Compounds, methods and pharmaceutical compositions for regulating the expression of DUX4

Abstract

The present invention aims to provide compounds, methods and pharmaceutical compositions for normalizing double homeobox 4 (DUX4) gene in individuals with abnormal expression of DUX4. The present invention provides a modified oligonucleotide consisting of 12 to 30 residues, comprising at least 8 consecutive nucleic acid base sequences, which are complementary to the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 at positions 126 to 147, 232 to 248, 1306 to 1325 or 1472 to 1495 of the same length from the 5' end, and the nucleic acid base sequence of the modified oligonucleotide has at least 90% complementarity with the nucleic acid base sequence of the mature mRNA of DUX4 of sequence number 1 at the same length.

Description

Technical Field

[0001] The present invention relates to compounds for reducing the expression of DUX4 mRNA and protein in animals, methods for using the compounds, and pharmaceutical compositions containing the compounds. The methods of the present invention are useful for treating, preventing, or alleviating DUX4-related diseases, such as facioscapulohumeral muscular dystrophy (FSHD). Background Art

[0002] Facioscapulohumeral muscular dystrophy (FSHD) is a muscular dystrophy with an estimated incidence of 1 in 20,000 worldwide and 1 in 7,500 in Europe. It is the third most common disease among muscular dystrophy after Duchenne muscular dystrophy and myotonic dystrophy. The initial symptoms are decreased muscle strength of the face or upper limbs, scapula, and upper limb girdle. As the disease progresses, the lower limb girdle and lower limbs also become impaired. In about 20% of cases, patients live in a wheelchair before the age of 40 (even in middle age, there are cases where facial muscle weakness is only mild). There are also many complications of pain, sensorineural hearing loss, and retinopathy. About 90% of patients develop the disease before the age of 20. In severe patients (about 4%), decreased muscle strength is shown from infancy.

[0003] FSHD is classified into two types, FSHD1 and FSHD2, based on the causative gene. FSHD1 accounts for about 95% of all FSHD patients. In FSHD1 patients, the D4Z4 repeat region of 4q35 is hereditarily shortened (1 to 10 D4Z4 repeats), which causes abnormal expression of DUX4 encoded by the D4Z4 repeat region (DUX4 is not expressed in healthy people). FSHD2 accounts for about 5% of all FSHD patients, and abnormal expression of DUX4 is caused by mutation of SMCHD1 (DNA methylase). DUX4 has a transcription factor-like function, which causes the expression of the downstream encoded genome that causes apoptosis or muscle atrophy of muscle cells. The abnormal expression of DUX4 is caused by the possession of the allele called 4qA among the two alleles of 4qA and 4qB. The polyadenylation site present in 4qA is essential for the stabilization of DUX4 mRNA (Non-Patent Documents 1, 2, and 3).

[0004] Antisense technology is becoming increasingly recognized as an effective means for modulating the expression of certain gene products and therefore has the potential to be very useful in several therapeutic, diagnostic and research applications for modulating DUX4.

[0005] A method for suppressing the expression of the DUX4 gene using an adeno-associated virus encoding DUX4 miRNA has been reported (Patent Document 1). However, the preparation of the adeno-associated virus is complicated and it is difficult to deliver the virus to the required muscles throughout the body.

[0006] A method for inhibiting the expression of the DUX4 gene using a lentivirus encoding DUX4 shRNA has been reported (Non-Patent Document 2), but the preparation of the lentivirus is complicated and difficult to deliver to the required muscles throughout the body. Furthermore, the gene inhibition effect in vitro in quadriceps or trapezius muscle cells showed 21% and 44% residual activity, which cannot be considered a sufficient effect.

[0007] A compound in which a plurality of antisense oligonucleotides against DUX4 are linked has been reported (Patent Document 2), but it is not a modified oligonucleotide and its inhibitory effect cannot be said to be sufficient.

[0008] Antisense oligonucleotide compounds that bind to the splicing site of DUX4 mRNA have been reported (Patent Document 3). However, these compounds are selective for pre-mRNA containing introns and therefore have weak inhibitory effects on mature mRNA. In addition, they are difficult to administer to living organisms due to knockdown by liposome transfection.

[0009] The current treatment methods for FSHD include rehabilitation (stretching, exercise), administration of NSAIDs, respiratory care, etc. as symptomatic treatment, but the effects are insufficient and the burden on patients is large. Therefore, the purpose of this specification is to provide compounds, compositions, and methods for treating FSHD.

