the antisense oligonucleotides of the present invention are administered parenterally, administration methods such as intravenous administration, intrathecal administration, or intraventricular administration can be used, and in this case, formulations such as sterile aqueous solutions can be used.

[0169] The effective dose of the antisense oligonucleotide of the present invention can be determined arbitrarily depending on the sex, age, body weight, symptoms, etc. of the individual to be administered. Furthermore, it can also be determined arbitrarily depending on the method, route, frequency, etc. of administration. For example, the dose may be 0.01 to 100 mg / kg body weight, preferably 0.1 to 50 mg / kg body weight, and more preferably 0.1 to 10 mg / kg body weight.

[0170] <<Method for regulating expression of the ATXN7 gene>> In this embodiment, the method for regulating expression of the ATXN7 gene includes a step of administering, as an active ingredient, the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof to a cell, tissue, or individual expressing the human ATXN7 gene.

[0171] <<Method for skipping exon 3 of ATXN7 gene>> The method for skipping exon 3 of the ATXN7 gene in this embodiment includes a step of administering, as an active ingredient, the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof to a cell, tissue, or individual expressing the ATXN7 gene.

[0172] In this embodiment, the antisense oligonucleotide or the like may be administered to a cell, tissue, or individual in vitro or in vivo. When administered in vivo, the administration route is the same as that described above.

[0173] In this embodiment, examples of "cells expressing the ATXN7 gene" include nerve cells that make up the central nervous system, nerve cells that make up the peripheral nervous system, photoreceptor cells that make up the optic nerve, and other cells that make up skin tissue.

[0174] In this embodiment, the method for treating or preventing spinocerebellar ataxia type 7 comprises the step of administering, as an active ingredient, the single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex or a pharmaceutically acceptable salt thereof to an individual suffering from spinocerebellar ataxia type 7.

[0175] The antisense oligonucleotide according to this embodiment has been described above. The antisense oligonucleotide having the above-described configuration is capable of regulating the expression of the human mutant ATXN7 gene and / or skipping the third exon of the human mutant ATXN7 gene. Here, the inhibitory activity (knockdown activity) on the expression of the human mutant ATXN7 gene can be measured by known methods. Examples of methods for measuring knockdown activity include the method described in Nature. 2015 Feb 19; 518(7539): 409-12. (Non-Patent Document 11). Furthermore, knockdown activity can also be measured by transfecting HEK293T cells or the like with the antisense oligonucleotide, as described below.

[0176] <<Introduction of Antisense Oligonucleotides into Cells>> "ATXN7 gene-expressing cells" are treated with antisense oligonucleotides for 6 hours to 7 days using methods such as lipofection, electroporation, or in-culture addition. Any cell expressing the ATXN7 gene may be used, including HEK293T cells, fibroblasts derived from patients with spinocerebellar ataxia type 7 (hereinafter referred to as "Spinocerebellar ataxia type 7 patient fibroblasts"), iPS cells induced from fibroblasts derived from patients with spinocerebellar ataxia type 7 (induced pluripotent stem cells; hereinafter referred to as "Spinocerebellar ataxia type 7 patient iPSCs"), and neural progenitor cells induced to differentiate from the spinocerebellar ataxia type 7 patient iPS cells (hereinafter referred to as "Spinocerebellar ataxia type 7 patient iPS-derived neural progenitor cells"). The cells treated with the antisense oligonucleotide are harvested by continuing to culture with or without removing the antisense oligonucleotide.

[0177] <<Method for Evaluating Inhibitory Activity Against ATXN7 Gene Expression>> Total RNA extracted from recovered cells is subjected to a reverse transcription reaction, and the resulting complementary DNA is subjected to real-time PCR or other methods using an ATXN7 gene-specific probe to measure the amount of ATXN7 mRNA. Examples of probes used in real-time PCR include Taqman probes. Reaction methods include, for example, a three-step process (cDNA denaturation, annealing, and extension) or a two-step process (cDNA denaturation, annealing, and extension) repeated any number of times. The two or three steps are repeated, for example, 25 to 45 times, preferably 35 to 40 times. The cDNA denaturation temperature is, for example, 90°C to 98°C, preferably 92°C to 95°C. The annealing temperature is, for example, 40°C to 70°C, preferably 50°C to 60°C. The temperature (for extension reaction) is, for example, 65°C to 75°C, preferably the optimum temperature for the polymerase used in the reaction. The temperature (for annealing and extension reaction) is, for example, 55°C to 70°C.

[0178] <Quantitative Evaluation of ATXN7 Protein> The collected cells are lysed to obtain an extract. The amount of ATXN7 protein contained in the extract is evaluated using immunochemical techniques such as Western blotting and ELISA (Enzyme-Linked Immunosorbent Assay). In Western blotting, any equipment can be used for the electrophoresis, transfer, and detection steps. The reaction time and temperature of the membrane with the primary or secondary antibody can be set as desired, for example, overnight at 4°C or 1 to 3 hours at room temperature.

[0179] <Evaluation of exon 3 skipping activity of the ATXN7 gene> Total RNA extracted from the recovered cells is subjected to reverse transcription, and the region surrounding exon 3 of the ATXN7 gene is amplified by PCR using the resulting cDNA. The PCR amplification product can be detected after electrophoresis on agarose gel or acrylamide gel, or by sequence analysis to confirm exon 3 skipping of the ATXN7 gene. The skipping activity can also be evaluated by performing real-time PCR on the polynucleotide of the PCR product containing the exon 3 region and comparing it with a negative control treated with a vehicle or administered with a drug.

[0180] The present invention is not limited to the above-described embodiments. For example, the antisense oligonucleotide includes the following embodiments.

[0181] One embodiment of the single-stranded antisense oligonucleotide (gapmer) of the present invention is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that regulates the expression and / or function of the human ATXN7 gene, wherein each nucleotide in the single-stranded antisense oligonucleotide is linked via a phosphate group and / or a modified phosphate group, the single-stranded antisense oligonucleotide comprises a gap region, a 3' wing region linked to the 3' end of the gap region, and a 5' wing region linked to the 5' end of the gap region, the gap region is a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the 3' wing region and the 5' wing region are modified nucleic acids, the base length of the single-stranded antisense oligonucleotide is 12 to 30 mer, and the base sequence of the single-stranded antisense oligonucleotide is The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof has a base sequence that has 90% to 100% sequence identity with respect to a base sequence complementary to at least one target region of the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, and that is composed of the same base length as the single-stranded antisense oligonucleotide.

