Antisense oligonucleotides that alter splicing of DUX4 pre-mRNA

Abstract

The purpose of the present invention is to establish a novel method for treating facioscapulohumeral muscular dystrophy.　An oligonucleotide which can alter the splicing of DUX4 gene from DUX4-fl to DUX4-s, and which is composed of 15 to 30 nucleotides and comprises a nucleotide sequence complementary to a region lying between nucleotide Nos. 502 to 556 or 578 to 612 in DUX4-fl mRNA comprising the nucleotide sequence represented by SEQ ID NO: 1, wherein the 5'-terminal and / or the 3'-terminal may be chemically modified; or a pharmaceutically acceptable salt thereof. A drug (e.g., a therapeutic agent for facioscapulohumeral muscular dystrophy, which comprises the oligonucleotide or a pharmaceutically acceptable salt thereof.

Description

[Technical field]

[0001] The present invention relates to antisense oligonucleotides that alter the splicing of DUX4 pre-mRNA. [Background technology]

[0002] Facioscapulohumeral muscular dystrophy (FSHD) is a muscular dystrophy that shows severe muscle atrophy and muscle weakness in the facial cheeks, scapular region, and upper arm muscles (Non-Patent Document 1: Tawil R. and Van Der Maarel SM Muscle Nerve 2006, 34:1-15). The onset period is very wide, from 0 to 60 years old, but generally begins in the teens with symptoms such as difficulty in lifting the arms, inability to whistle, and poor facial expression. The progression of symptoms is slow, but gradually the lower limbs are also affected, and in many cases walking is impaired. In addition, the degree of muscle impairment often differs between the left and right sides. Muscle pathological findings include necrosis and regeneration of muscle fibers, which are commonly seen in muscular dystrophies, as well as infiltration of inflammatory cells (Non-Patent Document 2: Arahata et al., Muscle Nerve 1995, 2:S56-S66). In addition to skeletal muscle symptoms, nerve hearing loss and retinopathy are often observed (Non-Patent Document 3: Padberg et al., Muscle Nerve 1995, 2:S73-S80). It is the third most common type of muscular dystrophy after Duchenne muscular dystrophy and myotonic muscular dystrophy. HereditaryIt is considered to be a muscle disease, and its prevalence has been reported to be 1 in 14,000 to 1 in 20,000 people in Europe and the United States (Non-Patent Document 4: Padberg et al., Muscle Nerve 1995, 2:S81-S84; Flanigan et al., Neuromuscul Disord 2001, 11:525-529; Non-Patent Document 5: Mostacciuolo et al., Clin Genet 2009, 75:550-555; Non-Patent Document 6: Norwood et al., Brain 2009, 132:3175-3186; Non-Patent Document 7: Deenen et al., Neurology 2014, 83:1056-1059).

[0003] The genetic factors that cause FSHD are very complex, but the "DUX4 gene derepression" hypothesis, published by Lemmers et al. in 2010, is now widely believed (Non-Patent Document 8: Lemmers et al., Science 2010, 329:1650-1653). FSHD is a dominant genetic disorder, and the disease is linked to the end of the long arm of chromosome 4 (4q35 region) (Non-Patent Document 9: Wijmenga et al., Nat Genet 1992, 2:26-30). This 4q35 region contains a 3.3kb repeat region called D4Z4 (D4Z4 repeat), which is normally repeated 11-100 times and is in a highly DNA methylated heterochromatin state. In about 95% of FSHD patients, the D4Z4 repeats on one chromosome 4 are shortened to 10 or less, resulting in reduced DNA methylation in the 4q35 region (Non-Patent Document 10: van Overveld et al., Nat Genet 2003, 35:315-317). The type in which this shortening of the D4Z4 repeat is observed is called FSHD type 1. In addition, about 5% of FSHD patients have a normal number of D4Z4 repeats, but reduced DNA methylation occurs, and are called FSHD type 2. It is currently known that the cause of reduced methylation in FSHD type 2 is a loss-of-function mutation in the SMCHD1 gene, which is involved in the methylation of the D4Z4 repeat (Non-Patent Document 11: Lemmers et al., Nat Genet 2012, 44:1370-1374).

[0004] In the D4Z4 sequence, there is an open reading frame (ORF) that codes for a transcription factor protein, and it is called DUX4 (Non-Patent Document 12: Gabriels et al., Gene 1999, 236:25-32.). Expression of DUX4 in skeletal muscle occurs at high frequency only when the following two conditions are met. The first is that DNA methylation in the D4Z4 region is reduced and the heterochromatin structure is loosened. The second is that the haplotype called 4qA and 4qB present on the 3' side of the D4Z4 repeat is 4qA (Non-Patent Document 13: Lemmers et al., Am J Hum Genet 2007, 81:884-894). The 4qA sequence contains a polyA addition sequence (ATTAAA) and acts as a polyA addition sequence that stabilizes the DUX4 mRNA (Non-Patent Document 14: Lemmers et al., Science 2010, 329:1650-1653). Because the 4qB sequence does not contain a polyA addition sequence, people with an allele that has the 4qB sequence and whose D4Z4 repeats are shortened to 10 or less do not develop FSHD.

[0005] DUX4 encodes a transcription factor, and alternative splicing can produce DUX4-fl and DUX4-s transcripts (Non-Patent Document 15: Snider et al., PLoS Genet 2010, 6: e1001181). In FSHD patients, full-length DUX4-fl is frequently expressed (Non-Patent Document 16: Jones et al., Hum Mol Genet 2012, 21: 4419-4430), and exerts strong transcription activation ability via the C-terminal transcription activation domain (Non-Patent Document 17: Choi et al., Nucleic Acids Res 2016, 44: 5161-5173; Non-Patent Document 18: Mitsuhashi et al., Biol Open 2018, 7: bio033977). On the other hand, the short form DUX4-s is slightly expressed in cells derived from healthy individuals, but since it does not have a transcription activation domain, it does not function as a transcription factor. The DUX4-fl protein activates the expression of genes expressed in early embryos and testes, such as PRAMEF2, TRIM43, and ZSCAN4 (Non-Patent Document 19: Geng et al., Dev Cell 2012, 22:38-51). Expression of DUX4-fl is associated with apoptosis (Kowaljow et al., Neuromuscul Disord 2007, 17:611-623; Wallace et al., Ann Neurol 2011, 69:540-552), abnormal muscle development (Bosnakovski et al., EMBO J 2008, 27:2766-2779; Banerji et al., Nat Commun 2017, 8:2152), inhibition of nonsense codon-mediated mRNA decay (Feng et al., eLife 2015, 4:e04996), formation of intracellular double-stranded RNA (Shadle et al., PLoS Genet 2017, 13:e1006658), and protein aggregation. (Non-Patent Document 26: Homma et al., Ann Clin Transl Neurol 2015, 2:151-166) and the like.DUX4-fl has also been shown to induce cytotoxicity and symptoms similar to those of FSHD in individual models expressing Drosophila (Non-Patent Document 27: Jones et al., PLoS One 2016, 11: e0150938), Xenopus (Non-Patent Document 28: Wuebbles et al. Int J Clin Exp Pathol 2010, 3: 386-400), zebrafish (Non-Patent Document 29: Mitsuhashi et al., Hum Mol Genet 2013, 22: 568-577), and mice (Non-Patent Document 30: Jones and Jones, PLoS One 2018, 13: e0192657; Non-Patent Document 31: Bosnakovski et al., Nat Commun 2017, 8: 550), and it is believed that DUX4-fl is involved in the development of FSHD. In addition, the physiological function of DUX4 has been shown to be that it is a master transcription factor that is expressed specifically at the 4-cell stage during early embryo development and activates a group of cleavage stage-specific genes including the above-mentioned ZSCAN4, but the details are not yet fully understood (Non-Patent Document 32: De laco et al., Nat Genet 2017, 49:941-945; Non-Patent Document 33: Hendrickson et al., Nat Genet 2017, 49:925-934).

[0006] DUX4-fl and DUX4-s have a common DNA-binding domain on the N-terminus. Therefore, when DUX4-fl and DUX4-s are present at the same time, they are thought to competitively bind to the same DNA. In an experiment using cultured cells, when DUX4-fl and DUX4-s were transfected at a 1:1 ratio, the activation of luciferase by DUX4-fl was suppressed to about 20%. (Non-Patent Document 34: Geng et al., Dev Cell 2012, 22:38-51) In addition, in an experiment in which 20 times the amount of DUX4-s was transfected into zebrafish simultaneously with DUX4-fl, the muscular dystrophy-like symptoms caused by DUX4-fl were alleviated (Non-Patent Document 35: Mitsuhashi et al., Hum Mol Genet 2013, 22:568-577). Since DUX4-s does not have transcription-promoting activity, this is thought to be the result of competitive inhibition of the target gene. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Tawil R. and Van Der Maarel SM Muscle Nerve 2006, 34:1-15 [Non-Patent Document 2] Arahata et al., Muscle Nerve 1995, 2:S56-S66 [Non-Patent Document 3] Padberg et al., Muscle Nerve 1995, 2:S73-S80 [Non-Patent Document 4] Padberg et al., Muscle Nerve 1995, 2:S81-S84; Flanigan et al., Neuromuscul Disord 2001, 11:525-529 [Non-Patent Document 5] Mostacciuolo et al., Clin Genet 2009, 75:550-555; Norwood et al, Brain 2009, 132:3175-3186

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[0008] An object of the present invention is to establish a new treatment method for facioscapulohumeral muscular dystrophy. [Means for solving the problem]

[0009] Since DUX4-fl and DUX4-s are different splicing isoforms transcribed from one DUX4 gene, it is possible to suppress cell death by converting the splicing of the DUX4 gene from DUX4-fl to DUX4-s using antisense oligonucleotides (Figure 1) and reducing the relative transcription amount of DUX4-fl. The present invention was completed based on this idea.