[0010] Prior art literature

[0011] Patent Literature

[0012] Patent Document 1: International Publication No. 2013 / 016352

[0013] Patent Document 2: International Publication No. 2017 / 007886

[0014] Patent Document 3: U.S. Publication No. 2012 / 0225034

[0015] Non-patent literature

[0016] Non-patent document 1: Snider et al., PLoS 2010, Vol. 6(10) p1

[0017] Non-patent document 2: Ferreboeuf et al., Human Molecular Genetics 2013, Vol. 23(1), p171

[0018] Non-patent document 3: Sacconi et al., Biochim. Biophys. Acta 2015, p607 Summary of the invention

[0019] The present invention provides compounds, methods and pharmaceutical compositions for inhibiting the expression of DUX4 and for treating, that is, treating, preventing, delaying or ameliorating DUX4-related diseases and / or their symptoms. The compounds and pharmaceutical compositions disclosed in this specification also inhibit variant DUX4 such as SNP or splice variants of DUX4.

[0020] One embodiment provides a method for reducing the expression of DUX4 in an animal (including a human), comprising administering to the animal a compound comprising a modified oligonucleotide targeting DUX4 described in the present specification or a pharmaceutical composition comprising the compound.

[0021] In one embodiment, a method is provided, wherein a compound or pharmaceutical composition containing a modified oligonucleotide targeting DUX4 is administered to an animal to perform nuclear ribonuclease (RNase H, etc.)-mediated knockdown. In addition, a method is provided, wherein a compound containing the modified oligonucleotide is administered to inhibit transcription of DUX4 mRNA or translation of DUX4 protein. The modified oligonucleotide is preferably distributed in muscle, particularly preferably in skeletal muscle.

[0022] In one embodiment, a method for treating an animal with FSHD is provided. In one embodiment, the method of the present invention further comprises administering to the animal a therapeutically effective amount of a compound or pharmaceutical composition comprising a modified oligonucleotide targeting DUX4 as described in the specification. In one embodiment, the method of the present invention comprises determining an animal with FSHD1 and / or FSHD2.

[0023] In one embodiment, a method for treating, preventing, delaying or improving muscle atrophy and muscle weakness is provided. Lack of expression / sleeping with eyes open / difficulty in lifting upper limbs / winged scapula caused by muscle weakness in the cheeks, shoulders and upper arms is alleviated, or the deterioration is delayed. In addition, it is preferred to prevent muscle weakness in the waist and lower limbs, and it is further preferred to prevent complications of sensorineural hearing loss and retinopathy.

[0024] In a certain embodiment, DUX4 mRNA has a sequence recorded in GenBank Accession No. NM_001293798.2 (incorporated into this specification as Sequence No. 1 in the sequence listing). The splice variant of DUX4 mRNA of Sequence No. 1 in the sequence listing is also referred to as DUX4-FL1 or mature mRNA of DUX4. In a certain embodiment, DUX4 mRNA has a sequence recorded in GenBank Accession No. NM_001306068.2 (incorporated into this specification as Sequence No. 5 in the sequence listing). The splice variant of DUX4 mRNA of Sequence No. 5 in the sequence listing is also referred to as DUX4-FL2. In a certain embodiment, DUX4 has a sequence recorded in GenBank Accession No. NM_001363820.1 (incorporated into this specification as Sequence No. 6 in the sequence listing). The splice variant of DUX4 mRNA of Sequence No. 6 in the sequence listing is also referred to as DUX4-s. In a certain embodiment, DUX4 is referred to as the SNP of the above splice variant.

[0025] The present invention relates to the non-limiting numbered embodiments described in the following items [1] to

[27] , but is not limited thereto.

[0026] Item [1] A modified oligonucleotide consisting of 12 to 30 residues,

[0027] The invention comprises at least 8 consecutive nucleic acid base sequences, which are complementary to the nucleic acid base sequence of the mature mRNA of DUX4 of SEQ ID NO: 1 at positions 126 to 147, 232 to 248, 1306 to 1325 or 1480 to 1495 from the 5' end thereof,

[0028] The nucleic acid base sequence of the modified oligonucleotide has at least 90% complementarity with an equal length portion of the nucleic acid base sequence of the mature mRNA of DUX4 of SEQ ID NO: 1,

[0029] When the above-mentioned at least 8 consecutive nucleic acid base sequences contain a nucleic acid base sequence that is complementary to an equal length portion of the nucleic acid base sequence of sequence number 1 at positions 1480 to 1495 from the 5' end, the above-mentioned modified oligonucleotide is composed of a nucleic acid base sequence having a complementary base to the base at position 1480 from the 5' end of the nucleic acid base of sequence number 1 at the 3' end.