[0182] In another embodiment of the single-stranded antisense oligonucleotide (gapmer) of the present invention, the base sequence of the single-stranded antisense oligonucleotide is a base sequence that has 95% to 100% sequence identity with respect to a base sequence complementary to at least one target region of the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, which has the same base length as the single-stranded antisense oligonucleotide.

[0183] In another embodiment of the single-stranded antisense oligonucleotide (gapmer) of the present invention, the base sequence of the single-stranded antisense oligonucleotide is a base sequence complementary to at least one target region in the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, the target region having the same base length as the single-stranded antisense oligonucleotide.

[0184] In another embodiment of the single-stranded antisense oligonucleotide (gapmer) of the present invention, the gap region is a 5-20mer nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the 3' wing region is a 2-5mer modified nucleic acid, and the 5' wing region is a 2-5mer modified nucleic acid.

[0185] In another embodiment of the single-stranded antisense oligonucleotide (gapmer) of the present invention, the single-stranded antisense oligonucleotide has a base length of 15 to 22 mer, the gap region is an 8 to 12 mer nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the 3' wing region is a 3 to 5 mer modified nucleic acid, the modified nucleic acid in the 3' wing region is selected from the group consisting of 2'-O-alkylated nucleic acids or cross-linked modified nucleic acids, the 5' wing region is a 3 to 5 mer modified nucleic acid, the modified nucleic acid in the 5' wing region is selected from the group consisting of 2'-O-alkylated nucleic acids or cross-linked modified nucleic acids, and at least one internucleotide bond in the single-stranded antisense oligonucleotide is a phosphorothioate bond.

[0186] One embodiment of the single-stranded antisense oligonucleotide (SSO) of the present invention is a single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that skips exon 3 in the human ATXN7 gene, wherein each nucleotide in the single-stranded antisense oligonucleotide is linked via a phosphate group and / or a modified phosphate group, the single-stranded antisense oligonucleotide comprises a modified nucleic acid having at least one modified sugar, the single-stranded antisense oligonucleotide has a base length of 15 to 30 mer, and the base sequence of the single-stranded antisense oligonucleotide has a sequence identity of 90% or more and 100% or less with respect to a base sequence complementary to at least one target region of the base sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7, which has the same base length as the single-stranded antisense oligonucleotide.

[0187] In another embodiment of the single-stranded antisense oligonucleotide (SSO) of the present invention, the base sequence of the single-stranded antisense oligonucleotide is a base sequence complementary to at least one target region in the base sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7, the target region having the same base length as the single-stranded antisense oligonucleotide (SSO).

[0188] In another embodiment of the single-stranded antisense oligonucleotide (SSO) of the present invention, the base length of the single-stranded antisense oligonucleotide is 18 to 25 mer, the number of bases in the gap region is 8 to 12 mer, and the gap region is a nucleic acid composed of deoxyribose which may contain a nucleic acid whose sugar moiety is modified, the sugar modification constituting the single-stranded antisense oligonucleotide is represented by the above formula (A2), and the R ４ are each independently a methyl group, a methoxyethyl group, or an N-methylpropanamide group.

[0189] Examples of the present invention will be described below, but the present invention is not limited to these examples.

[0190] <<Preparation of Single-Stranded Antisense Oligonucleotides Against the ATXN7 Gene>> First, the single-stranded antisense oligonucleotides shown in Tables 3-1 to 3-10, Tables 4-1 to 4-9, Table 5, and Tables 6-1 to 6-3 were designed. For some of the designed oligonucleotides, single-stranded antisense oligonucleotides against the ATXN7 gene were prepared by the following procedure.

[0191] Single-stranded antisense oligonucleotides containing 2'-O-methyl nucleic acid, 2'-MOE nucleic acid, and / or LNA as modified nucleic acid were synthesized on a 0.2 μmol scale using an automated nucleic acid synthesizer (nS-8 model, manufactured by Gene Design Co., Ltd.). Chain elongation was carried out using a standard phosphoramidite protocol. CPG resin was used as the solid support. DDTT (((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazaoline-3-thione) or the like was used for sulfuration to form the phosphorothioated (PS) backbone. After oligomerization on the solid support was completed, the single-stranded antisense oligonucleotide was cleaved from the solid support by alkali treatment and recovered in solution. The solvent was then evaporated from the recovered solution to obtain a crude product. The resulting crude product was purified by reverse-phase HPLC to obtain purified single-stranded antisense oligonucleotides. The purity and structure of each single-stranded antisense oligonucleotide were confirmed by LC-MS (Waters).

[0192] The single-stranded antisense oligonucleotides prepared by the above-described method are listed in Tables 3-1 to 3-4, Tables 4-1 to 4-9, Table 5, and Tables 6-1 to 6-3 below. The single-stranded antisense oligonucleotides shown in Tables 3-1 to 3-10 are single-stranded antisense oligonucleotides against human ATXN7 mRNA that function as gapmers. Note that the underlined bases indicate mismatched bases with respect to human ATXN7 mRNA, and the single-stranded antisense oligonucleotides are single-stranded antisense oligonucleotides with 100% sequence identity to mouse atxn7 mRNA (SEQ ID NO: 8).

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203] The single-stranded antisense oligonucleotides shown in Tables 4-1 to 4-9 are single-stranded antisense oligonucleotides against the human ATXN7 pre-mRNA that function as SSOs.

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213] The single-stranded oligonucleotide shown in Table 5 is a second-stranded oligonucleotide that forms a complementary strand to the single-stranded antisense oligonucleotide that functions as an SSO. Figure 6 shows the structural formula of the sequence SP-AX7-180.

[0214]

[0215] The double-stranded oligonucleotides shown in Tables 6-1 to 6-3 are double-stranded oligonucleotides obtained by forming a complementary strand (hybridizing) with the single-stranded antisense oligonucleotides (SSOs) listed in Tables 4-1 to 4-9 and the second-strand oligonucleotides listed in Table 5. Figure 7 shows an electrophoresis photograph after double-strand formation.

[0216]

[0217]

[0218]

[0219] In this specification, the following symbols or notations may be used to represent corresponding structures. Note that no specific symbol is assigned to a phosphodiester bond. Therefore, the symbols representing each nucleoside are written adjacent to the portions linked via a phosphodiester bond.

[0220]

[0221]

[0222] <<ATXN7 gene expression evaluation>> The expression evaluation of the ATXN7 gene was carried out using human fetal kidney cells and mouse primary cultured neurons according to the single-stranded antisense oligonucleotide produced. In addition, the expression evaluation of the ATXN7 gene can also be carried out using various cells differentiated from human iPS cells. In this example, gene expression evaluation means evaluating the amount of mRNA by measuring the amount of complementary DNA (cDNA) obtained by reverse transcription reaction. The specific procedures for each expression evaluation are described below.