[0010] The gist of the present invention is as follows. (1) An oligonucleotide comprising an oligonucleotide having 15 to 30 bases and consisting of a nucleotide sequence complementary to the region of nucleotide numbers 502 to 556 or 578 to 612 of DUX4-fl mRNA consisting of the nucleotide sequence of SEQ ID NO: 1, the 5'-end and / or 3'-end of which may be chemically modified, said oligonucleotide or a pharma- ceutical acceptable salt thereof capable of converting the splicing of the DUX4 gene from DUX4-fl to DUX4-s. (2) The oligonucleotide according to (1), which contains a sequence of at least 15 consecutive nucleotides in any one of SEQ ID NOs: 2 to 85 (wherein t in the sequence may be u, and u may be t), or a pharma- ceutically acceptable salt thereof. (3) The oligonucleotide according to (1) or (2), or a pharma- ceutically acceptable salt thereof, wherein the number of bases of the oligonucleotide is 16 to 18. (4) The oligonucleotide according to (3) or a pharma- ceutically acceptable salt thereof, wherein the number of bases of the oligonucleotide is 18. (5) The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of (1) to (4), wherein at least one of the sugars and / or phosphodiester bonds constituting the oligonucleotide is modified. (6) The oligonucleotide or a pharma- ceutically acceptable salt thereof according to (5), wherein the sugar constituting the oligonucleotide is D-ribofuranose, and the sugar modification is modification of the hydroxyl group at the 2'-position of D-ribofuranose. (7) The oligonucleotide or a pharma- ceutically acceptable salt thereof according to (6), wherein the sugar modification is 2'-O-alkylation and / or 2'-O, 4'-C-alkylenation of D-ribofuranose. (8) The oligonucleotide or a pharma- ceutically acceptable salt thereof according to (6), wherein the sugar modification is 2'-O-methylation and / or 2'-O,4'-C-ethylation of D-ribofuranose. (9) The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of (5) to (8), wherein the modification of the phosphodiester bond is phosphorothioate. (10) The oligonucleotide according to any one of (1) to (9), or a pharma- ceutical acceptable salt thereof, characterized in that the oligonucleotide is an oligonucleotide having 15 to 30 bases and consisting of a nucleotide sequence complementary to the region from nucleotide number 506 to 549 of the nucleotide sequence of SEQ ID NO: 1. (11) The oligonucleotide according to (10), which contains a sequence of at least 15 consecutive nucleotides in any one of SEQ ID NOs: 5 to 31 (wherein t in the sequence may be u, and u may be t), or a pharma- ceutically acceptable salt thereof. (12) An oligonucleotide having any one of the following sequences, or a pharma- ceutically acceptable salt thereof: H.O.G. m1s -G e2s -G m1s -A m1s -G e2s -C m1s -A m1s -G e2s -G m1s -G m1s -T e2s -G m1s -A m1s -C e2s -C m1s -C m1s -C e2s -C m1t -H(DUX4-006); H.O.G. m1s -A e2s -C m1s -C m1s -C e2s -A m1s -C m1s -G e2s -A m1s -G m1s -G e2s -G m1s -A m1s -G e2s -C m1s -A m1s -G e2s -G m1t -H(DUX4-009); H.O.G. m1s -A e2s -A m1s -G m1s -G e2s -C m1s -Gm1s -A e2s -C m1s -C m1s -C e2s -A m1s -C m1s -G e2s -A m1s -G m1s -G e2s -G m1t -H(DUX4-011); HO-G m1s -G e2s -U m1s -G m1s -T e2s -G m1s -G m1s -G e2s -C m1s -G m1s -A e2s -A m1s -G m1s -G e2s -C m1s -G m1s -A e2s -C m1t -H(DUX4-014); HO-G m1s -A e2s -G m1s -C m1s -A e2s -G m1s -G m1s -G e2s -U m1s -G m1s -A e2s -C m1s -C m1s -C e2s -C m1s -C m1s -G e2s -C m1t -H(DUX4-036); HO-G m1s -G e2s -A m1s -G m1s -C e2s -A m1s -G m1s -G e2s -G m1s -U m1s G e2s -A m1s -C m1s -Ce2s -C m1s -C m1s -C e2s -G m1t -H(DUX4-037); HO-A m1s -C e2s -G m1s -A m1s -G e2s -G m1s -G m1s -A e2s -G m1s -C m1s -A e2s -G m1s -G m1s -G e2s -U m1s -G m1s -A e2s -C m1t -H(DUX4-040); HO-C m1s -G e2s -A m1s -C m1s -C e2s -C m1s -A m1s -C e2s -G m1s -A m1s -G e2s -G m1s -G m1s -A e2s -G m1s -C m1s -A e2s -G m1t -H(DUX4-044); HO-A m1s -A e2s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1s -C m1s -A e2s -C m1s -G m1s -A e2s -G m1s -G m1s -G e2s -A m1t -H(DUX4-047); HO-Cm1s -G e2s -A m1s -A m1s -G e2s -G m1s -C m1s -G e2s -A m1s -C m1s -C e2s -C m1s -A m1s -C e2s -G m1s -A m1s -G e2s -G m1t -H(DUX4-048); HO-G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1s -C m1s -A e2s -C m1s -G m1s -A e2s -G m1t -H(DUX4-049); HO-U m1s -G e2s -U m1s -G m1s -G e2s -G m1s -C m1s -G e2s -A m1s -A m1s -G e2s -G m1s -C m1s -G e2s -A m1s -C m1s -C e2s -C m1t -H(DUX4-052); HO-G m1s -T e2s -G m1s -U m1s -G e2s -G m1s -G m1s -Ce2s -G m1s -A m1s -A e2s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1t -H(DUX4-053); HO-C e2s -G m1s -A m1s -A e2s -G m1s -G m1s -C e2s -G m1s -A m1s -C m1s -C m1s -C m1s -A e2s -C m1s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.7); HO-C m1s -G m1s -A m1s -A e2s -G m1s -G m1s -C m1s -G m1s -A m1s -C m1s -C m1s -C e2s -A m1s -C e2s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.10); HO-C m1s -G m1s -A e2s -A m1s -G m1s -G m1s -C m1s -G m1s -A e2s -C m1s -C e2s -C m1s -A m1s -C m1s -Gm1s -A e2s -G m1s -G m1t -H(DUX4-48.11) HO-C e2s -G m1s -A e2s -A m1s -G m1s -G m1s -C m1s -G m1s -A m1s -C m1s -C m1s -C m1s -A m1s -C e2s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.12); HO-C m1s -G m1s -A m1s -A e2s -G m1s -G m1s -C m1s -G m1s -A m1s -C e2s -C m1s -C m1s -A m1s -C e2s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.14); HO-C m1s -G m1s -A m1s -A e2s -G m1s -G m1s -C m1s -G m1s -A e2s -C m1s -C m1s -C m1s -A m1s -C e2s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.15); HO-C m1s-G m1s -A e2s -A m1s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1s -C m1s -A m1s -C m1s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.19); HO-C m1s -G m1s -A m1s -A e2s -G m1s -G m1s -C m1s -G m1s -A m1s -C m1s -C m1s -C m1s -A m1s -C e2s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.20); HO-T e2s -G m1s -T e2s -G m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A m1s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1s -C e2t -H(DUX4-52.1); HO-T e2s -G m1s -T e2s -G m1s -G m1s -G m1s -C e2s -Gm1s -A m1s -A e2s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1s -C m1t -H(DUX4-52.2); H.O.T. e2s -G m1s -U m1s -G m1s -G m1s -G m1s -C m1s -G m1s -A m1s -A m1s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1s -C e2t -H(DUX4-52.7); HO-U m1s -G m1s -T e2s -G m1s -G m1s -G m1s -C m1s -G m1s -A m1s -A m1s -G m1s -G m1s -C e2s -G m1s -A e2s -C m1s -C e2s -C m1t -H(DUX4-52.9) [In the above sequence, A e2s , G e2s , C e2s and T e2s represents the corresponding ENA (where the base position of C is 5-methylcytosine) phosphorothioate-linked to the 3' adjacent structure. m1s , G m1s , C m1s , U m1s, represents the corresponding 2'-OMe-RNA linked to the 3' adjacent structure via a phosphorothioate bond. e2t represents the corresponding ENA (where the base position of C is 5-methylcytosine) in a phosphodiester bond with the adjacent structure on the 3' side. m1t , G m1t , C m1t represents the corresponding 2'-OMe-RNA phosphodiester-linked to the 3' adjacent structure. (13) The oligonucleotide according to any one of (1) to (12), or a pharma- ceutically acceptable salt thereof, further comprising an aminoalkyl phosphate group containing a fatty acid bonded to the 5'-end or 3'-end of the oligonucleotide. (14) The oligonucleotide or a pharma- ceutically acceptable salt thereof according to (13), wherein the fatty acid is at least one selected from the group consisting of myristic acid, palmitic acid, stearic acid, arachidic acid and behenic acid. (15) The oligonucleotide or a pharma- ceutical acceptable salt thereof according to any one of (1) to (14) for use in the treatment of a disease or symptom caused by expression of the DUX4-fl gene. (16) A disease or symptom caused by expression of the DUX4-fl gene, face The oligonucleotide or a pharma- ceutical acceptable salt thereof according to (15), which is for facioscapulohumeral muscular dystrophy. (17) A medicine comprising the oligonucleotide according to any one of (1) to (16) or a pharma- ceutically acceptable salt thereof. (18) A therapeutic agent for a disease or symptom caused by expression of the DUX4-fl gene, comprising the oligonucleotide according to any one of (1) to (16) or a pharma- ceutical acceptable salt thereof. (19) A disease or symptom caused by expression of the DUX4-fl gene, face The therapeutic agent according to (18), which is for facet-scapulohumeral muscular dystrophy. (20) A drug for converting splicing of DUX4 gene from DUX4-fl to DUX4-s, comprising the oligonucleotide according to any one of (1) to (16) or a pharma- ceutical acceptable salt thereof. (21) A method for treating a disease or symptom caused by expression of DUX4-fl gene in a subject, comprising administering to the subject the oligonucleotide according to any one of (1) to (16) or a pharma- ceutical acceptable salt thereof. (22) A disease or symptom caused by expression of the DUX4-fl gene, face The method for treating facet-scapulohumeral muscular dystrophy according to (21). (23) Use of the oligonucleotide or a pharma- ceutical acceptable salt thereof according to any one of (1) to (16) for the manufacture of a therapeutic agent for a disease or symptom caused by expression of the DUX4-fl gene. (24) A disease or symptom caused by expression of the DUX4-fl gene, face The use according to (22), which is for facet-scapulohumeral muscular dystrophy. Effect of the Invention

[0011] According to the present invention, the transcription amount of DUX4-fl can be reduced by converting the splicing of the DUX4 gene from DUX4-fl to DUX4-s using an antisense oligonucleotide. This specification includes the contents described in the specification and / or drawings of Japanese patent application No. 2019-129735, which is the priority basis of this application. [Brief description of the drawings]

[0012] [Figure 1] A diagram showing a method for converting the splicing of the DUX4 gene from DUX4-fl to DUX4-s using antisense oligonucleotides. [Diagram 2] Gel electrophoretic analysis of DUX4 transcripts obtained after transfection of HeLa cells with a DUX4 minigene construct and the compounds of the examples (DUX4-004 to DUX4-010). [Diagram 3] Gel electrophoretic analysis of DUX4 transcripts obtained after transfection of HeLa cells with a DUX4 minigene construct and the compounds of the examples (DUX4-011 to DUX4-016). [Figure 4] Gel electrophoretic analysis of DUX4 transcripts obtained after transfection of HeLa cells with a DUX4 minigene construct and the compounds of the examples (DUX4-022 to DUX4-030). [Diagram 5] Gel electrophoretic analysis of DUX4 transcripts obtained after transfection of HeLa cells with a DUX4 minigene construct and the compounds of the examples (DUX4-031 to DUX4-040). [Figure 6] Gel electrophoretic analysis of DUX4 transcripts obtained after transfection of HeLa cells with a DUX4 minigene construct and the compounds of the examples (DUX4-041 to DUX4-050). [Figure 7] Gel electrophoretic analysis of DUX4 transcripts obtained after transfection of HeLa cells with a DUX4 minigene construct and the compounds of the examples (DUX4-051 to DUX4-058). [Figure 8A] This is a graph showing the expression levels of target genes (upper row: ZSCAN4, lower row: MBD3L2, TRIM43) whose transcription is activated by DUX4. The vertical axis indicates the relative expression level to the expression level of the RPL13A gene, which is an internal standard. In the figure, "No" indicates the relative expression levels of cells that were not transfected with either the DUX4 minigene construct or the compounds of the examples (DUX4-009, DUX4-031, DUX4-036, DUX4-048), "D4Z4" indicates the cells that were transfected with only the DUX4 minigene construct, and "DUX4-009", "DUX4-031", "DUX4-036" and "DUX4-048" indicate the relative expression levels of cells that were transfected with both the DUX4 minigene construct and the compounds of the examples (DUX4-009, DUX4-031, DUX4-036, DUX4-048). The experiment was performed with N=3, and the results were analyzed by Dunnett's test in comparison with D4Z4, with p<0.05 marked with "*", p<0.01 marked with "**", and p<0.001 marked with "***". [Figure 8B]Graphs showing the expression levels of target genes whose transcription is activated by DUX4 (upper row: ZSCAN4, lower row: MBD3L2, TRIM43). The vertical axis indicates the relative expression level to the expression level of the RPL13A gene, which is an internal standard. In the figure, "No" indicates the relative expression level of cells that were not transfected with the DUX4 minigene construct or the compounds of the examples (DUX4-48.7, DUX4-48.11, DUX4-48.12, DUX4-48.13, DUX4-52.2), "D4Z4" indicates the relative expression level of cells that were transfected with only the DUX4 minigene construct, and "DUX4-48.7", "DUX4-48.11", "DUX4-48.12", "DUX4-48.13" and "DUX4-52.2" indicate the relative expression level of cells that were transfected with both the DUX4 minigene construct and the compounds of the examples (DUX4-48.7, DUX4-48.11, DUX4-48.12, DUX4-48.13, DUX4-52.2). The experiment was performed with N=3, and the results were analyzed by Dunnett's test in comparison with D4Z4, with p<0.05 marked with "*", p<0.01 marked with "**", and p<0.001 marked with "***". [Figure 9] This is a diagram showing PCR products in zebrafish fertilized eggs injected with D4Z4 mRNA. RT(1) indicates the results of the RNA sample before reverse transcription reaction, and RT(+) indicates the results after reverse transcription reaction. "D4" indicates the PCR products of a fertilized egg injected with only D4Z4 mRNA, and "D4 + DUX4-048" indicates the PCR products of a fertilized egg injected with both D4Z4 mRNA and the compound of the embodiment (DUX4-048). "M1" and "M2" indicate markers, and [UI] indicates the PCR products from a fertilized egg that was not injected. [Figure 10] Gel electrophoretic analysis of DUX4 transcripts obtained after transfection of HeLa cells with a DUX4 minigene construct and the compounds of the examples (DUX4-48.1 to DUX4-48.10). [Figure 11]Gel electrophoretic analysis of DUX4 transcripts obtained after transfection of HeLa cells with DUX4 minigene constructs and compounds of the examples (DUX4-48.11 to DUX4-48.13, DUX4-52.1 and DUX4-52.2). [Figure 12] Gel electrophoretic analysis of DUX4 transcripts obtained after transfection of HeLa cells with DUX4 minigene constructs and compounds of the examples (DUX4-48.14 to DUX4-48.23, and DUX4-048). [Figure 13] Gel electrophoretic analysis of DUX4 transcripts obtained after transfection of HeLa cells with DUX4 minigene constructs and compounds of the examples (DUX4-52.3 to DUX4-52.12, and DUX4-052). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Hereinafter, the embodiment of the present invention will be described in more detail.