[0030] Item [2] The modified oligonucleotide according to Item [1], wherein one or more modified nucleotides in the modified oligonucleotide contain a modified sugar.

[0031] Item [3] The modified oligonucleotide according to Item [2], wherein the modified sugar is selected from a bicyclic sugar, a sugar modified with a 2'-O-methoxyethyl group, and a sugar modified with a 2'-O-methyl group.

[0032] Item [4] A modified oligonucleotide according to Item [3], wherein the bicyclic sugar is selected from LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz] and ALNA[Trz].

[0033] Item [5] A modified oligonucleotide consisting of 12 to 30 residues,

[0034] The invention comprises at least 8 consecutive nucleic acid base sequences, which are complementary to the nucleic acid base sequence of the mature mRNA of DUX4 of SEQ ID NO: 1, which is equal to the length of the nucleic acid base sequence of positions 1472 to 1495 from the 5' end.

[0035] The nucleic acid base sequence of the modified oligonucleotide has at least 90% complementarity with an equal length portion of the nucleic acid base sequence of the mature mRNA of DUX4 of SEQ ID NO: 1,

[0036] The modified oligonucleotide contains at least one nucleoside containing a modified sugar selected from the group consisting of GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz] and ALNA[Trz].

[0037] Item [6] The modified oligonucleotide according to Item [5], wherein the modified oligonucleotide further comprises a sugar modified with a 2'-O-methoxyethyl group and / or a sugar modified with a 2'-O-methyl group.

[0038] Item [7] The modified oligonucleotide according to any one of Items [1] to [6], wherein at least one modified nucleotide in the modified oligonucleotide comprises a modified nucleic acid base.

[0039] Item [8] The modified oligonucleotide according to Item [7], wherein the modified nucleic acid base is 5-methylcytosine.

[0040] Item [9] The modified oligonucleotide according to any one of Items [1] to [8], wherein at least one internucleoside bond is a modified internucleoside bond.

[0041] Item

[10] The modified oligonucleotide according to Item [9], wherein the modified internucleoside bond is a phosphorothioate internucleoside bond.

[0042] Item

[11] The modified oligonucleotide according to any one of Items [1] to

[10] , wherein the modified oligonucleotide comprises:

[0043] 1) Gap fragments,

[0044] 2) 5' flanking fragment, and

[0045] 3) 3' flanking fragment;

[0046] The gap fragment is located between the 5' wing fragment and the 3' wing fragment.

[0047] The nucleosides of the 5' wing fragment and the 3' wing fragment each contain at least one modified sugar,

[0048] The nucleosides in the gap fragments are exclusively nucleosides not containing modified sugars, or are nucleosides containing 1 or 2 nucleosides containing modified sugars and no other modified sugars.

[0049] Item

[12] A modified oligonucleotide according to any one of Items [1] to

[11] , wherein the modified oligonucleotide is composed of a nucleic acid base sequence complementary to the nucleic acid base sequence at positions 128 to 143 from the 5' end, the nucleic acid base sequence at positions 232 to 247 from the 5' end, the nucleic acid base sequence at positions 233 to 248 from the 5' end, the nucleic acid base sequence at positions 1309 to 1323 from the 5' end, or the nucleic acid base sequence at positions 1480 to 1495 from the 5' end of the nucleic acid base sequence of the mature mRNA of DUX4 of SEQ ID NO: 1.

[0050] Item

[13] A modified oligonucleotide according to any one of Items [1] to

[12] , wherein the modified oligonucleotide is composed of a base sequence of gtggcgatgc ccgggt (SEQ ID NO: 75), gagattcccg cnggtg (SEQ ID NO: 78: n represents 5-methylcytosine), ngagattcccgccggt (SEQ ID NO: 2: n represents 5-methylcytosine), gnagttctccgcggt (SEQ ID NO: 3: n represents 5-methylcytosine) or gnntagacagcgtngg (SEQ ID NO: 4: n represents 5-methylcytosine).