[0223] <Expression Evaluation Using Human Embryonic Kidney Cells> Human embryonic kidney cells HEK293T (ATCC® CRL-3216™) were cultured in a culture medium at 37°C and 5% CO ２ The HEK293T cells were cultured under the following conditions. The culture medium used had the following composition:

[0224] (Culture medium composition for HEK293T cells) Dulbecco's modified Eagle's medium (DMEM): Sigma, Cat# D6429 10% Fetal bovine serum (FBS): BioWest, Cat# S1820 100-fold diluted penicillin-streptomycin mixed solution: Nacalai Tesque, Cat# 09367-34 (penicillin 10,000 units / ml, streptomycin 10,000 μg / ml, stabilizer included)

[0225] The day before measuring the expression level of the human ATXN7 gene, HEK293T cells (10,000 cells / well) were seeded in a 96-well plate and incubated at 37°C, 5% CO ２ The cells were cultured overnight under the conditions of 100°C / 200°F. Then, each single-stranded antisense oligonucleotide (final concentration 2-50 nM) diluted in RNase-free water or phosphate-buffered saline (PBS) was transfected into the above cells by lipofection. As a negative control, cells transfected with a solvent containing no single-stranded antisense oligonucleotide were used. The transfected cells were incubated in growth medium at 37°C and 5% CO. ２The cells were cultured for 48 hours under the conditions of 95°C for 3 seconds and 60°C for 30 seconds. The growth medium was then removed, and the extracted total RNA was reverse-transcribed using the Taqman Fast Cells-to-CT Kit (Thermo Fisher Scientific, Cat# 4399003). The complementary DNA (cDNA) obtained from this reverse transcription reaction was used to perform real-time PCR using pre-designed gene-specific probes (see below) in Taqman gene expression assays (Applied Biosystems) (40 cycles of 95°C for 3 seconds, 60°C for 30 seconds).

[0226] List of gene-specific probes used in the evaluation of human ATXN7 gene expression: Human ATXN7 (used for gapmer evaluation): Hs00165660_m1 Human ATXN7 (used for SSO evaluation): Hs00998066_g1 Human GAPDH: 4326317E (internal control)

[0227] The expression ratios of human ATXN7 mRNA for each single-stranded antisense oligonucleotide determined by the above-described method are shown in Table 7, and the expression ratios of human ATXN7 mRNA containing exon 3 for each single-stranded antisense oligonucleotide (SSO) are shown in Tables 8-1 to 8-3. The expression ratio of human ATXN7 mRNA determined in the negative control group was set to 1.00. Single-stranded antisense oligonucleotides with an expression ratio of 0.80 or less were determined to be single-stranded antisense oligonucleotides capable of suppressing human ATXN7 mRNA expression. Generally, it is believed that suppression of mRNA expression also suppresses subsequent translation into protein. Therefore, single-stranded antisense oligonucleotides with an expression ratio of 0.80 or less can be determined to be single-stranded antisense oligonucleotides capable of regulating the function of the human ATXN7 gene.

[0228]

[0229]

[0230]

[0231]

[0232] <Expression evaluation using mouse primary cultured neurons> Mouse primary cultured neurons were cultured in culture medium at 37°C and 5% CO ２ The culture medium for primary cultured mouse neurons was prepared as follows:

[0233] (Culture medium composition for mouse primary cultured neurons) B-27 Electrophysiology Kit: Gibco, Cat. #A1413701 100-fold diluted 200 mM L-glutamine solution: Nacalai Tesque, Cat. #16948-04 100-fold diluted penicillin-streptomycin mixed solution: Nacalai Tesque, Cat. #09367-34 (penicillin 10,000 units / ml, streptomycin 10,000 μg / ml, stabilizer included)

[0234] Primary cultured mouse neurons (derived from mouse fetal cerebrum) were seeded in a 96-well plate at 40,000 cells / well and incubated at 37°C, 5% CO ２ The cells were cultured for 1 to 5 days under these conditions. Then, antisense oligonucleotides (final concentration 0.2 to 5 μM) diluted with RNase-free water or phosphate-buffered saline (PBS) were added to the culture medium. As a negative control, the solvent for the single-stranded antisense oligonucleotide, e.g., RNase-free water or PBS, was added to the culture medium. The cells were cultured in the culture medium at 37°C and 5% CO ２ The cells were cultured for 2 to 7 days under these conditions. The culture medium was then removed, and the extracted total RNA was reverse-transcribed using the Taqman Fast Cells-to-CT Kit (Thermo Fisher Scientific, Cat# 4399003). The complementary DNA (cDNA) obtained from this reverse transcription reaction was used to perform real-time PCR using predesigned gene-specific probes (see below) in Taqman gene expression assays (Applied Biosystems) (40 cycles of 95°C for 3 seconds, 60°C for 30 seconds).

[0235] List of gene-specific probes used in evaluating the expression of the mouse ATXN7 gene: Mouse ATXN7 (used for evaluating gapmer: Mm01315281_m1) Mouse ATXN7 (used for evaluating SSO): Mm01315283_g1 Mouse GAPDH: 4352339E (internal control)

[0236] The expression ratios of mouse atxn7 mRNA for each single-stranded antisense oligonucleotide (gapmer) determined using the above method are shown in Table 9, and the expression ratios of human ATXN7 mRNA containing exon 3 for each single-stranded antisense oligonucleotide (SSO) are shown in Table 10. The expression ratio of mouse atxn7 mRNA determined in the negative control group was set to 1.00. Single-stranded antisense oligonucleotides with an expression ratio of 0.80 or less were determined to be capable of suppressing mouse atxn7 mRNA expression. Since it is generally believed that suppression of mRNA expression will also suppress subsequent protein translation, an expression ratio of 0.80 or less can be determined to be a single-stranded antisense oligonucleotide capable of regulating the function of the mouse atxn7 gene. In addition, when the number of mismatches in the target region of the target RNA to which the single-stranded antisense oligonucleotide binds for both the human ATXN7 gene (SEQ ID NO: 1) and the mouse atxn7 gene (SEQ ID NO: 8) is "0" (for example, sequence name SP-AX7-007 in Table 9), this means that the base sequence of the target region is conserved between the human ATXN7 gene and the mouse atxn7 gene. When the number of mismatches in the target region of the target RNA to which the single-stranded antisense oligonucleotide binds for the mouse atxn7 gene (SEQ ID NO: 8) is "2" (for example, sequence name SP-AX7-070 in Table 9), this means that the single-stranded antisense oligonucleotide hybridizes with the mouse atxn7 gene via two mismatches. In Table 9, the base positions that become mismatches when the single-stranded antisense oligonucleotide hybridizes to the mouse atxn7 gene are underlined (N).