[0014] The present invention provides an oligonucleotide comprising 15 to 30 bases and consisting of a nucleotide sequence complementary to the region of nucleotide numbers 502 to 556 or 578 to 612 (preferably the region of nucleotide numbers 506 to 549) of DUX4-fl mRNA consisting of the nucleotide sequence of SEQ ID NO: 1, the 5'-end and / or 3'-end of which may be chemically modified, and capable of converting the splicing of the DUX4 gene from DUX4-fl to DUX4-s, as well as a pharma- ceutically acceptable salt or solvate thereof.

[0015] The sequence information of DUX4-fl mRNA is registered in GenBank, and the accession number is HQ266761. The base sequence is shown in SEQ ID NO: 1 in the sequence listing. The sequence of DUX4-s mRNA is the sequence of base numbers 1 to 477 of the base sequence of SEQ ID NO: 1.

[0016] DUX4-fl and DUX4-s are different splicing isoforms transcribed from a single DUX4 gene, with DUX4-fl being the full-length form (approximately 55 kDa) and DUX4-s being the short form (approximately 20 kDa). DUX4-fl exerts strong transcriptional activation ability via the C-terminal transcriptional activation domain, and it is thought that DUX4-fl is involved in the development of FSHD. On the other hand, the short form DUX4-s is expressed slightly in cells derived from healthy individuals, but does not function as a transcription factor because it does not have a transcriptional activation domain.

[0017] The oligonucleotide of the present invention can convert the splicing of the DUX4 gene from DUX4-fl to DUX4-s. By converting the splicing of the DUX4 gene from DUX4-fl to DUX4-s, the relative transcription amount of DUX4-fl can be reduced, thereby suppressing cell death, and a therapeutic effect on facioscapulohumeral muscular dystrophy can be expected. DUX4-fl does not usually contain an intron, but there are patients who express one with intron 1 remaining. Therefore, DUX4-fl may or may not contain an intron. The degree of conversion from DUX4-fl to DUX4-s does not matter, but the conversion rate is preferably 10% or more, and more preferably 25% or more. The degree of conversion from DUX4-fl to DUX4-s can be measured by the following method. The oligonucleotide of the present invention is transfected into cells, total RNA is extracted from the transfected cells, and a reverse transcription reaction is performed, and then double-stranded DNA is made by PCR reaction using primers that amplify both DUX4-fl and DUX4-s. The double-stranded DNA amplified from DUX4-fl and DUX4-s is separated into two bands by gel electrophoresis, and the bands are visualized to measure the degree of conversion. In this case, when using ethidium bromide, SYBR Green, etc., the degree of visualization varies depending on the size of the amplified double-stranded DNA, so care must be taken to determine the degree of conversion. In addition, the degree of conversion can be measured by creating TaqMan probes specific to DUX4-fl and DUX4-s and performing quantitative PCR to quantify the amount of DUX4-fl and DUX4-s mRNA. The degree of conversion can be measured by detecting DUX4-fl and DUX4-s proteins in the sample by Western blotting, or by detecting peptide fragments specific to DUX4-fl and DUX4-s proteins by mass spectrometry.

[0018] Examples of oligonucleotides of the present invention include those containing a sequence of at least 15 consecutive nucleotides in any of SEQ ID NOs: 2 to 85 (preferably, SEQ ID NOs: 5 to 31) (however, in the sequence, t may be u, and u may be t).

[0019] The number of bases in the oligonucleotide of the present invention is suitably 15 to 30, preferably 16 to 18, and more preferably 18.

[0020] A preferred example of the oligonucleotide of the present invention is an oligonucleotide having 15 to 30 bases and consisting of a nucleotide sequence complementary to the region from nucleotide number 506 to 549 of the nucleotide sequence of SEQ ID NO: 1, and a more preferred example of the oligonucleotide of the present invention is an oligonucleotide containing a sequence of at least 15 consecutive nucleotides in any of the sequences of SEQ ID NOs: 5 to 31 (however, in the sequence, t may be u, and u may be t).

[0021] The oligonucleotide (antisense oligonucleotide) of the present invention may be natural DNA, natural RNA, a DNA / RNA chimera, or a modified form thereof, but it is preferable that at least one of the nucleotides constituting the oligonucleotide is a modified nucleotide.

[0022] The modified nucleotides in the present invention include those in which the sugar has been modified (for example, those in which the hydroxyl group at the 2' position of D-ribofuranose has been modified (D-ribofuranose is 2'-O-alkylated), those in which D-ribofuranose has been 2'-,4'-bridged (D-ribofuranose is 2'-O, Examples of such oligonucleotides include those in which at least one D-ribofuranose constituting the antisense oligonucleotide is 2'-O-alkylated (e.g., 2'-O-methylated) or 2'-O,4'-C-alkylenated (e.g., 2'-O,4'-C-ethylenated) have a high binding strength to RNA and are highly resistant to nucleases, and therefore can be expected to have a higher therapeutic effect than natural nucleotides (i.e., oligoDNA, oligoRNA). In addition, oligonucleotides in which at least one phosphodiester bond constituting the oligonucleotide is thioated can also be expected to have a higher therapeutic effect than natural nucleotides (i.e., oligoDNA, oligoRNA) because they are highly resistant to nucleases. Oligonucleotides containing both modified sugars and modified phosphates as described above can be expected to have an even higher therapeutic effect because they are more resistant to nucleases.

[0023] Examples of sugar modifications for the oligonucleotides (antisense oligonucleotides) of the present invention include 2'-O-alkylation of D-ribofuranose (e.g., 2'-O-methylation, 2'-O-aminoethylation, 2'-O-propylation, 2'-O-allylation, 2'-O-methoxyethylation, 2'-O-butylation, 2'-O-pentylation, 2'-O-propargylation, etc.); 2'-O,4'-C-alkylenation of D-ribofuranose (e.g., 2'-O,4'-C-ethylenation, 2'-O,4'-C-propylenation, 2'-O,4'-C-tetramethylenation, 2'-O,4'-C-pentamethyleneation, etc.), S-cEt (2',4'-constrained ethyl), AmNA (amide-bridged nucleic acid and combinations of 2'-deoxylation and other modifications such as 2'-deoxy-2'-C,4'-C-methyleneoxymethylenation of D-ribofuranose, 3'-deoxy-3'-amino-2'-deoxy-D-ribofuranose, and 3'-deoxy-3'-amino-2'-deoxy-2'-fluoro-D-ribofuranose.

[0024] With respect to the oligonucleotides (antisense oligonucleotides) of the present invention, examples of modifications of the phosphodiester bond include phosphorothioate bond, methylphosphonate bond, methylthiophosphonate bond, phosphorodithioate bond, phosphoramidate bond, and the like.

[0025] With respect to the oligonucleotides (antisense oligonucleotides) of the present invention, examples of base modifications include 5-methylation, 5-fluoronation, 5-bromination, 5-iodination, and N4-methylation of cytosine; 5-demethylation (uracil), 5-fluoronation, 5-bromination, and 5-iodination of thymine; N6-methylation and 8-bromination of adenine; and N2-methylation and 8-bromination of guanine.

[0026] The nucleotide residues constituting the oligonucleotide of the present invention are, for example, t、G t 、5meC t 、C t 、T t 、U t 、A p 、G p 、5meC p 、C p 、T p 、U p 、A s 、G s 、5meC s 、C s 、T s 、U s 、A m1t 、G m1t 、C m1t 、5meC m1t 、U m1t 、A m1p 、G m1p 、C m1p 、5meC m1p 、U m1p 、A m1s 、G m1s 、C m1s 、5meC m1s 、U m1s 、A 2t 、G 2t 、C 2t 、T 2t 、A e2p 、G e2p 、C e2p 、T e2p 、A e2s 、G e2s 、C e2s 、T e2s 、A 1t 、G 1t 、C 1t 、T 1t 、A e1p 、G e1p 、C e1p 、T e1p 、A e1s 、G e1s 、C e1s 、T e1s 、A 3t 、G 3t 、C 3t 、T 3t 、A e3p 、G e3p 、C e3p 、T e3p 、A e3s 、Ge3s , C e3s , T e3s , A m2t , G m2t , 5meC m2t , T m2t , A m2p , G m2p , 5meC m2p , T m2p , A m2s , G m2s , 5meC m2s , T m2s Examples include:

[0027] Preferred oligonucleotides of the present invention are those shown in Tables 1 to 3, and more preferred are the oligonucleotides shown below. H.O.G. m1s -G e2s -G m1s -A m1s -G e2s -C m1s -A m1s -G e2s -G m1s -G m1s -T e2s -G m1s -A m1s -C e2s -C m1s -C m1s -C e2s -C m1t -H(DUX4-006); H.O.G. m1s -A e2s -C m1s -C m1s -C e2s -A m1s -C m1s -G e2s -A m1s -G m1s -G e2s -G m1s -A m1s -G e2s -C m1s -A m1s -G e2s -G m1t -H(DUX4-009); H.O.G. m1s -A e2s -Am1s -G m1s -G e2s -C m1s -G m1s -A e2s -C m1s -C m1s -C e2s -A m1s -C m1s -G e2s -A m1s -G m1s -G e2s -G m1t -H(DUX4-011); HO-G m1s -G e2s -U m1s -G m1s -T e2s -G m1s -G m1s -G e2s -C m1s -G m1s -A e2s -A m1s -G m1s -G e2s -C m1s -G m1s -A e2s -C m1t -H(DUX4-014); HO-G m1s -A e2s -G m1s -C m1s -A e2s -G m1s -G m1s -G e2s -U m1s -G m1s -A e2s -C m1s -C m1s -C e2s -C m1s -C m1s -G e2s -C m1t -H(DUX4-036); HO-G m1s -G e2s -A m1s -G m1s -C e2s -A m1s -G m1s -G e2s -G m1s -Um1s G e2s -A m1s -C m1s -C e2s -C m1s -C m1s -C e2s -G m1t -H(DUX4-037); HO-A m1s -C e2s -G m1s -A m1s -G e2s -G m1s -G m1s -A e2s -G m1s -C m1s -A e2s -G m1s -G m1s -G e2s -U m1s -G m1s -A e2s -C m1t -H(DUX4-040); HO-C m1s -G e2s -A m1s -C m1s -C e2s -C m1s -A m1s -C e2s -G m1s -A m1s -G e2s -G m1s -G m1s -A e2s -G m1s -C m1s -A e2s -G m1t -H(DUX4-044); HO-A m1s -A e2s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1s -C m1s -A e2s -C m1s -G m1s -A e2s -G m1s -G m1s -Ge2s -A m1t -H(DUX4-047); HO-C m1s -G e2s -A m1s -A m1s -G e2s -G m1s -C m1s -G e2s -A m1s -C m1s -C e2s -C m1s -A m1s -C e2s -G m1s -A m1s -G e2s -G m1t -H(DUX4-048); HO-G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1s -C m1s -A e2s -C m1s -G m1s -A e2s -G m1t -H(DUX4-049); HO-U m1s -G e2s -U m1s -G m1s -G e2s -G m1s -C m1s -G e2s -A m1s -A m1s -G e2s -G m1s -C m1s -G e2s -A m1s -C m1s -C e2s -C m1t -H(DUX4-052); HO-G m1s -T e2s -G m1s -Um1s -G e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1t -H(DUX4-053); HO-C e2s -G m1s -A m1s -A e2s -G m1s -G m1s -C e2s -G m1s -A m1s -C m1s -C m1s -C m1s -A e2s -C m1s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.7); HO-C m1s -G m1s -A m1s -A e2s -G m1s -G m1s -C m1s -G m1s -A m1s -C m1s -C m1s -C e2s -A m1s -C e2s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.10); HO-C m1s -G m1s -A e2s -A m1s -G m1s -G m1s -C m1s -G m1s -A e2s -C m1s -Ce2s -C m1s -A m1s -C m1s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.11); HO-C e2s -G m1s -A e2s -A m1s -G m1s -G m1s -C m1s -G m1s -A m1s -C m1s -C m1s -C m1s -A m1s -C e2s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.12); HO-C m1s -G m1s -A m1s -A e2s -G m1s -G m1s -C m1s -G m1s -A m1s -C e2s -C m1s -C m1s -A m1s -C e2s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.14); HO-C m1s -G m1s -A m1s -A e2s -G m1s -G m1s -C m1s -G m1s -A e2s -C m1s -C m1s -C m1s -A m1s -C e2s -G m1s -A e2s -G m1s-G m1t -H(DUX4-48.15); HO-C m1s -G m1s -A e2s -A m1s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1s -C m1s -A m1s -C m1s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.19); HO-C m1s -G m1s -A m1s -A e2s -G m1s -G m1s -C m1s -G m1s -A m1s -C m1s -C m1s -C m1s -A m1s -C e2s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.20); HO-T e2s -G m1s -T e2s -G m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A m1s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1s -C e2t -H(DUX4-52.1); HO-T e2s -G m1s -T e2s -Gm1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1s -C m1t -H(DUX4-52.2); HO-T e2s -G m1s -U m1s -G m1s -G m1s -G m1s -C m1s -G m1s -A m1s -A m1s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1s -C e2t -H(DUX4-52.7); HO-U m1s -G m1s -T e2s -G m1s -G m1s -G m1s -C m1s -G m1s -A m1s -A m1s -G m1s -G m1s -C e2s -G m1s -A e2s -C m1s -C e2s -C m1t -H(DUX4-52.9)。

[0028] The oligonucleotide (antisense oligonucleotide) of the present invention can be synthesized using a commercially available synthesizer (e.g., Perkin Elmer's Model 392 using the phosphoramidite method) according to the method described in the literature (Nucleic Acids Research, 12, 4539 (1984)). The phosphoramidite reagent used in this case can be a commercially available reagent for natural nucleosides and 2'-O-methyl nucleosides (i.e., 2'-O-methyl guanosine, 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl uridine). The following is a list of 2'-O-alkylguanosine, adenosine, cytidine, and uridine with alkyl groups having 2 to 6 carbon atoms.

[0029] 2'-O-aminoethylguanosine, adenosine, cytidine, and uridine can be synthesized according to the literature (Blommers et al. Biochemistry (1998), 37, 17714-17725.).

[0030] 2'-O-propylguanosine, adenosine, cytidine, and uridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838.).

[0031] Commercially available reagents can be used for 2'-O-allylguanosine, adenosine, cytidine, and uridine.

[0032] 2'-O-Methoxyethylguanosine, adenosine, cytidine, and uridine can be synthesized according to the patent (US6261840) or the literature (Martin, P. Helv. Chim. Acta. (1995) 78, 486-504.

[0033] 2'-O-Butylguanosine, adenosine, cytidine, and uridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838.).

[0034] 2'-O-pentylguanosine, adenosine, cytidine, and uridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838.).

[0035] Commercially available reagents can be used for 2'-O-propargylguanosine, adenosine, cytidine, and uridine.

[0036] 2'-O,4'-C-methyleneguanosine, adenosine, cytidine, 5-methylcytidine and thymidine can be produced according to the method described in WO99 / 14226, and 2'-O,4'-C-alkyleneguanosine, adenosine, cytidine, 5-methylcytidine and thymidine having an alkylene group with 2 to 5 carbon atoms can be produced according to the method described in WO00 / 47599.

[0037] 2'-Deoxy-2'-C,4'-C-methyleneoxymethylenated nucleosides of D-ribofuranose can be synthesized according to the literature (Wang, G. et al. Tetrahedron (1999), 55, 7707-7724).

[0038] S-cEt (constrained ethyl) can be synthesized according to the literature (Seth, PP et al. J. Org. Chem (2010), 75, 1569-1581.).

[0039] AmNA can be synthesized according to the literature (Yahara, A. et al. ChemBioChem (2012), 13, 2513-2516.) or WO2014 / 109384.

[0040] In the present invention, the nucleic acid base sequence can be described as adenine (A) or (a), guanine (G) or (g), cytosine (C) or (c), thymine (T) or (t), and uracil (U) or (u). 5-methylcytosine can be used instead of cytosine. Among the nucleic acid bases, uracil (U) or (u) and thymine (T) or (t) are interchangeable. Either uracil (U) or (u) or thymine (T) or (t) can be used to form a base pair with adenine (A) or (a) in the complementary strand.

[0041] After coupling with a phosphoramidite reagent, an antisense oligonucleotide having a phosphorothioate bond can be synthesized by reacting with a reagent such as sulfur, tetraethylthiuram disulfide (TETD, Applied Biosystems), Beaucage reagent (Glen Research), or xanthan hydride (Tetrahedron Letters, 32, 3005 (1991), J. Am. Chem. Soc. 112, 1253 (1990), PCT / WO98 / 54198).

[0042] The controlled pore glass (CPG) used in the synthesizer can be a commercially available product bound to a 2'-O-methyl nucleoside. For 2'-O,4'-C-methylene guanosine, adenosine, 5-methyl cytidine and thymidine, the nucleoside produced according to the method described in WO99 / 14226 can be bound to the CPG, and for 2'-O,4'-C-alkylene guanosine, adenosine, 5-methyl cytidine and thymidine with an alkylene group having 2 to 5 carbon atoms, the nucleoside produced according to the method described in WO00 / 47599 can be bound to the CPG according to the literature (Oligonucleotide Synthesis, Edited by MJGait, Oxford University Press, 1984). By using a modified CPG (described in Example 12b of JP-A-7-87982), an oligonucleotide bound to a 2-hydroxyethyl phosphate group at the 3' end can be synthesized. In addition, 3'-amino-Modifier C3 CPG, 3'-amino-Modifier C7 CPG, Glyceryl CPG, (Glen Research), 3'-specer C3 SynBase CPG 1000, and 3'-specer C9 SynBase CPG 1000 (link technologies) can be used to synthesize oligonucleotides with a hydroxyalkyl phosphate group or an aminoalkyl phosphate group attached to the 3' end.

[0043] The oligonucleotides of the present invention can be chemically modified by linking the 3'-end and / or 5'-end to a molecular structure other than a nucleotide via a phosphodiester bond or a phosphorothioate bond. The present invention also provides such chemically modified oligonucleotides.

[0044] After chain elongation of the oligonucleotide having the desired sequence is completed, an oligonucleotide having an aminoalkyl phosphate group attached to the 5' end can be synthesized by using 5'-Amino-Modifier C6 (Glen Research), 5'-TFA-Amino-Modifier C6-CE Phosphoramidite, 5'-TFA-Amino-Modifier-C5-CE Phosphoramidite (Link Technologies), etc.

[0045] The 5'-end and / or 3'-end of the oligonucleotide (antisense oligonucleotide) of the present invention may have a hydrophobic group. The 5'-end and / or 3'-end of the oligonucleotide (antisense oligonucleotide) of the present invention may further have an aminoalkyl phosphate group containing a fatty acid bonded thereto. For example, the 5'-end and / or 3'-end of the oligonucleotide may have, instead of a hydroxyl group, JPEG0007627495000001.jpg17101 JPEG0007627495000002.jpg18108 JPEG0007627495000003.jpg17116 JPEG0007627495000004.jpg17123 JPEG0007627495000005.jpg17130 After chain elongation of an oligonucleotide having a target sequence is completed, an amidite unit corresponding to a fatty acid, such as 2-cyanoethyl (6-palmitamidohexyl) diisopropylphosphoramidite (Nucleic Acids Res. (2020) 47, 6029-6044, Link Technologies), can be coupled to synthesize the compound.

[0046] It can be synthesized by reacting an active ester such as a pentafluorophenyl ester of a fatty acid such as myristic acid, palmitic acid, stearic acid, arachidic acid, or behenic acid with an oligonucleotide having an aminoalkyl phosphate group (e.g., an alkyl group having 3 to 9 carbon atoms) at the 5' end and / or 3' end and a target sequence (Nucleic Acids Res. (2020) 47, 6029-6044). In addition, an oligonucleotide having a target sequence can be synthesized by condensing a fatty acid such as myristic acid, palmitic acid, stearic acid, arachidic acid, or behenic acid using a condensing agent such as HATU on a solid support having an aminoalkyl phosphate group (e.g., an alkyl group having 3 to 9 carbon atoms) at the 5' end during solid-phase synthesis, followed by deprotection and purification (PCT / WO2017 / 192679).

[0047] In addition, oligonucleotides with cholesterol or tocopherol bound to the 5' end can be synthesized by using 5'-Tocopherol-CE Phosphoramidite, 5'-Cholesterol-CE Phosphoramidite, 5'-Cholesterol-TEG-CE Phosphoramidite (Link Technologies), etc.

[0048] The oligonucleotide (antisense oligonucleotide) of the present invention can be used as a medicine, specifically, for the treatment of facioscapulohumeral muscular dystrophy. The treatment may be performed before the onset (prevention), simultaneously with the onset, or after the onset.

[0049] The oligonucleotide (antisense oligonucleotide) of the present invention may be used in the form of its pharma- ceutically acceptable salt. The term "pharma-ceutically acceptable salt" refers to a salt of an oligonucleotide (antisense oligonucleotide), and examples of such salts include metal salts such as alkali metal salts (e.g., sodium salt, potassium salt, and lithium salt), alkaline earth metal salts (e.g., calcium salt, and magnesium salt), aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; inorganic salts (e.g., ammonium salt), t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzoyl ...phenylglycine alkyl ester salt, phenylglycine alkyl ester salt, phenylglycine alkyl ester salt, phenylglycine alkyl ester salt, phenylglycine alkyl ester salt, phenylglycine alkyl ester salt, phenylglycine alkyl ester salt, phenylglycine alkyl ester salt, phenylglycine alkyl ester salt, phenylglycine alkyl ester salt, phenylglycine alkyl ester salt, phenylglycine alkyl ester salt, phenylglycine alkyl ester salt, phenylglycine alkyl ester salt, phenylglycine alkyl ester salt, phenylglycine alkyl ester salt, phenylglycine alkyl ester salt, phenylglycine alkyl ester salt, phenyl Examples of the salts include amine salts such as organic salts such as di-phenethylamine salts, piperazine salts, tetramethylammonium salts, and tris(hydroxymethyl)aminomethane salts; hydrohalides such as hydrofluoride, hydrochloride, hydrobromide, and hydroiodide; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; lower alkanesulfonates such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonates such as benzenesulfonate and p-toluenesulfonate; organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate. These salts can be produced by known methods.