[0051] Item

[14] The modified oligonucleotide according to Item

[13] , represented by the following formula:

[0052] GlsMlsMlsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMlsGlsGl.

[0053] In the formula,

[0054] Each nucleic acid base is represented by the following symbols:

[0055] A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine,

[0056] The sugar moieties are represented by the following symbols:

[0057] l=LNA, d=2'-deoxyribose,

[0058] The internucleoside bonds are represented by the following symbols:

[0059] s = phosphorothioate.

[0060] Item

[15] The modified oligonucleotide according to Item

[13] , represented by the following formula:

[0061] GmsMmsMmsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMmsGmsGm.

[0062] In the formula,

[0063] Each nucleic acid base is represented by the following symbols:

[0064] A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine,

[0065] The sugar moieties are represented by the following symbols:

[0066] m=ALNA[Ms], d=2'-deoxyribose,

[0067] The internucleoside bonds are represented by the following symbols:

[0068] s = phosphorothioate.

[0069] Item

[16] The modified oligonucleotide according to Item

[13] , represented by the following formula:

[0070] GmsMmsAmsGdsTdsTdsCdsTdsCdsCdsGdsCdsGmsGmsTm.

[0071] In the formula,

[0072] Each nucleic acid base is represented by the following symbols:

[0073] A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine,

[0074] The sugar moieties are represented by the following symbols:

[0075] m=ALNA[Ms], d=2'-deoxyribose,

[0076] The internucleoside bonds are represented by the following symbols:

[0077] s = phosphorothioate.

[0078] Item

[17] The modified oligonucleotide according to Item

[13] , represented by the following formula:

[0079] MlsGlsAlsGdsAdsTdsTdsCdsCdsCdsGdsCdsCdsGlsGlsTl.

[0080] In the formula,

[0081] Each nucleic acid base is represented by the following symbols:

[0082] A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine;

[0083] The sugar moieties are represented by the following symbols:

[0084] l=LNA, d=2'-deoxyribose,

[0085] The internucleoside bonds are represented by the following symbols:

[0086] s = phosphorothioate.

[0087] Item

[18] The modified oligonucleotide or a salt thereof according to Item

[14] , represented by the following formula:

[0088]

[0089] Item

[19] The modified oligonucleotide or a salt thereof according to Item

[15] , represented by the following formula:

[0090]

[0091] Item

[20] The modified oligonucleotide or a salt thereof according to Item

[16] , represented by the following formula:

[0092]

[0093] Item

[21] The modified oligonucleotide or a salt thereof according to Item

[17] , represented by the following formula:

[0094]

[0095] Item

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

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

[0096] Item

[23] A pharmaceutical composition according to Item

[22] , which is used for treating, preventing or delaying the progression of a DUX4-related disease.

[0097] Item

[24] The pharmaceutical composition according to Item

[23] , wherein the DUX4-related disease is facioscapulohumeral muscular dystrophy.

[0098] Item

[25] A method for treating, preventing or delaying the progression of a DUX4-related disease in a subject, characterized in that an effective amount of the modified oligonucleotide described in any one of Items [1] to

[21] is administered to a subject in need thereof.

[0099] Item

[26] A use of the modified oligonucleotide described in any one of Items [1] to

[21] for the manufacture of a pharmaceutical for treating, preventing or delaying the progression of a DUX4-related disease.

[0100] Item

[27] A use of the modified oligonucleotide described in any one of Items [1] to

[21] for treating, preventing or delaying the progression of a DUX4-related disease.

[0101] According to the present invention, it is possible to provide a modified oligonucleotide that is effective for treating a disease such as facioscapulohumeral muscular dystrophy caused by abnormal expression of the DUX4 gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1 This is a drawing showing the inhibitory effect of DUX4 gene expression by administration of various DUX4-modified oligonucleotides.

[0103] Figure 2 This is a drawing showing the inhibitory effect of DUX4 gene expression by administration of various DUX4-modified oligonucleotides.

[0104] Figure 3 This is a drawing showing the dose-dependent inhibitory effect of DUX4 gene expression by administration of DUX4 modified oligonucleotide.

[0105] Figure 4 This is a drawing showing the inhibitory effect of DUX4 gene expression by administration of various DUX4-modified oligonucleotides.