[0237]

[0238]

[0239] <<Evaluation of Exon 3 Skipping of the ATXN7 Gene>> Evaluation of the induction of exon 3 skipping of the ATXN7 gene was carried out using human embryonic kidney HEK293T cells and fibroblasts from a patient with spinocerebellar ataxia type 7 that contain an abnormally expanded CAG repeat sequence in exon 3, depending on the single-stranded antisense oligonucleotide produced. Specific procedures are described below.

[0240] <Evaluation of exon 3 skipping using HEK293T cells> Human embryonic kidney cells, HEK293T cells (ATCC (registered trademark) CRL-3216 (trademark)), were cultured in a culture medium at 37°C, 5% CO ２ The HEK293T cells were cultured under the following conditions. The culture medium used had the following composition:

[0241] Culture medium composition for HEK293T cells: Dulbecco's modified Eagle's medium (DMEM): Thermo Fisher Scientific, Cat. #11995 10% fetal bovine serum (FBS): Thermo Fisher Scientific, Cat. #10437028

[0242] The day before transfection, HEK293T cells (12,000 cells / well) were seeded in a 96-well plate and incubated at 37°C, 5% CO ２ The cells were cultured overnight under the conditions of 0.1% ethanol. Then, each single-stranded antisense oligonucleotide (10-200 nM) diluted in RNase-free water was transfected into the above cells by lipofection. As a negative control, cells transfected with RNase-free water in which the single-stranded antisense oligonucleotide was not dissolved were used. The transfected cells were cultured in culture medium at 37°C and 5% CO. ２The cells were cultured for 48 hours under the conditions of

[0100] . The culture medium was then removed, and the extracted total RNA was reverse-transcribed using the Taqman Fast Cells-to-CT Kit (Thermo Fisher Scientific, Cat# 4399003). The complementary DNA (cDNA) obtained from this reverse transcription reaction was used to perform PCR spanning exons 1 to 5 of the ATXN7 gene using predesigned specific primers (see below).

[0243] The PCR program used was as follows: 94°C, 1 minute: heat denaturation [98°C, 10 seconds; 65°C, 15 seconds; 68°C, 60 seconds] x 40 cycles: PCR amplification

[0244] The gene-specific primers used were ATXN7_Fw and ATXN7_Rv. ATXN7_Fw: 5'-AGCCGCCAGATCTGAACAG-3' (SEQ ID NO: 311) ATXN7_Rv: 5'-ATTGGCATGTCTTCCCGACA-3' (SEQ ID NO: 312)

[0245] The PCR reaction products were separated by 2-4% agarose gel electrophoresis, and the gel was photographed using a photography system combining an LED transilluminator Gel Mieru (Wako) and an iPhone SE (Apple).

[0246] The experimental results of measuring the exon 3 skipping activity of each antisense oligonucleotide against human ATXN7 pre-mRNA, determined by the above-mentioned method, are shown in FIG.

[0247] In HEK293T cells, cDNA derived from the HEK293T cells to which each antisense oligonucleotide had been added yielded two amplification products of the same size as those obtained from cells not transfected with the antisense oligonucleotide, as well as a smaller amplification product (Figure 9, top). Sequence analysis of these two amplification products revealed that the amplification products were a 527-base polynucleotide consisting of exons 1, 2, 3, 4, and 5, and a 187-base polynucleotide consisting of exons 1, 2, 4, and 5 (Figure 9, bottom). Thus, antisense oligonucleotides with exon 3 skipping activity were obtained.

[0248] (Evaluation of exon 3 skipping using fibroblasts from patients with spinocerebellar ataxia type 7) GM03561 cells or GM07492 cells (1,000,000 cells / dish) were seeded on a 10 cm dish and incubated at 37°C, 5% CO ２ The cells were cultured overnight under these conditions. Then, each single-stranded antisense oligonucleotide (final concentration: 50 nM or 200 nM) diluted in RNase-free water or phosphate-buffered saline (PBS) was transfected into the above cells using the lipofection method. As a negative control, cells transfected with RNase-free water or PBS containing no dissolved single-stranded antisense oligonucleotide were used. The transfected cells were incubated in growth medium at 37°C and 5% CO. ２The cells were cultured for 48 hours under these conditions. The culture medium was then removed, and RNA extraction was performed using the RNeasy Mini Kit (QIAGEN, Cat#74106). Reverse transcription of the extracted mRNA was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cat#4368814). For the reverse transcription reaction, 0.1-1 μg of mRNA was diluted to 20 μL. Using the cDNA obtained from this reverse transcription reaction, PCR spanning exons 1 to 5 of the ATXN7 gene was performed using predesigned specific primers (see below). The PCR program used was as follows:

[0249] The PCR program used was as follows: 94°C, 1 minute: heat denaturation [98°C, 10 seconds; 65°C, 15 seconds; 68°C, 60 seconds] x 40 cycles: PCR amplification

[0250] The gene-specific primers used were ATXN7_Fw and ATXN7_Rv. ATXN7_Fw: 5'-AGCCGCCAGATCTGAACAG-3' (SEQ ID NO: 311) ATXN7_Rv: 5'-ATTGGCATGTCTTCCCGACA-3' (SEQ ID NO: 312)

[0251] The reaction products of the PCR reaction were separated by electrophoresis using agarose gel or acrylamide gel, and gel photographs were taken using a photography system combining an LED Transilluminator Gel Mieru (Wako) and an iPhone SE (Apple).

[0252] When the region from exon 1 to exon 5 of the ATXN7 cDNA derived from fibroblasts of a patient with spinocerebellar ataxia type 7 obtained by the method described above was amplified, a 661-base band (mutant allele in Figure 10) not observed in fibroblasts derived from healthy individuals was obtained in a sample that had not been transfected with antisense oligonucleotide (labeled "vehicle" in Figure 10). The base sequence of this amplified product was determined by sequencing, and it was shown that this band contained an abnormal expansion of a CAG repeat in exon 3. This result was in good agreement with the results of genetic analysis of the patient.

[0253] The cDNA from fibroblasts of a patient with spinocerebellar ataxia type 7 treated with antisense oligonucleotides yielded a product smaller in size than that obtained from cells not transfected with antisense oligonucleotides (exon skipping product in Figure 10), indicating that exon 3 was skipped by antisense oligonucleotide treatment.