[0050] Furthermore, oligonucleotides (antisense oligonucleotides) may exist as solvates (e.g., hydrates), and such solvates are also included in the pharma- ceutically acceptable salts of the oligonucleotides of the present invention.

[0051] Therefore, the present invention provides a medicine comprising the above-mentioned oligonucleotide or a pharma- ceutical acceptable salt thereof. The present invention also provides a therapeutic agent for a disease or symptom caused by expression of the DUX4-fl gene, particularly a therapeutic agent for facioscapulohumeral muscular dystrophy, comprising the above-mentioned oligonucleotide or a pharma- ceutical acceptable salt thereof.

[0052] DUX4-fl protein activates the expression of genes expressed in early embryos and testes, such as PRAMEF2, TRIM43, and ZSCAN4 (Non-Patent Document 19: Geng et al., Dev Cell 2012, 22:38-51). Expression of DUX4-fl is associated with apoptosis (Kowaljow et al., Neuromuscul Disord 2007, 17:611-623; Wallace et al., Ann Neurol 2011, 69:540-552), abnormal muscle development (Bosnakovski et al., EMBO J 2008, 27:2766-2779; Banerji et al., Nat Commun 2017, 8:2152), inhibition of nonsense codon-mediated mRNA decay (Feng et al., eLife 2015, 4:e04996), formation of intracellular double-stranded RNA (Shadle et al., PLoS Genet 2017, 13:e1006658), and protein aggregation. (Non-Patent Document 26: Homma et al., Ann Clin Transl Neurol 2015, 2:151-166), etc., which may be the cause of various diseases and symptoms. The oligonucleotide of the present invention converts the splicing of the DUX4 gene from DUX4-fl, which has strong transcriptional activity, to DUX4-s, which has no transcriptional activity, and is therefore effective in treating diseases or symptoms caused by the transcriptional activity of DUX4-fl.

[0053] The therapeutic agent for facioscapulohumeral muscular dystrophy of the present invention may be targeted to patients expressing DUX4-fl. Various studies, including the report by Lemmers et al. (Non-Patent Document 8: Lemmers et al., Science 2010, 329:1650-1653), have shown that DUX4-fl is expressed in patients with haplotype 4qA, whether they have FSHD type 1 or FSHD type 2, so the present invention is applicable to both FSHD type 1 and FSHD type 2.

[0054] When the oligonucleotide (antisense oligonucleotide) of the present invention or a pharma- ceutical acceptable salt thereof is used to treat facioscapulohumeral muscular dystrophy, it can be administered orally in the form of tablets, capsules, granules, powders, syrups, etc., or parenterally in the form of injections, suppositories, patches, topical preparations, etc., either by itself or mixed with an appropriate pharma- ceutical acceptable excipient, diluent, etc.

[0055] These preparations may contain excipients (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, bayerian starch, alpha starch, and dextrin; cellulose derivatives such as crystalline cellulose; organic excipients such as gum arabic; dextran; and pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminometasilicate; phosphates such as calcium hydrogen phosphate; carbonates such as calcium carbonate; and sulfates such as calcium sulfate), lubricants, and the like. lubricants (e.g., stearic acid; metal stearates such as calcium stearate and magnesium stearate; talc; colloidal silica; waxes such as beeswax and gay wax; boric acid; adipic acid; sulfates such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL-leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; the above-mentioned starch derivatives, etc.), binders (e.g., hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, etc.), lydon, macrogol, compounds similar to the above excipients, etc.), disintegrants (e.g., cellulose derivatives such as low-substituted hydroxypropylcellulose, carboxymethylcellulose, carboxymethylcellulose calcium, and internally crosslinked sodium carboxymethylcellulose; chemically modified starches and celluloses such as carboxymethyl starch, carboxymethyl starch sodium, and crosslinked polyvinylpyrrolidone, etc.), emulsifiers (e.g., colloidal clays such as bentonite and veegum; metal hydroxides such as magnesium hydroxide and aluminum hydroxide; anionic surfactants such as sodium lauryl sulfate and calcium stearate; cationic surfactants such as benzalkonium chloride; nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, and sucrose fatty acid esters, etc.), stabilizers (paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal;It is produced by a well-known method using additives such as dehydroacetic acid, sorbic acid, flavoring agents (e.g., commonly used sweeteners, acidulants, flavors, etc.), diluents, etc.;

[0056] The therapeutic agent of the present invention may contain 0.1 to 250 μmoles / mL of an oligonucleotide (antisense oligonucleotide) or a pharma- ceutical acceptable salt thereof, and preferably may contain 1 to 50 μmoles / mL of an oligonucleotide (antisense oligonucleotide) or a pharma- ceutical acceptable salt thereof, 0.02 to 10% w / v of a carbohydrate or polyhydric alcohol, and 0.01 to 0.4% w / v of a pharma- ceutical acceptable surfactant.

[0057] As the carbohydrate, monosaccharides or disaccharides are particularly preferred. Examples of these carbohydrates and polyhydric alcohols include glucose, galactose, mannose, lactose, maltose, mannitol and sorbitol. These may be used alone or in combination.

[0058] In addition, preferred examples of the surfactant in the present invention include polyoxyethylene sorbitan mono- to tri-esters, alkylphenyl polyoxyethylenes, sodium taurocholate, sodium cholate, and polyhydric alcohol esters. Of these, polyoxyethylene sorbitan mono- to tri-esters are particularly preferred, and oleate, laurate, stearate, and palmitate are particularly preferred as esters. These may be used alone or in combination.

[0059] Furthermore, the therapeutic agent of the present invention may further preferably contain 0.03 to 0.09 M of a pharma- ceutical acceptable neutral salt, such as sodium chloride, potassium chloride and / or calcium chloride.

[0060] Furthermore, the therapeutic agent of the present invention may more preferably contain a pharma- ceutically acceptable buffer at 0.002 to 0.05 M. Preferred examples of the buffer include sodium citrate, sodium glycinate, sodium phosphate, and tris(hydroxymethyl)aminomethane. These buffers may be used alone or in combination.

[0061] Furthermore, the above-mentioned therapeutic agent may be supplied in a solution state. However, in cases where it is necessary to store the agent for a certain period of time, it is usually preferable to freeze-dry the oligonucleotide (antisense oligonucleotide) in order to stabilize it and prevent a decrease in therapeutic effect. In this case, the agent may be reconstituted with a dissolving solution (such as distilled water for injection) when used, that is, in a liquid state to be administered. Therefore, the therapeutic agent of the present invention also includes a freeze-dried agent that is used by reconstituting the agent with a dissolving solution so that each component has a predetermined concentration range. In order to promote the solubility of the freeze-dried agent, amino acids such as albumin and glycine may be further contained.

[0062] When the oligonucleotide (antisense oligonucleotide) of the present invention or a pharma- ceutical acceptable salt thereof is administered to a human, it may be administered, for example, at a daily dose of about 0.01 to 100 mg / kg (body weight), preferably 0.1 to 20 mg / kg (body weight) for an adult, once or several times as a subcutaneous injection, intravenous drip injection, or intravenous injection; however, the dose and number of administrations may be appropriately changed depending on the type of disease, symptoms, age, administration method, etc.

[0063] The oligonucleotide (antisense oligonucleotide) of the present invention or its pharmacologic acceptable salt can be administered to a human, for example, a patient expressing DUX4-fl, for example, as follows. That is, the oligonucleotide (antisense oligonucleotide) or its pharmacologic acceptable salt is produced by a method well known to those skilled in the art, and sterilized by a conventional method to prepare an injectable solution of, for example, 125 mg / mL. This solution is administered intravenously to a patient, for example, in the form of an infusion, so that the dose of the oligonucleotide (antisense oligonucleotide) is, for example, 10 mg per kg of body weight. The administration is performed, for example, at intervals of one week, and the treatment is then appropriately repeated while checking the therapeutic effect.

[0064] In addition, the oligonucleotide (antisense oligonucleotide) of the present invention and its pharma- ceutical acceptable salt can be used to convert splicing of the DUX4 gene from DUX4-fl to DUX4-s. Thus, the present invention provides an agent for converting splicing of the DUX4 gene from DUX4-fl to DUX4-s, comprising the above-mentioned oligonucleotide or its pharma-ceutical acceptable salt. As an agent for converting splicing of the DUX4 gene from DUX4-fl to DUX4-s, an oligonucleotide or its pharma-ceutical acceptable salt can be used that can increase the expression level of DUX4-s in cells expressing DUX4-fl by about 10% or more, preferably 30% or more, more preferably 50% or more compared to a control, but is not limited to the above range as long as it has the function of converting DUX4-fl to DUX4-s. The agent of the present invention can be used as a medicine or an experimental reagent.

[0065] When used as an experimental reagent, cells, tissues or organs expressing DUX4-fl can be treated with the oligonucleotide (antisense oligonucleotide) of the present invention and its pharma- ceutical acceptable salt to convert splicing of the DUX4 gene from DUX4-fl to DUX4-s. The oligonucleotide (antisense oligonucleotide) of the present invention and its pharma- ceutical acceptable salt may be used in an amount effective for converting splicing of the DUX4 gene from DUX4-fl to DUX4-s. Examples of cells expressing DUX4-fl include naturally occurring cells such as ES cells, iPS cells, and myoblasts derived from FSHD patients. In addition to naturally occurring cells, recombinant cells into which the DUX4 gene has been introduced can also be exemplified. Examples of tissues and organs expressing DUX4-fl include testes, early developmental embryos at the 4-cell stage, and skeletal muscles of FSHD patients. The expression of DUX4-fl can be analyzed by analyzing DUX4-fl (transcript) in a sample by RT-PCR, detecting DUX4-fl protein in a sample by Western blotting, or detecting peptide fragments specific to DUX4-fl protein by mass spectrometry. EXAMPLES

[0066] The present invention will be specifically described below with reference to examples. Note that these examples are for the purpose of explaining the present invention and are not intended to limit the scope of the present invention. Example 1 HO-C m1s -T e2s -G m1s -U m1s -G e2s -G m1s -G m1s -A e2s -G m1s -A m1s -G e2s -C m1s -C m1s -C e2s -C m1s -A m1s -G e2s -G m1tSynthesis of -H(DUX4-035) (SEQ ID NO:2) The title oligonucleotide was synthesized using an automatic nucleic acid synthesizer ("MerMade 192X" BioAutomation) by the phosphoramidite method (Nucleic Acids Research, 12, 4539 (1984)). However, for the condensation, a solution of Activator Solution-3 (0.25 mol / L 5-benzylthio-1H-tetrazole·acetonitrile solution, Wako Pure Chemical Industries, Ltd., product No. 013-20011) containing 0.4% 1-methylimidazole (Wako Pure Chemical Industries, Ltd., product No. 134-12801) was used, and the reaction time was about 10 minutes. As a thiolation reagent for forming phosphorothioate bonds, phenylacetyl disulfide (manufactured by CARBOSYNTH, product No. FP07495) was dissolved to a concentration of 0.2 M in a mixture of dehydrated acetonitrile (manufactured by Kanto Chemical, product No. 01837-05): dehydrated pyridine (manufactured by Kanto Chemical, product No. 11339-05) = 1:1 (v / v) and used. Other reagents used were CAP A for AKTA (1-methylimidazole / acetonitrile solution, Sigma-Aldrich, product No. L040050), Cap B1 for AKTA (acetic anhydride / acetonitrile solution, Sigma-Aldrich, product No. L050050), Cap B2 for AKTA (pyridine / acetonitrile solution, Sigma-Aldrich, product No. L050150), and DCA Deblock (dichloroacetic acid / toluene solution, Sigma-Aldrich, product No. L023050). As amidite reagents, 2'-O-Me phosphoramidites of nucleosides (adenosine product No. ANP-5751, cytidine product No. ANP-5752, guanosine product No. ANP-5753, uridine product No. ANP-5754) were from ChemGenes.The non-natural phosphoramidites are those of Example 14 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-6-N-benzoyl adenosine-3'-O-(2-cyanoethyl N,N-diisopropyl) phosphoramidite), Example 27 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-2-N-isobutyrylguanosine-3'-O-(2-cyanoethyl N,N-diisopropyl) phosphoramidite), Example 22 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-4-N-benzoyl-5-methylcytidine-3'-O-(2-cyanoethyl) phosphoramidite) and Example 23 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-4-N-benzoyl-5-methylcytidine-3'-O-(2-cyanoethyl) phosphoramidite) in JP-A 2000-297097. The compounds of Example 10 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-5-methyluridine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite) and Example 9 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-5-methyluridine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite) were used. The title compound was synthesized using Glen Unysupport FC 96 well format 0.2 μmol (manufactured by GlenResearch) as a solid phase support.