[0106] Figure 5 The figure shows the inhibitory effect of creatine kinase and DUX4 mRNA in DUX4 Tg mice by administration of DUX4 modified oligonucleotide (Compound No. 3).

[0107] Figure 6 The figure shows the inhibitory effect of administration of DUX4 modified oligonucleotides (Compound No. 123, Compound No. 247, and Compound No. 113) on creatine kinase and DUX4 mRNA in DUX4 Tg mice. DETAILED DESCRIPTION

[0108] Both the above summary and the following detailed description are exemplary and illustrative only and should be understood as not limiting the claimed invention. In this specification, unless otherwise specified, the use of the singular includes the plural. In this specification, unless otherwise specified, the use of "or" refers to "and / or". Furthermore, the use of the term "including" and other forms, such as "includes" and "included", is not restrictive. Furthermore, unless otherwise specified, terms such as "element" include elements containing one unit and elements containing more than one subunit.

[0109] The chapter titles used in this specification are for structural purposes only and should not be interpreted as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books and papers, and portions and entireties of documents discussed in this specification are expressly incorporated into this specification by reference.

[0110] (definition)

[0111] As long as concrete definition is not given, the nomenclature and their order and practice that are associated and utilized with analytical chemistry, organic synthesis chemistry and medical chemistry and pharmaceutical chemistry recorded in this specification are well known in the art and are commonly used. In the chemical synthesis and chemical analysis used in this specification, the practice of standard can be used. Where permitted, for all patents, applications, open applications and other publications mentioned throughout the disclosure of this specification, the GenBank accession number and the associated sequence information and other data that can be obtained through the database of the National Center for Biotechnology Information (NCBI) etc., about a part of the file discussed in this specification and its entirety, clearly incorporated into this specification by reference.

[0112] In addition, this specification is filed together with a sequence listing in an electronic format, and the information of the sequence listing described in the electronic format is incorporated in its entirety into this specification by reference.

[0113] Unless otherwise specified, the following terms have the following meanings.

[0114] "Nucleic acid" refers to a molecule composed of monomeric nucleotides. Examples of nucleic acids include ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acid, double-stranded nucleic acid, small interfering RNA (siRNA) and microRNA (miRNA), but are not limited to these. Nucleic acids may also contain a combination of these elements in a single molecule.

[0115] "Nucleobase" refers to a heterocyclic moiety that can pair with the base of another nucleic acid. Nucleobases include "modified nucleic acid bases" and "unmodified nucleic acid bases".

[0116] The "nucleobase sequence" refers to any order of consecutive nucleobases that is not related to sugar bonds or nucleobase modifications.

[0117] "Nucleoside" refers to a nucleic acid base linked to a sugar. In certain embodiments, the nucleoside is linked to a phosphate group.

[0118] "Nucleotide" refers to a nucleoside having a phosphate group or the like covalently bonded to the sugar portion of the nucleoside. The sugar portion of a naturally occurring nucleotide is ribose or deoxyribose, which is covalently bonded to the phosphate group via a phosphodiester bond.

[0119] "Oligomeric compound" or "oligomer" refers to a polymer of linked monomeric subunits capable of hybridizing to at least a region of a nucleic acid molecule.

[0120] "Oligonucleotide" refers to a polymer of linked nucleosides wherein the individual nucleosides and the internucleoside linkages may or may not be modified independently of each other.

[0121] "Non-modified nucleotides" refer to nucleotides composed of naturally occurring nucleic acid bases, sugar moieties, and internucleoside bonds. In certain embodiments, the non-modified nucleotides are RNA nucleotides (i.e., β-D-ribonucleosides) or DNA nucleotides (i.e., β-D-deoxyribonucleosides), but are not limited thereto.

[0122] "Modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleoside linkage, or a modified nucleic acid base. "Modified nucleoside" refers to a nucleoside that independently has a modified sugar moiety or a modified nucleic acid base.

[0123] "Internucleoside linkage" refers to the chemical bond between nucleosides.

[0124] "Linked nucleosides" refers to contiguous nucleosides that are bonded or linked by an internucleoside linkage.

[0125] A "naturally occurring internucleoside linkage" refers to a 3'-5' phosphodiester linkage.