[0254] <<Expression evaluation of mutant ATXN7 protein>> Expression evaluation of ATXN7 protein was performed using human fibroblasts according to the single-stranded antisense oligonucleotide produced. In this example, protein expression level evaluation refers to evaluation of the amount of protein translated from mRNA. The specific procedure for expression evaluation is described below.

[0255] (Quantitative Analysis of Proteins by Western Blotting) GM03561 cells or GM07492 cells (1,000,000 cells / dish) were seeded on a 10 cm dish and incubated at 37°C, 5% CO ２ The cells were cultured overnight under the conditions of 0.1% ribosomal acid (0.1%), 0.1% ribosomal acid (0.1%), and 0.2% ribosomal acid (0.1%). Antisense oligonucleotides diluted in RNase-free water or phosphate-buffered saline (PBS) (final concentration: 50 nM or 200 nM) were then transfected into the cells using the lipofection method. As a negative control, cells transfected with RNase-free water or PBS containing no dissolved single-stranded antisense oligonucleotides were used. The transfected cells were incubated in growth medium at 37°C and 5% CO. ２The cells were cultured for 48 hours under the conditions of

[0000] . The growth medium was removed, washed with PBS, and then lysis buffer (60 mM Tris-HCl, 2% sodium dodecyl sulfate, 10% sucrose) containing 1 / 100 volume of protease inhibitor (ThermoFisher Scientific, Cat# 1860932) was added and allowed to stand at room temperature for 5 minutes. The cells were collected with a cell scraper, and the collected solution was disrupted using a 1 ml tuberculin syringe and a 27G injection needle and allowed to stand at room temperature for 5 minutes. The cells were then centrifuged at 15,000 x g for 10 minutes at 20°C, and the supernatant was collected as a sample. The collected samples were subjected to protein quantification using the Pierce™ BCA Protein Assay kit (ThermoScientific, Cat# 23225). Pierce™ NuPAGE LDS Sample Buffer (ThermoFisher Scientific, Cat# NP0008) and Pierce™ NuPAGE Sample Reducing Agent (ThermoFisher Scientific, Cat# NP0009) were added to the samples, and the samples were heated at 70°C for 10 minutes. The prepared samples were layered so that the protein amount was 5 μg to 18 μg per lane, and electrophoresis was performed. Electrophoresis was performed for 90 minutes using Pierce™ NuPAGE Tris-Acetate Midi Protein Gels 3-8% (ThermoFisher Scientific, Cat# WG1602A) at a constant voltage of 150 V. The running buffer used was Pierce™ NuPAGE Tris-Acetate SDS Running Buffer (20x) (ThermoFisher Scientific, Cat# LA0041) diluted to 1x.

[0256] After electrophoresis, transfer was performed using a semi-dry method. The membrane used was a Trans-Blot Turbo Transfer Pack (BIO-RAD, Cat# 1704157), and the transfer device used was a BIO-RAD Trans-Blot Turbo Transfer System. The transfer protocol used was the Standard protocol (30 minutes) described in the transfer device's instruction manual. After transfer, the membrane was washed with TBST. The composition of TBST was Tween-containing Tris-buffered saline (pH 7.4) (Cell Signaling, Cat# 9997) diluted to 1x concentration. After washing, the membrane was blocked by shaking it in Blocking One (Nacalai Tesque, Cat. No. 03953-95) at room temperature for 1 hour.

[0257] After blocking, the membrane was washed with TBST, diluted primary antibodies were added, and the membrane was shaken overnight at 4°C. The primary antibodies and dilution solvents were as follows: ATXN7 Primary antibody: Anti-ATXN7 antibody (ThermoFisher Scientific, Cat# A302-638A) Dilution solvent: Blocking One α-tubulin Primary antibody: α-tubulin antibody (11H10) (Cell Signaling, Cat# 2125) Dilution solvent: Blocking One

[0258] The primary antibody was added to the membrane, shaken, and then washed with TBST. A diluted secondary antibody was added and shaken at room temperature for 1 hour. The secondary antibody and dilution solvent were as follows: Secondary antibody: Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody (Invitrogen, Cat# A24537) Dilution solvent: Blocking One

[0259] The secondary antibody was added to the membrane, shaken, washed with TBST, and then detected using ECL select (Amersham, Cat# RPN2235). Amersham Imager 680 was used for detection and expression quantification analysis.

[0260] The results of the evaluation obtained by the above method showed that the expression of mutant Ataxin-7 protein was suppressed in fibroblasts from patients with spinocerebellar ataxia type 7 to which antisense oligonucleotides had been added ( Figure 11 ), indicating that the expression of mutant Ataxin-7 protein expressed from the mutant allele containing the CAG repeat expansion mutation was suppressed by antisense oligonucleotide treatment.

[0261] <<In vivo expression evaluation of mouse atxn7 gene>> The expression evaluation of the ATXN7 gene was performed by intracerebroventricular administration to mice and measuring the amount of mRNA in each region, such as the cerebral cortex, cerebellum, and brainstem. In this example, gene expression evaluation refers to evaluating the amount of mRNA by measuring the amount of complementary DNA (cDNA) obtained by reverse transcription. Specific procedures for each expression evaluation are described below.

[0262] ICR mice (Jackson Laboratories Japan) were anesthetized with isoflurane (Pfizer, Cat# 114133403). Next, antisense oligonucleotides dissolved in artificial cerebrospinal fluid (Tocris Bioscience, Cat# 3525 / 25 mL) were administered to the anesthetized ICR mice (Jackson Laboratories Japan) at 10 μL / individual using a two-stage needle (Top, Medical Device Approval Number 15800BZZ01460000) attached to a 50 μL Hamilton syringe (Hamilton, Cat# 705LT). Negative control mice received only artificial cerebrospinal fluid at 10 μL / individual.

[0263] After a certain period of rearing following administration, the ICR mice were euthanized and samples were collected from the cerebral cortex, cerebellum, and brainstem. The collected tissues were immersed in RNA Later (Applied Biosystems, Cat# AM7024) overnight and then stored at -80°C. RNA was extracted from the stored tissue samples using an RNeasy Mini Kit (QIAGEN, Cat# 74106). Reverse transcription of the extracted mRNA was performed using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cat# 4368814). For the reverse transcription reaction, 0.1-1 μg of mRNA was diluted to 20 μL and used. The resulting complementary DNA (cDNA) was used for real-time PCR using Taqman expression assays (Applied Biosystems) with predesigned gene-specific probes (see below) (40 cycles of 95°C for 3 seconds, 60°C for 30 seconds).