[0067] The protected oligonucleotide analogue having the target sequence was treated with 600 μL of concentrated aqueous ammonia to cleave the oligomer from the support, and the protecting group cyanoethyl group on the phosphorus atom and the protecting group on the nucleic acid base were removed. The mixed solution of the oligomer was mixed with 300 μL of Clarity QSP DNA Loading Buffer (Phenomenex) and charged onto a Clarity SPE 96 well plate (Phenomenex). 1 mL of Clarity QSP DNA Loading Buffer:water=1:1 solution, 2 mL of 0.1 M tetrabutylammonium bromide aqueous solution:acetonitrile=8:2 (v / v) solution, 3 mL of 3% dichloroacetic acid (DCA) aqueous solution, 4 mL of water, and 2 mL of 20 mM Tris aqueous solution were added in this order, and then the components extracted with the 20 mM Tris aqueous solution:acetonitrile=9:1 solution were collected. After distilling off the solvent, the target compound was obtained. This compound was analyzed by reversed-phase HPLC (column (Phenomenex, Clarity 2.6 μm Oligo-MS 100A (2.1 × 50 mm)), solution A: 100 mM hexafluoroisopropanol (HFIP), 8 mM triethylamine aqueous solution, solution B: methanol, B%: 10% → 25% (4 min, linear gradient); 60 °C; 0.5 mL / min; 260 nm), and eluted at 3.384 minutes. The compound was identified by negative ion ESI mass spectrometry (calculated value: 6446.76, measured value: 6446.75).

[0068] The base sequence of this compound is complementary to nucleotide numbers 595-612 of Homo spiens clone 60-1 double homeodomain protein DUX4-fl (DUX4) mRNA (NCBI-GenBank accession No. HQ266761).

[0069] In the sequence listing, the base sequences are shown without distinguishing between natural nucleosides and 2'-O,4'-C-ethylene nucleosides.

[0070] (Examples 2 to 47) The compounds of Examples 2 to 47 shown in Table 1 were synthesized in the same manner as in Example 1. [Table 1] In the sequences in the table, lowercase letters indicate 2'-OMe-RNA, and underlined capital letters indicate ENA. However, the base site of C in ENA is 5-methylcytosine. Each nucleoside is linked by phosphorothioate. The target region indicates the nucleotide number of Homo spiens clone 60-1 double homeodomain protein DUX4-fl (DUX4) mRNA (NCBI-GenBank accession No. HQ266761). The molecular weight is the actual value measured by negative ion ESI mass spectrometry.

[0071] (Examples 48-95) By synthesizing under the same conditions as in Example 1, the compounds listed in Table 2 below can also be synthesized. [Table 2] In the sequences in the table, lowercase letters indicate 2'-OMe-RNA, and underlined capital letters indicate ENA. However, the base site of C in ENA is 5-methylcytosine. Each nucleoside is linked by phosphorothioate. The target region indicates the nucleotide number of Homo spiens clone 60-1 double homeodomain protein DUX4-fl (DUX4) mRNA (NCBI-GenBank accession No. HQ266761). The molecular weight is the actual value measured by negative ion ESI mass spectrometry.

[0072] In this specification, A t , G t , 5meC t , C t , T t , U t , A p , G p , 5meC p , C p , T p、U p 、A s 、G s 、5meC s 、C s 、T s 、U s 、A m1t 、G m1t 、C m1t 、5meC m1t 、U m1t 、A m1p 、G m1p 、C m1p 、5meC m1p 、U m1p 、A m1s 、G m1s 、C m1s 、5meC m1s 、U m1s 、A 2t 、G 2t 、C 2t 、T 2t 、A e2p 、G e2p 、C e2p 、T e2p 、A e2s 、G e2s 、C e2s 、T e2s 、A 1t 、G 1t 、C 1t 、T 1t 、A e1p 、G e1p 、C e1p 、T e1p 、A e1s 、G e1s 、C e1s 、T e1s 、A 3t 、G 3t 、C 3t 、T 3t 、A e3p 、G e3p 、C e3p 、T e3p 、A e3s 、G e3s 、C e3s 、T e3s 、A m2t 、G m2t 、5meC m2t 、T m2t 、A m2p 、G m2p 、5meC m2p 、Tm2p , A m2s , G m2s , 5meC m2s , T m2s is a group having the structure shown below. JPEG0007627495000008.jpg232147 JPEG0007627495000009.jpg232150 JPEG0007627495000010.jpg207148 JPEG0007627495000011.jpg213144 JPEG0007627495000012.jpg213141 JPEG0007627495000013.jpg213145

[0073] (Test Example 1) (A) Construction of DUX4 minigene construct Recombinant E. coli carrying the BAC clone RP11-242C23 (Morioka et al., PLoS One 2016, 11: e0151963) containing the DUX4 gene was purchased from the BAC PAC Resources Center at Children's Hospital Oakland. The strain was cultured on LB / chloramphenicol agar medium, and the BAC DNA was purified using the QIAGEN plasmid midi kit (QIAGEN, cat.no.12143) according to protocol QP01.

[0074] A region of 2163 bp including DUX4 exon 1 to exon 3 was amplified by PCR using BAC DNA as a template, primer 207 (5'-CGCGTCCGTCCGTGAAATTCC-3') (sequence number 86), primer 209 (5'-CAGGGGATATTGTGACATATCTCTGCAC-3') (sequence number 87), and PrimeStar GXL (Takara; cat. no. R050A). The PCR product was purified using MinElute Gel Extraction Kit (QIAGEN, cat. no. 28606) and ligated into pCR blunt vector (Thermofisher scientific; cat. no. K275020) using Mighty mix (Takara; cat. no. 6023). The recombinant plasmid DNA was transformed into E. coli TOP10 competent cells (Thermofisher; cat. no. C404010) and selected on LB / kanamycin agar medium. The transformant colonies were cultured in liquid culture, and the recombinant plasmid DNA was purified using the GenElute plasmid miniprep kit (SIGMA; cat. no. PLN350). The nucleotide sequence of the resulting recombinant plasmid DNA was determined using an ABI 3500xL Genetic Analyzer (Applied Biosystems), and it was confirmed that exons 1 to 3 of DUX4 were inserted. The plasmid was named plasmid 318.

[0075] Using plasmid 318 as a template, primer 216 (5'-GAATTCTGCCACCATGGCCCTCCCG-3') (SEQ ID NO: 88) containing an EcoRI recognition sequence and a Kozak sequence, and primer 218 (5'-CTCGAGCTATAGGATCCACAGGGAGG-3') (SEQ ID NO: 89) containing an XhoI recognition sequence, PCR was performed again using PrimeStar GXL to add an EcoRI recognition sequence and a Kozak sequence to the 5' end of the region containing DUX4 exon 1 to exon 3, and an XhoI recognition sequence to the 3' end. The DUX4 gene DNA to which the restriction enzyme recognition sequence and the Kozak sequence were added was ligated to the pCR blunt vector and named plasmid 320.

[0076] Plasmid 320 and expression vector pcDNA3.1(+) (Thermofisher scientific; cat. no. V79020) were treated with EcoRI and XhoI at 37°C for 2 hours, respectively, and the bands were confirmed by agarose electrophoresis. DNA was purified from the bands corresponding to the DUX4 gene sequence and the pcDNA3.1(+) vector using MinElute Gel Extraction Kit (QIAGEN, cat. no. 28606), and the DUX4 gene sequence and vector were ligated using Mighty mix. The base sequence was confirmed with an ABI 3500xL Genetic Analyzer, and the construct was named DUX4 minigene construct.

[0077] (B) Transfection of DUX4 minigene constructs and example compounds into HeLa cells HeLa cells, a cultured cell line derived from human cervical cancer, were purchased from the Riken BRC Cell Bank (cat. RCB0007, Tsukuba, Japan). HeLa cells were cultured in DMEM (SIGMA; cat. no. D5796) containing 10% FBS (Thermofisher scientific; cat. no. 10270-106) at 37°C and 5% CO2. HeLa cells were plated at 56 x 10 4The next day, 1 μg of the DUX4 minigene construct and the compound of the example (final concentration 100 nM) were introduced using Lipofectamine 2000 (Thermofisher Scientific; cat. no. 11668027).

[0078] (C) Detection of transcripts from DUX4 minigene constructs After transfection with the DUX4 minigene construct, cells were harvested 24 hours later, and RNA was extracted using the RNeasy mini kit (QIAGEN; cat. no. 74104). Reverse transcription was performed using PrimeScript (Takara; cat. no. 2680A), and PCR was performed using primers that amplify both DUX4-fl and DUX4-s (primer 222: 5'-GGATTCAGATCTGGTTTCAGAATCGAAGG-3' (sequence number 90), primer 225: 5'-CCAGGAGATGTAACTCTAATCCAGGTTTGC-3' (sequence number 91)). Bands of DUX4-fl and DUX4-s were separated by 4.8% polyacrylamide gel electrophoresis, and band intensities were visualized using LAS3000 (Fujifilm). The markers used were λ / EcoT14I marker (Takara; cat. no. 3010 treated with Takara; cat. no. 1038A, used as M1 in the figure) and 100 bp marker (Takara; cat. no. 3422B, used as M2 in the figure).

[0079] The results of the gel electrophoresis are shown in Figures 2 to 7. In the figures, "control" indicates cells that were not transfected with either the DUX4 minigene construct or the compounds of the examples. "D4" indicates cells that were transfected with only the DUX4 minigene construct. In cells transfected with the compounds of the examples (DUX4-004 to DUX4-016, DUX4-025 to DUX4-058), a band of about 300 bp that was thought to be derived from DUX4-s was observed, which was stronger than the D4 lane.

[0080] (D) Confirmation of the base sequence of the DUX4-s band generated by the compound of the example After transfection with the DUX4 minigene construct, cells were harvested 24 hours later, and RNA was extracted using the RNeasy mini kit. Reverse transcription was performed using PrimeScript, and PCR was performed using primers that amplify both DUX4-fl and DUX4-s (primers 222 and 225). The bands of DUX4-fl and DUX4-s were separated by 2% agarose gel electrophoresis, and a band of approximately 300 bp was excised, and the DNA was purified using the MinElute Gel Extraction kit and ligated into the pCR blunt vector using Mighty mix. The recombinant plasmid DNA was transformed into E. coli TOP10 competent cells and selected on LB / kanamycin agar medium. Colonies of the transformants were cultured in liquid, and the recombinant plasmid DNA was purified using the GenElute plasmid miniprep kit. The resulting recombinant plasmid DNA was used as a template for Sanger reaction with M13 forward primer (5'- CGACGTTGTAAAACGACGGCCAGT-3' (SEQ ID NO: 92)) and M13 reverse primer (5'- ggaaacagctatgaccatgattac-3' (SEQ ID NO: 93)), and the nucleotide sequence was analyzed using an ABI 3500xL Genetic Analyzer. The result was that the sequence was deleted at the 3' end of exon 2, 10 bp, compared to the DUX4-s sequence (GenBank HQ266762). Since the stop codon of DUX4-s is located at the beginning of exon 2, it is considered that the full-length DUX4-s protein (GenBank: ADN68617.1) will be produced from the sequence confirmed in this experiment.