[0126] "Modified internucleoside bonds" refer to substitutions or arbitrary changes from naturally occurring internucleoside bonds (i.e., phosphodiester internucleoside bonds), such as phosphorothioate internucleoside bonds, but are not limited thereto.

[0127] "Phosphorothioate internucleoside bond" refers to an internucleoside bond in which a phosphodiester bond is modified by replacing one non-crosslinking oxygen atom with a sulfur atom. A phosphorothioate bond is an example of such a modified internucleoside bond.

[0128] "Modified nucleic acid base" refers to any nucleic acid base other than adenine, cytosine, guanine, thymine or uracil. For example, there is 5-methylcytosine, but it is not limited to this. "Non-modified nucleic acid base" refers to adenine (A) and guanine (G) of purine bases, and thymine (T), cytosine (C) and uracil (U) of pyrimidine bases.

[0129] "Modified oligonucleotide" refers to an oligonucleotide comprising at least one modified nucleoside and / or a modified internucleoside bond.

[0130] "Salt" is a general term for compounds obtained by replacing one or more dissociable hydrogen ions contained in an acid with cations such as metal ions or ammonium ions. Salts of modified oligonucleotides include salts (e.g., sodium salts, magnesium salts) formed with inorganic ions (e.g., sodium ions, magnesium ions) on the sulfur (S) group of the thiophosphate bond or the functional group (e.g., amino group) in the modified nucleic acid base, but are not limited to these.

[0131] "Sugar" or "sugar moiety" refers to a natural sugar moiety or a modified sugar moiety.

[0132] "Natural sugar moiety" refers to the sugar found in DNA (2'-H) or RNA (2'-OH).

[0133] "Modified sugar" refers to a substitution or change from a natural sugar moiety. Examples of modified sugars include substituted sugar moieties and substitute sugar moieties.

[0134] "Substituted sugar moiety" refers to a furanose group other than the native sugar of RNA or DNA.

[0135] "2'-O-methoxyethyl" (the same also applies to 2'-MOE and 2'-O(CH2)2-OCH3) refers to an O-methoxy-ethyl modification of the 2'-position of the furanosyl ring. Sugars modified with 2'-O-methoxyethyl are modified sugars.

[0136] "2'-O-methoxyethyl nucleotide" refers to a nucleotide comprising a sugar moiety modified with a 2'-O-methoxyethyl group.

[0137] "2'-O-methyl" (the same applies to 2'-OMe and 2'-OCH3) refers to the O-methyl modification of the 2'-position of the furanosyl ring. Sugars modified with 2'-O-methyl are modified sugars.

[0138] "2'-O-methyl nucleotide" refers to a nucleotide comprising a sugar moiety modified with a 2'-O-methyl group.

[0139] "Sugar substitute" used for "substitute sugar moiety" refers to the substitution of only sugar unit (furanose ring). Sugar substitutes can replace the naturally occurring sugar moiety of nucleosides, and as a result, the obtained nucleoside subunits can be connected to each other and / or to other nucleosides to form oligomer compounds that can hybridize with complementary oligomer compounds. Such structures include: rings containing atoms of different numbers from furanose groups (e.g., 4, 6 or 7-membered rings); substitution of oxygen and non-oxygen atoms (e.g., carbon, sulfur or nitrogen) of furanose groups; or both changes in the number of atoms and substitution of oxygen. Such structures may also include substitutions corresponding to the contents recorded in the substituted sugar moiety (e.g., 6-membered carbocyclic bicyclic sugar substitutes optionally containing further substituents). Sugar substitutes also include more complex sugar substitutions (e.g., non-cyclic peptide nucleic acids). As sugar substitutes, morpholino, cyclohexenyl and cyclohexanehexol can be cited, but are not limited to them.

[0140] "Bicyclic sugar" refers to a furanosyl ring modified by crosslinking two different carbon atoms present on the same ring. Preferably, "bicyclic sugar" refers to a modified sugar in which the 2' and 4' positions of the furanosyl ring are modified by crosslinking. "Bicyclic nucleic acid" refers to a nucleoside or nucleotide whose furanose moiety contains a "bicyclic sugar".

[0141] "LNA" refers to a nucleoside or nucleotide generally referred to as 2',4'-locked nucleic acid, for example, of the general formula:

[0142]

[0143] [Wherein,

[0144] B is a nucleic acid base,

[0145] X and Y are each independently a hydrogen atom, a protective group for a hydroxyl group, a phosphate group which may be substituted, a covalent bond to a phosphorus moiety or a support, etc.]