[0264] List of gene-specific probes used in evaluating the expression of the mouse ATXN7 gene Mouse ATXN7 (used for gapmer evaluation): Mm01315281_m1 Mouse ATXN7 (used for SSO evaluation): Mm01315283_g1 Mouse GAPDH: 4352339E (internal control)

[0265] The mouse atxn7 mRNA expression ratios for each antisense oligonucleotide determined using the above method are shown in Table 11. The mouse atxn7 mRNA expression ratio determined in the negative control group was set to 1.00. Antisense oligonucleotides with an expression ratio of 0.80 or less were determined to be single-stranded antisense oligonucleotides capable of suppressing mouse atxn7 mRNA expression. Generally, suppression of mRNA expression is thought to also suppress subsequent protein translation, and therefore, an expression ratio of 0.80 or less can be determined to be a single-stranded antisense oligonucleotide capable of regulating the function of the mouse atxn7 gene. The target regions to which the single-stranded antisense oligonucleotides listed in Table 11 bind are regions with 90% or more sequence identity between the human ATXN7 gene and the mouse ATXN7 gene.

[0266]

[0267] <Evaluation of serum stability of single-stranded antisense oligonucleotides> Mouse serum (20 μL) or human serum (20 μL) was mixed with 4 μL of Tris-EDTA buffer solution (pH = 8.0) containing 400 pmol of single-stranded antisense oligonucleotide, followed by the addition of mineral oil (15 μL). This solution was incubated at 37°C and then mixed with 10 μL of 8 mol / L urea solution to inactivate the nucleases in the serum. Ultrapure water (10 μL) was added, and the mixture was centrifuged to separate the aqueous layer containing the single-stranded antisense oligonucleotide from the mineral oil layer, and the aqueous layer was recovered. The recovered aqueous layer was analyzed by LC-MS (Waters), and the remaining single-stranded antisense oligonucleotide was calculated from the area intensity of the UV-chromatogram of the single-stranded antisense oligonucleotide obtained. "Remaining oligonucleotide (%)" indicates the percentage of undegraded single-stranded antisense oligonucleotide remaining after 72 hours relative to the undegraded single-stranded antisense oligonucleotide at the time of analysis immediately after mixing with serum.

[0268] Those with a survival rate of 50% or more after 72 hours are judged to be stable single-stranded antisense oligonucleotides.

[0269] <Evaluation of Cytotoxicity of Single-Stranded Antisense Oligonucleotides> HepG2 cells, a human hepatoma-derived cell line, were cultured in growth medium at 37°C and 5% CO. ２ The growth medium used is of the following composition:

[0270] Composition of growth medium used for cytotoxicity evaluation: Eagle's minimum essential medium: SIGMA, Cat. #M5650-500ML 10% fetal bovine serum (FBS): Biowest, Cat. #S1400-500 200mM L-Glutamine: Gibco, Cat. #25030-081 100-fold diluted penicillin-streptomycin mixed solution (ThermoFisher Scientific, Cat. #15140-122)

[0271] The day before the experiment, the above cells (1.5 × 10 ４ The seeded cells were incubated at 37°C and 5% CO ２ After overnight culture under these conditions, antisense oligonucleotides (final concentration: 0.05-50 nM) complexed with Lipofectamine RNAiMAX (Invitrogen: Cat# 31985-070) in Opti-Minimum Essential Medium (Thermo Fisher Scientific, Cat# 31985070) were added, and the cells were incubated at 37°C, 5% CO ２ The cells are cultured for 24 hours under the conditions of

[0100] . Thereafter, Caspase-Glo 3 / 7 Assay System (Promega, Cat# G8093) or Celltiter-Glo 2.0 Assay (Promega, Cat# G9242) is added to the growth medium to assess caspase activity and cell viability.

[0272] <<In Vivo Evaluation of Central Toxicity of Single-Stranded Antisense Oligonucleotides>> The central toxicity of each single-stranded antisense oligonucleotide was evaluated by intraventricular administration to mice, which were then housed immediately to 1 hour after administration and for up to 28 days after administration. The mice were then observed for general condition and body weight, and pathological examination (hematoxylin-eosin staining) of tissue samples was performed at the time of collection. Acute central toxicity findings observed immediately after intraventricular administration were calculated as a "score" based on the following evaluation criteria. The highest score among the findings observed for items 1-5 was recorded for each individual, and the scores for each item were totaled. For example, if ptosis, lateral recumbency, bradypnea, and convulsions were observed in one individual, the total score would be 1 + 4 + 4 = 9. Since a higher score indicates a more severe finding, a higher total score indicates a more severe acute central toxicity finding. The average score for each group was used as the score. Evaluation items Item 1: Hyperactivity (1 point), stereotypic behavior (grooming, circling) (1 point), rearing behavior (1 point), vocalization (1 point), irritability / tail lift (1 point) Item 2: Ptosis, eye closure, decreased (absent) spontaneous movement (1 point) Item 3: Staggering (1 point), ataxic gait (2 points) Item 4: Abnormal posture / irregular breathing (1 point), lateral / prone position (2 points), bradypnea / abnormal breathing (4 points) Item 5: Jumping, tremors, muscle spasms (2 points), convulsions (4 points)

[0273] In addition, delayed central toxicity findings observed one day or more after intracerebroventricular administration to mice are calculated as a "score" based on the following evaluation criteria. Each score is the average value for each group. Clinical sign score: 0 points: no abnormalities 1 point: abnormal hind limb function, tremors, decreased spontaneous movement 2 points: dragging of hind limbs, weakness of tail or hind limbs 3 points: complete hind limb dysfunction, paralysis of hind limbs, recumbency, prone position 4 points: euthanasia Pathological score: 0 points: no abnormalities 1 point: abnormalities (single cell necrosis, vacuolation, etc.)

[0274] Nucleic acid candidates for clinical use are desirable if (1) the designed oligonucleotide has low acute toxicity and sufficient expression suppression is observed even at low doses, (2) there is no delayed toxicity even at high doses, and (3) the expression suppression is dose-dependent. To identify oligonucleotides that meet these requirements, as described above, single-stranded antisense oligonucleotides expected to have low expression suppression or regulation of off-target genes are selected and evaluated using cultured cells including fibroblasts from patients with spinocerebellar ataxia type 7. In addition, as necessary, evaluation of the serum stability of single-stranded antisense oligonucleotides, evaluation of the cytotoxicity of single-stranded antisense oligonucleotides, evaluation of the potency of target gene expression suppression (including dose-dependency) in in vivo tests, evaluation of the persistence of expression suppression, and observation of the condition of experimental animals after administration of the antisense oligonucleotides are also performed.

[0275] Although the embodiments and examples of the present invention have been described above, it is also planned from the beginning that the configurations of the above-described embodiments and examples may be appropriately combined.