[0081] (E) Measurement of expression levels of DUX4 target genes After transfection with the DUX4 minigene construct and the compound of the embodiment (DUX4-009), the cells were harvested 24 hours later, and RNA was extracted using the RNeasy mini kit. cDNA was synthesized by reverse transcription using PrimeScript. Using the cDNA as a template, the expression levels of ZSCAN4, MBD3L2, and TRIM43, which are known as target genes whose transcription is activated by DUX4, were measured by real-time PCR using PowerUP SYBR Green PCR master mix (Thermofisher scientific; cat.no. A25742). PCR was performed using StepOne Plus (Thermofisher scientific), and quantified by the ΔΔCt method using the RPL13A gene as an internal standard. The sequences of the primers used are as follows. ZSCAN4-Fw: 5'-TGGAAATCAAGTGGCAAAAA-3' (SEQ ID NO: 94); ZSCAN4-Rv: 5'-CTGCATGTGGACGTGGAC-3' (SEQ ID NO: 95) MBD3L2-Fw: 5'-GCGTTCACCTCTTTTCCAAG-3' (SEQ ID NO: 96); MBD3L2-Rv: 5'-GCCATGTGGATTTCTCGTTT-3' (SEQ ID NO: 97) TRIM43-Fw: 5'-ACCCATCACTGGACTGGTGT-3' (SEQ ID NO: 98); TRIM43-Rv: 5'-CACATCCTCAAAGAGCCTGA-3' (SEQ ID NO: 99) The results are shown in Figure 8A and 8B. The cells transfected with the DUX4 minigene construct and the compounds of the examples (Figure 8A: DUX4-009, DUX4-031, DUX4-036, DUX4-048, Figure 8B: DUX4-48.7, DUX4-48.11, DUX4-48.12, DUX4-48.13, DUX4-52.2) showed a decrease in the expression levels of ZSCAN4, MBD3L2, and TRIM43 compared to cells transfected with only the DUX4 minigene construct. (Test Example 2) (A) Preparation of DUX4 pre-mRNA (D4Z4 mRNA) The DUX4 minigene construct used in the HeLa cell experiments was digested with EcoRI and XhoI, and the fragments were separated by agarose gel electrophoresis. The DNA was purified from the gel and ligated into the pCS2-V5 vector carrying the SP6 promoter. The resulting plasmid, DUX4_pCS2, was linearized with NotI, and mRNA was synthesized in vitro using it as a template with the mMessage mMachine SP6 transcription kit (ThermoFisher scientific; cat.no.AM1340) and purified with the RNeasy MinElute Cleanup kit (QIAGEN; cat.no.74204). (B) Injection of D4Z4 mRNA and the compound of the present invention into zebrafish, and detection of changes in splicing of DUX4 pre-mRNA Approximately 100 fertilized eggs of wild-type zebrafish strain RIKEN WT were injected with 1 nL of DUX4 pre-mRNA (final concentration: 10 ng / μl) alone or a mixture of DUX4 pre-mRNA and example compound DUX-048 (final concentration: 500 μM) using a microinjector (Eppendorf). After 5 hours, TRIzol reagent (ThermoFisher scientific; cat. no. 15596026) was added to homogenize the fertilized eggs, and RNA was purified using an RNeasy Mini kit. DNase treatment was performed using an RNase-free DNase set (QIAGEN; cat. no. 79254). A portion of the obtained RNA was used as a sample for RT, and 1.2 μg of the RNA was purified using PrimeScript 1. stThe cDNA was reverse transcribed using a double strand cDNA synthesis kit. PCR was performed using the resulting cDNA as a template and primers 222 (sequence number 90) and 225 (sequence number 91) with PrimeSTAR GXL DNA polymerase (Takara; cat. no. R050A). DUX4-fl and DUX4-s bands were separated by 4.8% polyacrylamide gel electrophoresis, and the band intensities were visualized with an LAS3000 (Fujifilm). λ / EcoT14I marker (Takara; cat. no. 3010 treated with Takara; cat. no. 1038A, used as M1 in the figure) and 100bp marker (Takara; cat. no. 3422B, used as M2 in the figure) were used. The results are shown in Figure 9. As expected, no bands were detected in the negative control (RT-) in which reverse transcription was not performed, confirming the specificity of the primers. On the other hand, in the sample (RT+) in which reverse transcription was performed, the band corresponding to DUX4-fl decreased in the sample injected with the example compound (DUX-048), while the band corresponding to DUX4-s increased. (Examples 96-130) The compounds shown in Table 3 below were synthesized under the same synthesis conditions as in Example 1. [Table 3] In the sequences in the table, lowercase letters indicate 2'-OMe-RNA and uppercase letters indicate ENA. However, the base site of C in ENA is 5-methylcytosine. Each nucleoside is linked by phosphorothioate. The target region indicates the nucleotide number of Homo spiens clone 60-1 double homeodomain protein DUX4-fl (DUX4) mRNA (NCBI-GenBank accession No. HQ266761). The molecular weight is the actual value measured by negative ion ESI mass spectrometry. (Test Example 3) (A) Transfection of DUX4 minigene construct and compound of the embodiment into HeLa cells, and detection of transcription products from the DUX4 minigene construct Transfection of the DUX4 minigene construct and the compounds of the examples (DUX4-048, DUX4-48.1 to DUX4-48.23, DUX4-052, DUX4-52.1 to DUX4-52.12) into HeLa cells and detection of the transcription products from the DUX4 minigene construct were performed in the same manner as in Test Example 1 (B) and (C). The results of the gel electrophoresis are shown in Figures 10 to 13. In the figures, control indicates that neither the DUX4 minigene construct nor the compound of the example was transfected. D4 indicates that only the DUX4 minigene construct was transfected. In the cells transfected with the compounds of the example (DUX4-048, DUX4-48.1 to DUX4-48.23, DUX4-052, DUX4-52.1 to DUX4-52.12), a band of about 300 bp thought to be derived from DUX4-s was observed, which was stronger than the band in the lane of D4. In addition, in the cells transfected with the compounds of the example (DUX4-48.7, DUX4-48.12, and DUX4-52.2), the band thought to be derived from DUX4-fl was significantly reduced. (Reference example 1) 2-Cyanoethyl (6-stearamidohexyl)diisopropylphosphoramidite JPEG0007627495000015.jpg2181Stearic acid (5.00 g) in dichloromethane (100 mL) was cooled to about 10°C, 6-amino-1-hexanol (3.09 g), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.37 g), and diisopropylethylamine (3.06 mL) were added, and the mixture was stirred at room temperature for 17 hours. MeOH was added, and the mixture was concentrated to obtain a residue, which was dispersed in acetone-MeOH-hexane-toluene and ISOLUTE HM-N (bulk, Biotage). The solvent was removed under reduced pressure, and the residue was subjected to silica gel column chromatography (hexane / ethyl acetate / methanol) to obtain a crude intermediate (3.98 g). A suspension of this crude product (3.86 g) in dichloromethane (100 mL) was cooled to 0° C., diisopropylethylamine (7.01 mL) and 2-cyanoethyl diisopropylchlorophosphoramidite (3.37 mL) were added, and the mixture was stirred at room temperature for 3 hours. The solvent was distilled off under reduced pressure, and the resulting residue was subjected to NH-silica gel column chromatography (DCM / ethyl acetate) and further to NH-silica gel column chromatography (hexane / ethyl acetate) to obtain the title compound (785 mg) as a colorless solid. 1 H-NMR (CDCl3) δ: 0.88 (3H, t, J = 6.3 Hz), 1.15-1.20 (12H, m), 1.22-1.66 (38H, m), 2.14 (2H, t, J = 7.6 Hz), 2.65 (2H, t, J = 6.3 Hz), 3.24 (2H, dt, J = 6.7, 6.7 Hz), 3.53-3.92 (6H, m), 5.44 (1H, br s). MS (m / z): 423.6 (MN(iPr)2+OH+Na) + . (Examples 131-134) The compounds listed in Table 4 below were synthesized under the same conditions as in Example 1. However, after chain elongation of the oligonucleotide having the target sequence was completed, 2-Cyanoethyl (6-palmitamidohexyl)diisopropylphosphoramidite (Nucleic Acids Res. (2020) 47, 6029-6044) was dissolved in acetonitrile:dichloromethane (1:2 v / v) to a concentration of 0.1 M, and coupled. After coupling, the compound was oxidized using an oxidation solution [iodine solution (about 0.05 mol / L)] [pyridine:water (9:1)] (manufactured by Sigma-Aldrich). The protected oligonucleotide analogue having the target sequence was treated with 600 μL of concentrated aqueous ammonia to cut the oligomer from the support, and the protecting group cyanoethyl group on the phosphorus atom and the protecting group on the nucleic acid base were removed. The mixed solution of the oligomer was mixed with 300 μL of Clarity QSP DNA Loading Buffer (manufactured by Phenomenex) and charged onto a Clarity QSP cartridge 60 mg / 3 mL 30 μm (manufactured by Phenomenex). After adding 1 mL of Clarity QSP DNA Loading Buffer:water=1:1 solution, 2 mL of 0.1 M tetrabutylammonium bromide aqueous solution:acetonitrile=8:2 (v / v) solution, 4 mL of water, and 4 mL of 20 mM Tris aqueous solution in that order, the components extracted with 800 μL of 20 mM Tris aqueous solution:acetonitrile=8:2 solution were collected. After distilling off the solvent, the target compound was obtained. This compound was analyzed by reversed-phase HPLC (column (Phenomenex, Clarity 2.6 μm Oligo-MS 100A (2.1 × 50 mm)), solution A: 100 mM hexafluoroisopropanol (HFIP), 8 mM triethylamine aqueous solution, solution B: methanol, B%: 10% → 25% → 40% → 65% (4 min → 6 min → 8 min, linear gradient); 60 °C; 0.5 mL / min; 260 nm), and eluted at 5.7 minutes. The compound was identified by negative ion ESI mass spectrometry. [Table 4] In the sequences in the table, lowercase letters indicate 2'-OMe-RNA and uppercase letters indicate ENA. However, the base site of C in ENA is 5-methylcytosine. Each nucleoside is linked by phosphorothioate. The target region indicates the nucleotide number of Homo spiens clone 60-1 double homeodomain protein DUX4-fl (DUX4) mRNA (NCBI-GenBank accession No. HQ266761). The molecular weight is the actual value measured by negative ion ESI mass spectrometry. The compounds of Examples 131 to 134 each have, at the 5' end, It has a group shown in JPEG0007627495000017.jpg17109. (Examples 135-138) The compounds shown in Table 5 below were synthesized under the same conditions as in Example 1. However, after chain elongation of the oligonucleotide having the target sequence was completed, coupling was performed using a solution prepared by dissolving the compound of Reference Example 1 in acetonitrile:dichloromethane (1:2 v / v) to a concentration of 0.1 M. After coupling, oxidation was performed using an oxidation solution [iodine solution (about 0.05 mol / L)] [pyridine:water (9:1)] (manufactured by Sigma-Aldrich). The protected oligonucleotide analogue having the target sequence was treated with 600 μL of concentrated aqueous ammonia to cut the oligomer from the support, and the protecting group cyanoethyl group on the phosphorus atom and the protecting group on the nucleic acid base were removed. The mixed solution of the oligomer was mixed with 300 μL of Clarity QSP DNA Loading Buffer (manufactured by Phenomenex) and charged onto a Clarity QSP cartridge 60 mg / 3 mL 30 μL (manufactured by Phenomenex). After adding 1 mL of Clarity QSP DNA Loading Buffer:water=1:1 solution, 2 mL of 0.1 M tetrabutylammonium bromide aqueous solution:acetonitrile=8:2 (v / v) solution, 4 mL of water, and 4 mL of 20 mM Tris aqueous solution in that order, the components extracted with 800 μL of 20 mM Tris aqueous solution:acetonitrile=8:2 solution were collected. After distilling off the solvent, the target compound was obtained. This compound was analyzed by reversed-phase HPLC (column (Phenomenex, Clarity 2.6 μm Oligo-MS 100A (2.1 × 50 mm)), solution A: 100 mM hexafluoroisopropanol (HFIP), 8 mM triethylamine aqueous solution, solution B: methanol, B%: 10% → 25% → 40% → 65% (4 min → 6 min → 8 min, linear gradient); 60 °C; 0.5 mL / min; 260 nm), and eluted at 6.2 minutes. The compound was identified by negative ion ESI mass spectrometry. [Table 5] In the sequences in the table, lowercase letters indicate 2'-OMe-RNA and uppercase letters indicate ENA. However, the base site of C in ENA is 5-methylcytosine. Each nucleoside is linked by phosphorothioate. The target region indicates the nucleotide number of Homo spiens clone 60-1 double homeodomain protein DUX4-fl (DUX4) mRNA (NCBI-GenBank accession No. HQ266761). The molecular weight is the actual value measured by negative ion ESI mass spectrometry. The compounds of Examples 135 to 138 have, at the 5' end, It has a group shown in JPEG0007627495000019.jpg17116. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. [Industrial Applicability]