[0146] The nucleoside or nucleotide represented by (refer to WO98 / 39352). Typical specific examples include the following formula:

[0147]

[0148] Indicates nucleoside or nucleotide.

[0149] "GuNA" is the following formula:

[0150]

[0151] [wherein, B is a nucleic acid base, R3, R4, R5, and R6 are each independently a hydrogen atom or a C atom which may be substituted with one or more substituents. 1-6alkyl, R7 and R8 are each independently a hydrogen atom, a protective group for a hydroxyl group, a phosphate group which may be substituted, a covalent bond to a phosphorus portion or a support, and R9, R 10 , R 11 Each independently represents a hydrogen atom, which may be substituted with one or more substituents. 1-6 Alkyl or amino protecting group]

[0152] The nucleoside or nucleotide represented by (for example, see International Publication No. 2014 / 046212 and International Publication No. 2017 / 047816).

[0153] "ALNA[mU]" is the following general formula (I):

[0154]

[0155] [Wherein,

[0156] B is a nucleic acid base;

[0157] R1, R2, R3 and R4 are each independently a hydrogen atom, or a C atom which may be substituted with one or more substituents. 1-6 alkyl,

[0158] R5 and R6 are each independently a hydrogen atom, a protective group for a hydroxyl group, a phosphate group which may be substituted, a covalent bond to a phosphorus part or a support, etc.,

[0159] m is 1 or 2;

[0160] X is the following formula (II-1):

[0161]

[0162] The group represented by

[0163] Symbols described in formula (II-1):

[0164]

[0165] represents the bonding point to the 2'-amino group,

[0166] One of R7 and R8 is a hydrogen atom, and the other is a methyl group which may be substituted with one or more substituents]

[0167] A nucleoside or nucleotide represented by (for example, see Japanese Patent Application No. 2018-212424). A typical specific example is a nucleoside or nucleotide in which one of R7 and R8 is a hydrogen atom and the other is an unsubstituted methyl group.

[0168] "ALNA[ipU]" is a nucleoside or nucleotide represented by the general formula (I) defined in the above-mentioned "ALNA[mU]", wherein:

[0169] X is the following formula (II-1):

[0170]

[0171] The group represented by

[0172] One of R7 and R8 is a hydrogen atom, and the other is an isopropyl group which may be substituted with one or more substituents (for example, see Japanese Patent Application No. 2018-212424). A typical specific example is a nucleoside or nucleotide in which one of R7 and R8 is a hydrogen atom and the other is an unsubstituted isopropyl group.

[0173] "ALNA[Trz]" is a nucleoside or nucleotide represented by the general formula (I) defined in the above-mentioned "ALNA[mU]", wherein:

[0174] X is the following formula (II-2):

[0175]

[0176] The group represented by

[0177] A is a triazole group which may be substituted with one or more substituents (for example, see Japanese Patent Application No. 2018-212424). A typical specific example is that A is a triazole group which may have one or more methyl groups, more specifically a 1,5-dimethyl-1,2,4-triazole-3-yl nucleoside or nucleotide.

[0178] "ALNA[Oxz]" is a nucleoside or nucleotide represented by the general formula (I) defined in the above-mentioned "ALNA[mU]", wherein:

[0179] X is the following formula (II-2):

[0180]

[0181] The group represented by

[0182] A is substituted by one or more substituents. A is a diazole group (for example, see Japanese Patent Application No. 2018-212424). A typical specific example is A which may have one or more methyl groups. oxadiazolyl, more specifically 5-methyl-1,2,4- Oxazolyl-3-yl nucleosides or nucleotides.

[0183] "ALNA[Ms]" is a nucleoside or nucleotide represented by the general formula (I) defined in the above-mentioned "ALNA[mU]", wherein:

[0184] X is the following formula (II-3):

[0185]

[0186] The group represented by

[0187] M is a sulfonyl group which may be substituted with a methyl group, and the methyl group may be substituted with one or more substituents (for example, see Japanese Patent Application No. 2018-212424). A typical specific example is a nucleoside or nucleotide in which M is a sulfonyl group substituted with an unsubstituted methyl group.