[0276] The embodiments and examples disclosed herein are illustrative in all respects and should not be considered limiting. The scope of the present invention is defined by the claims, not by the embodiments and examples described above, and is intended to include any modifications within the scope of the claims and meanings equivalent to the claims.

Claims

1. A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that regulates the expression and / or function of the human ATXN7 gene, wherein each nucleotide in the single-stranded antisense oligonucleotide is linked via a phosphate group and / or a modified phosphate group, the single-stranded antisense oligonucleotide comprising a gap region, a 3' wing region linked to the 3' end of the gap region, and a 5' wing region linked to the 5' end of the gap region, the gap region being a nucleic acid composed of deoxyribose that may contain a nucleic acid whose sugar moiety is modified, the 3' wing region and the 5' wing region being modified nucleic acids, the sugar modification constituting the single-stranded antisense oligonucleotide being a modified nucleic acid represented by the following formula (A1), the base length of the single-stranded antisense oligonucleotide being 12 to 30 mer, and the base sequence of the single-stranded antisense oligonucleotide being: A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, which is a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to at least one target region of the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, which is the same base length as the single-stranded antisense oligonucleotide; a base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted, or added in the target region; or a base sequence which hybridizes under stringent conditions to an oligonucleotide having the target region. (wherein, base is a nucleic acid base, each independently being a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine, or uracil; R １ and R ２ are each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, or R １ and R ２ and X together form a ring, each X is independently a hydrogen atom, a hydroxyl group, a fluorine atom, an alkoxy group having 1 to 6 carbon atoms which may be substituted, or an amino group which may be substituted, and each Y is independently a hydrogen atom or an alkyl group having 1 to 8 carbon atoms which may have a carbon-oxygen double bond, or some carbon atoms of the alkyl group and X together form a ring.

2. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, wherein the base sequence of the single-stranded antisense oligonucleotide is a base sequence having a sequence identity of 95% or more and 100% or less based on a base sequence complementary to at least one target region of the same base length as the single-stranded antisense oligonucleotide in the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO:

5.

3. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, wherein the base sequence of the single-stranded antisense oligonucleotide is a base sequence complementary to at least one target region in the base sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, the target region having the same base length as the single-stranded antisense oligonucleotide.

4. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the gap region has a length of 5 to 20 nucleotides, the 3' wing region is composed of a 2 to 5 nucleotide modified nucleic acid, and the 5' wing region is composed of a 2 to 5 nucleotide modified nucleic acid.

5. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein the base length of the single-stranded antisense oligonucleotide is 15 to 22 mer.

6. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein the modified nucleic acid in the 3' wing region is selected from the group consisting of 2'-O-alkylated nucleic acids and cross-linked modified nucleic acids, and the modified nucleic acid in the 5' wing region is selected from the group consisting of 2'-O-alkylated nucleic acids and cross-linked modified nucleic acids.

7. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein the gap region has an 8-12 mer base length, the 3' wing region is composed of a 3-5 mer modified nucleic acid, and the 5' wing region is composed of a 3-5 mer modified nucleic acid.

8. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein the 3' wing region is composed of a 2'-MOE nucleic acid or a 2',4'-bridged modified nucleic acid, the 5' wing region is composed of a 2'-MOE nucleic acid or a 2',4'-bridged modified nucleic acid, and at least one internucleotide bond of the single-stranded antisense oligonucleotide is a phosphorothioate bond.

9. The base sequence of the single-stranded antisense oligonucleotide is, in the base sequence of SEQ ID NO: 1, the following bases, counting from the 5' end: 384 to 385, 457 to 460, 831 to 833, 1118 to 1129, 1904 to 1907, 2170 to 2176, 3109, 3654 to 3667, 4088, 4271 to 4275, 4528 to 4532, 4732, 4850, 4872 to 4876 9. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, wherein the single-stranded antisense oligonucleotide is a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to a target region consisting of a 15-22mer continuous base starting from bases at positions 78, 5462 to 5463, 5710 to 5712, 5783, 5814 to 5819, 5950, 6402 to 6404, 6805 to 6806, or 6851; a base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted, or added in the target region; or a base sequence which hybridizes under stringent conditions to an oligonucleotide having the target region.

10. The base sequence of the single-stranded antisense oligonucleotide has 90% to 100% sequence identity with respect to a base sequence complementary to a target region consisting of a consecutive 15-22mer starting from bases located at positions 384-385, 457-460, 831-833, 1118-1126, 2174-2176, 3654, 4088, 4271-4275, 4528, 4850, 4873, 5462, 5710-5712, 5816-5819, 6402-6404, 6805-6806, or 6851 in the base sequence set forth in SEQ ID NO: 1, counting from the 5' end; the 3' wing region is 2-5mer; The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, wherein the 5' wing region is a 2- to 5-mer.

11. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, wherein the base sequence of the single-stranded antisense oligonucleotide is a base sequence that has 90% to 100% sequence identity with respect to a base sequence complementary to a target region consisting of a consecutive 15-22mer starting from bases located at positions 1122, 2174, 4272, 4528, 5462, 5712, or 5818, counting from the 5' end, in the base sequence set forth in SEQ ID NO:

1.

12. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, wherein the base sequence of the single-stranded antisense oligonucleotide is one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 10 to 16, 18, 20, 21, 23 to 25, 27, 28, 31 to 35, 37, 41, 42, 45, 47 to 49, 52, 54 to 60, 63 to 70, 73 to 83, and 85 to 197.

13. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, wherein the base sequence of the single-stranded antisense oligonucleotide is one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 10 to 13, 15, 18, 21, 24, 27, 33 to 35, 37, 41, 45, 54 to 60, 65, 66, 68 to 70, 74 to 81, and 83.

14. A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof that induces skipping of exon 3 in the human ATXN7 gene, wherein each nucleotide in the single-stranded antisense oligonucleotide is linked via a phosphate group and / or a modified phosphate group, the single-stranded antisense oligonucleotide comprises a modified nucleic acid having at least one modified sugar, and the sugar modification constituting the single-stranded antisense oligonucleotide is a modified nucleic acid represented by the following formula (A2) and / or formula (A3), the base length of the single-stranded antisense oligonucleotide is 15 to 30 mer, and the base sequence of the single-stranded antisense oligonucleotide is: a base sequence having 90% to 100% sequence identity with respect to a base sequence complementary to at least one target region of the same base length as the single-stranded antisense oligonucleotide in the base sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7, a base sequence complementary to a base sequence in the target region in which one or several bases have been deleted, substituted, inserted or added, or A single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, which has a base sequence that hybridizes under stringent conditions to an oligonucleotide having the target region. (wherein, base is a nucleic acid base, each independently being a group represented by adenine, guanine, cytosine, 5-methylcytosine, thymine, or uracil; R １ and R ２ are each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, or R １ and R ２ and together form a ring, and R ３ are each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, and R ４ and R ５ are each independently a hydrogen atom or an optionally substituted alkyl group having 1 to 6 carbon atoms, or R ４ and R ５ and are taken together to form a carbon-oxygen double bond or ring, each Z is independently an oxygen atom or an optionally substituted nitrogen atom, and n is an integer of 0 to 2.

15. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 14, wherein the base sequence of the single-stranded antisense oligonucleotide is a base sequence complementary to at least one target region in the base sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7, the target region having the same base length as the single-stranded antisense oligonucleotide.

16. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 14 or 15, wherein the single-stranded antisense oligonucleotide has a base length of 18 to 25 mer.

17. The sugar modification constituting the single-stranded antisense oligonucleotide is represented by formula (A2), wherein R ３ The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 14 to 16, wherein each of the following is independently a methyl group, a methoxyethyl group, or an N-methylpropanamide group.

18. The base sequence of the single-stranded antisense oligonucleotide has a sequence identity of 90% to 100% based on a base sequence complementary to a target region consisting of a contiguous 18- to 25-mer base from the base located at positions 6, 8 to 15, 26, 282, 287, 292, 297, 302, or 317 from the 5' end in the base sequence set forth in SEQ ID NO: 6, or a target region consisting of a contiguous 18- to 25-mer base from the base located at positions 49450, 49452 to 49459, 49470, 49726, 49731, 49736, 49741, 49746, 49761, or 49781 from the 5' end in the base sequence set forth in SEQ ID NO: 7; The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 14 to 17, which is a base sequence complementary to a base sequence in which one or several bases have been deleted, substituted, inserted or added in the target region, or a base sequence which hybridizes under stringent conditions to an oligonucleotide having the target region.

19. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 14 to 18, wherein the base sequence of the single-stranded antisense oligonucleotide has a sequence identity of 90% to 100% based on a base sequence complementary to a target region consisting of an 18- to 25-mer contiguous base from bases located at positions 8 to 11, 282, 287, or 302, counting from the 5' end, in the base sequence of SEQ ID NO: 6, or a target region consisting of an 18- to 25-mer contiguous base from bases located at positions 49452 to 49455, 49726, 49731, or 49746, counting from the 5' end, in the base sequence of SEQ ID NO:

7.

20. The single-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 14 to 19, wherein the base sequence of the single-stranded antisense oligonucleotide is one base sequence selected from the group consisting of the base sequences of SEQ ID NOs: 236, 260 to 263, 265 to 267, 269, 271, 292, and 293.

21. A double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof, comprising: the single-stranded antisense oligonucleotide according to any one of claims 1 to 20; and a second-strand oligonucleotide hybridized to the single-stranded antisense oligonucleotide, wherein the base sequence of the second-strand oligonucleotide is complementary to the base sequence of the single-stranded antisense oligonucleotide and has a sequence identity of 90% or more and 100% or less with respect to the base sequence of the complementary strand.

22. The double-stranded antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 21, wherein the double-stranded antisense oligonucleotide is at least one double-stranded antisense oligonucleotide selected from the group consisting of double-stranded oligonucleotide sequences SP-AX7-195, SP-AX7-205, SP-AX7-206, and SP-AX7-209.

23. An oligonucleotide conjugate or a pharmaceutically acceptable salt thereof, comprising: the single-stranded antisense oligonucleotide according to any one of claims 1 to 20 or a pharmaceutically acceptable salt thereof; or the double-stranded antisense oligonucleotide according to claim 21 or 22 or a pharmaceutically acceptable salt thereof; and an additional substance bound to the single-stranded antisense oligonucleotide or the second strand oligonucleotide directly or via a linker bond, wherein the linker bonds are each independently a phosphodiester bond or a phosphorothioate bond, and the additional substance is at least one selected from the group consisting of polyethylene glycol, a peptide, an alkyl chain, a ligand compound, an antibody, a protein, a nucleic acid, and a sugar chain.

24. A pharmaceutical comprising, as an active ingredient, the single-stranded antisense oligonucleotide according to any one of claims 1 to 20 or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide according to claim 21 or claim 22 or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex according to claim 23 or a pharmaceutically acceptable salt thereof.

25. An agent for regulating the expression and / or function of the human ATXN7 gene, comprising as an active ingredient the single-stranded antisense oligonucleotide according to any one of claims 1 to 20 or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide according to claim 21 or claim 22 or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex according to claim 23 or a pharmaceutically acceptable salt thereof.

26. A therapeutic agent for spinocerebellar ataxia type 7, comprising as an active ingredient the single-stranded antisense oligonucleotide according to any one of claims 1 to 20 or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide according to claim 21 or 22 or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex according to claim 23 or a pharmaceutically acceptable salt thereof.

27. A preventive agent for spinocerebellar ataxia type 7, comprising as an active ingredient the single-stranded antisense oligonucleotide according to any one of claims 1 to 20 or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide according to claim 21 or 22 or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex according to claim 23 or a pharmaceutically acceptable salt thereof.

28. A method for treating or preventing spinocerebellar ataxia type 7, comprising the step of administering, as an active ingredient, the single-stranded antisense oligonucleotide according to any one of claims 1 to 20 or a pharmaceutically acceptable salt thereof, the double-stranded antisense oligonucleotide according to claim 21 or 22 or a pharmaceutically acceptable salt thereof, or the antisense oligonucleotide complex according to claim 23 or a pharmaceutically acceptable salt thereof to an individual suffering from spinocerebellar ataxia type 7.

29. A single-stranded antisense oligonucleotide according to any one of claims 1 to 20 or a pharmaceutically acceptable salt thereof, a double-stranded antisense oligonucleotide according to claim 21 or claim 22 or a pharmaceutically acceptable salt thereof, or an antisense oligonucleotide complex according to claim 23 or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of spinocerebellar ataxia type 7.

30. A single-stranded antisense oligonucleotide according to any one of claims 1 to 20 or a pharmaceutically acceptable salt thereof, a double-stranded antisense oligonucleotide according to claim 21 or claim 22 or a pharmaceutically acceptable salt thereof, or an antisense oligonucleotide complex according to claim 23 or a pharmaceutically acceptable salt thereof, for use in producing an agent for treating or preventing spinocerebellar ataxia type 7.

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