[0082] The present invention can be used to treat facioscapulohumeral muscular dystrophy. [Sequence List Free Text]

[0083] <SEQ ID NO: 1> Shows the base sequence of DUX4-fl accession number: HQ266761. <SEQ ID NOs: 2 to 85> Show the sequences of the antisense oligonucleotides synthesized in Examples 1 to 118, 131 to 133, and 135 to 137. The antisense oligonucleotide may be any of natural DNA, natural RNA, DNA / RNA chimera, and modified forms thereof, and at least one of the nucleotides constituting the antisense oligonucleotide may be a modified nucleotide. <SEQ ID NOs: 86 to 99> Shows the sequences of primers. <SEQ ID NOs: 100 to 102> Shown are the sequences of the antisense oligonucleotides synthesized in Examples 119 to 130, 134 and 138. The antisense oligonucleotide may be any of natural DNA, natural RNA, DNA / RNA chimera, and modified forms thereof, and at least one of the nucleotides constituting the antisense oligonucleotide may be a modified nucleotide.

Claims

1. An oligonucleotide having 18 to 30 bases and consisting of a nucleotide sequence complementary to the region of nucleotide numbers 502 to 556 or 578 to 612 of DUX4-fl mRNA consisting of the nucleotide sequence of SEQ ID NO: 1, comprising any sequence selected from the group consisting of SEQ ID NOs: 3 to 10, 13 to 48 and 100 to 102 (wherein, t may be u, and u may be t), the 5' end and / or 3' end of which may be chemically modified, and which is capable of converting the splicing of the DUX4 gene from DUX4-fl to DUX4-s, or a pharma- ceutically acceptable salt thereof.

2. 2. The oligonucleotide according to claim 1, or a pharma- ceutically acceptable salt thereof, wherein the oligonucleotide has 18 bases.

3. 3. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 1 or 2, wherein at least one of the sugar and / or phosphodiester bonds constituting the oligonucleotide is modified.

4. 4. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 3, wherein the sugar constituting the oligonucleotide is D-ribofuranose, and the sugar modification is modification of the hydroxyl group at the 2'-position of D-ribofuranose.

5. 5. The oligonucleotide or a pharma- ceutical acceptable salt thereof according to claim 4, wherein the sugar modification is 2'-O-alkylation and / or 2'-O, 4'-C-alkylenation of D-ribofuranose.

6. 5. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 4, wherein the sugar modification is 2'-O-methylation and / or 2'-O,4'-C-ethylation of D-ribofuranose.

7. 7. The oligonucleotide or a pharma- ceutical acceptable salt thereof according to any one of claims 3 to 6, wherein the modification of the phosphodiester bond is phosphorothioate.

8. The oligonucleotide according to any one of claims 1 to 7, comprising any one of the sequences of SEQ ID NOs: 5 to 31 (wherein t in the sequence may be u, and u may be t), or a pharma- ceutical acceptable salt thereof.

9. An oligonucleotide having any one of the following sequences or a pharma- ceutical acceptable salt thereof: HO-G m1s -G e2s -G m1s -A m1s -G e2s -C m1s -A m1s -G e2s -G m1s -G m1s -T e2s -G m1s -A m1s -C e2s -C m1s -C m1s -C e2s -C m1t -H(DUX4-006); HO-G m1s -A e2s -C m1s -C m1s -C e2s -A m1s -C m1s -G e2s -A m1s -G m1s -G e2s -G m1s -A m1s -G e2s -C m1s -A m1s -G e2s -G m1t -H(DUX4-009); HO-G m1s -A e2s -A m1s -G m1s -G e2s -C m1s -G m1s -A e2s -C m1s -C m1s -C e2s -A m1s -C m1s -G e2s -A m1s -G m1s -G e2s -G m1t -H(DUX4-011); HO-G m1s -G e2s -U m1s -G m1s -T e2s -G m1s -G m1s -G e2s -C m1s -G m1s -A e2s -A m1s -G m1s -G e2s -C m1s -G m1s -A e2s -C m1t -H(DUX4-014); HO-G m1s -A e2s -G m1s -C m1s -A e2s -G m1s -G m1s -G e2s -U m1s -G m1s -A e2s -C m1s -C m1s -C e2s -C m1s -C m1s -G e2s -C m1t -H(DUX4-036); HO-G m1s -G e2s -A m1s -G m1s -C e2s -A m1s -G m1s -G e2s -G m1s -U m1s G e2s -A m1s -C m1s -C e2s -C m1s -C m1s -C e2s -G m1t -H(DUX4-037); HO-A m1s -C e2s -G m1s -A m1s -G e2s -G m1s -G m1s -A e2s -G m1s -C m1s -A e2s -G m1s -G m1s -G e2s -U m1s -G m1s -A e2s -C m1t -H(DUX4-040); HO-C m1s -G e2s -A m1s -C m1s -C e2s -C m1s -A m1s -C e2s -G m1s -A m1s -G e2s -G m1s -G m1s -A e2s -G m1s -C m1s -A e2s -G m1t -H(DUX4-044); HO-A m1s -A e2s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1s -C m1s -A e2s -C m1s -G m1s -A e2s -G m1s -G m1s -G e2s -A m1t -H(DUX4-047); HO-C m1s -G e2s -A m1s -A m1s -G e2s -G m1s -C m1s -G e2s -A m1s -C m1s -C e2s -C m1s -A m1s -C e2s -G m1s -A m1s -G e2s -G m1t -H(DUX4-048); HO-G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1s -C m1s -A e2s -C m1s -G m1s -A e2s -G m1t -H(DUX4-049); HO-U m1s -G e2s -U m1s -G m1s -G e2s -G m1s -C m1s -G e2s -A m1s -A m1s -G e2s -G m1s -C m1s -G e2s -A m1s -C m1s -C e2s -C m1t -H(DUX4-052); HO-G m1s -T e2s -G m1s -U m1s -G e2s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1t -H(DUX4-053); HO-C e2s -G m1s -A m1s -A e2s -G m1s -G m1s -C e2s -G m1s -A m1s -C m1s -C m1s -C m1s -A e2s -C m1s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.7); HO-C m1s -G m1s -A m1s -A e2s -G m1s -G m1s -C m1s -G m1s -A m1s -C m1s -C m1s -C e2s -A m1s -C e2s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.10); HO-C m1s -G m1s -A e2s -A m1s -G m1s -G m1s -C m1s -G m1s -A e2s -C m1s -C e2s -C m1s -A m1s -C m1s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.11); HO-C e2s -G m1s -A e2s -A m1s -G m1s -G m1s -C m1s -G m1s -A m1s -C m1s -C m1s -C m1s -A m1s -C e2s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.12); HO-C m1s -G m1s -A m1s -A e2s -G m1s -G m1s -C m1s -G m1s -A m1s -C e2s -C m1s -C m1s -A m1s -C e2s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.14); HO-C m1s -G m1s -A m1s -A e2s -G m1s -G m1s -C m1s -G m1s -A e2s -C m1s -C m1s -C m1s -A m1s -C e2s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.15); HO-C m1s -G m1s -A e2s -A m1s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1s -C m1s -A m1s -C m1s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.19); HO-C m1s -G m1s -A m1s -A e2s -G m1s -G m1s -C m1s -G m1s -A m1s -C m1s -C m1s -C m1s -A m1s -C e2s -G m1s -A e2s -G m1s -G m1t -H(DUX4-48.20); HO-T e2s -G m1s -T e2s -G m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A m1s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1s -C e2t -H(DUX4-52.1); HO-T e2s -G m1s -T e2s -G m1s -G m1s -G m1s -C e2s -G m1s -A m1s -A e2s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1s -C m1t -H(DUX4-52.2); HO-T e2s -G m1s -U m1s -G m1s -G m1s -G m1s -C m1s -G m1s -A m1s -A m1s -G m1s -G m1s -C e2s -G m1s -A m1s -C e2s -C m1s -C e2t -H(DUX4-52.7); HO-U m1s -G m1s -T e2s -G m1s -G m1s -G m1s -C m1s -G m1s -A m1s -A m1s -G m1s -G m1s -C e2s -G m1s -A e2s -C m1s -C e2s -C m1t -H(DUX4-52.9) [In the above sequence, A e2s , G e2s , C e2s and T e2s represents the corresponding ENA (where the base position of C is 5-methylcytosine) in a phosphorothioate bond with the adjacent structure on the 3' side. m1s , G m1s , C m1s , U m1s , represents the corresponding 2'-OMe-RNA linked to the 3' adjacent structure via a phosphorothioate bond. e2t represents the corresponding ENA (where the base position of C is 5-methylcytosine) in a phosphodiester bond with the adjacent structure on the 3' side. m1t , G m1t , C m1t represents the corresponding 2'-OMe-RNA in a phosphodiester bond with the 3' adjacent structure.

10. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 9, further comprising an aminoalkyl phosphate group containing a fatty acid bonded to the 5'-end or 3'-end of the oligonucleotide.

11. 11. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to claim 10, wherein the fatty acid is at least one selected from the group consisting of myristic acid, palmitic acid, stearic acid, arachidic acid and behenic acid.

12. 12. The oligonucleotide or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 11 for use in treating a disease or condition caused by expression of the DUX4-fl gene.

13. The oligonucleotide or a pharma- ceutical acceptable salt thereof according to claim 12, wherein the disease or symptom caused by expression of the DUX4-fl gene is facioscapulohumeral muscular dystrophy.

14. A medicine comprising the oligonucleotide according to any one of claims 1 to 11 or a pharma- ceutically acceptable salt thereof.

15. A therapeutic agent for a disease or symptom caused by expression of DUX4-fl gene, comprising an oligonucleotide or a pharma- ceutically acceptable salt thereof capable of converting splicing of the DUX4 gene from DUX4-fl to DUX4-s, wherein the oligonucleotide is an oligonucleotide of 18 to 30 bases consisting of a nucleotide sequence complementary to a region of nucleotide numbers 502 to 556 or 578 to 612 of DUX4-fl mRNA consisting of the nucleotide sequence of SEQ ID NO: 1, and comprising any sequence selected from the group consisting of SEQ ID NOs: 3 to 48 and 100 to 102 (however, t in the sequence may be u, and u may be t), and the 5' end and / or 3' end of the therapeutic agent may be chemically modified.

16. The therapeutic drug according to claim 15, wherein the disease or symptom caused by expression of the DUX4-fl gene is facioscapulohumeral muscular dystrophy.

17. A drug for converting the splicing of DUX4 gene from DUX4-fl to DUX4-s, comprising an oligonucleotide or a pharma- ceutically acceptable salt thereof capable of converting the splicing of DUX4 gene from DUX4-fl to DUX4-s, wherein the oligonucleotide is an oligonucleotide of 18 to 30 bases consisting of a nucleotide sequence complementary to a region of nucleotide numbers 502 to 556 or 578 to 612 of DUX4-fl mRNA consisting of the nucleotide sequence of SEQ ID NO: 1, and comprising any sequence selected from the group consisting of SEQ ID NOs: 3 to 48 and 100 to 102 (however, t in the sequence may be u, and u may be t), and the 5' end and / or 3' end of the drug may be chemically modified.

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