[0188] "5-methylcytosine" refers to cytosine modified by a methyl group bonded to position 5. 5-methylcytosine is a modified nucleic acid base.

[0189] "Single-stranded oligonucleotide" refers to an oligonucleotide that has not hybridized to a complementary strand.

[0190] "DUX4" refers to a nucleic acid or protein of a transcription factor also called double homeobox 4. Examples of DUX4 include various splice variants transcribed from the DUX4 gene or single nucleotide substitutions (SNPs) thereof, and may also be the variants and / or SNPs.

[0191] A large number of splicing variants have been reported for DUX4 mRNA. Human DUX4-s (SEQ ID NO. 6 in the sequence listing) is composed of short exon 1 (exon 1s), exon 2, and exon 3 due to an atypical splicing donor site in exon 1, and encodes a short DUX4 protein without toxicity. Human DUX4-FL is composed of exon 1, exon 2, and exon 3, and encodes a full-length DUX4 protein. DUX4-FL has DUX4-FL1 (SEQ ID NO. 1 in the sequence listing), which is a mature mRNA without intron 1, and DUX4-FL2 (SEQ ID NO. 5 in the sequence listing), which contains intron 1. Both of them encode a full-length DUX4 protein, and when expressed in muscle, it is believed to be the cause of FSHD (see the above-mentioned non-patent document 2).

[0192] DUX4 mRNA is also expressed in normal testis, and in addition to DUX4-FL, splice variants of exon 1, exon 2, exon 6, and exon 7 and / or splice variants of exon 1, exon 2, exon 4, exon 5, exon 6, and exon 7 are also expressed (see the above-mentioned non-patent document 1).

[0193] The DUX4 protein functions as a transcription factor, and examples of genes whose transcription is regulated by DUX4 include MBD3L2, ZSCAN4, TRIM43, DEFB103, and ZNF217 (see Non-Patent Document 2 above).

[0194] The target DUX4 mRNA of the modified oligonucleotide of the present invention is preferably human DUX4, more preferably DUX4-FL, and even more preferably DUX4-FL1 described in SEQ ID NO: 1 in the sequence listing. In addition, the target site of the modified oligonucleotide of the present invention for DUX4 is preferably exon 1, intron 1, exon 2, intron 2, and exon 3.

[0195] It is known that the DUX4 gene is expressed by fusion with other genes due to chromosomal abnormalities such as translocation. As other genes, for example, IGH (Yasuda et al., Nature Genetics 48 (5), 569 (2016)), CIC (Yoshimoto et al., Cancer research 77, 2927 (2017)), and EWSR1 (Sirvent et al., Cancer Genetics and Cytogenetics 195, 12 (2009)) are reported, which are considered to be the causal genes of B-cell acute lymphoblastic leukemia, differentiated round cell sarcoma, embryonic rhabdomyosarcoma, etc. The modified oligonucleotides of the present invention also include compounds targeting their fusion genes.

[0196] "DUX4 expression" refers to the level of mRNA transcribed from a gene encoding DUX4, or the level of protein translated from the mRNA. DUX4 expression can be determined by a method known in the art, such as Northern blotting, Western blotting, or PCR.

[0197] "DUX4 nucleic acid" refers to any nucleic acid encoding DUX4. For example, in one embodiment, the DUX4 nucleic acid comprises a DNA sequence encoding DUX4, an RNA sequence transcribed from a DNA encoding DUX4 (including genomic DNA containing introns and exons), and an mRNA precursor or a spliced ​​mature mRNA encoding DUX4. In addition, in one embodiment, the DUX4 nucleic acid comprises a DNA sequence or an RNA sequence of a gene generated by fusion of the DUX4 gene with another gene.

[0198] "DUX4 mRNA" refers to mRNA encoding DUX4 protein.

[0199] “Consecutive nucleic acid bases” and “continuous nucleic acid bases” refer to nucleic acid bases that are directly adjacent to each other.

[0200] "Complementary" refers to the ability of a first nucleic acid to undergo base pairing with a second nucleic acid.

[0201] "Completely complementary (also called complementarity)" or "100% complementary (also called complementarity)" means that all nucleic acid bases of the nucleic acid base sequence of the first nucleic acid have complementary nucleic acid bases in the second nucleic acid base sequence of the second nucleic acid. In one embodiment, the first nucleic acid is a modified oligonucleotide and the target nucleic acid is the second nucleic acid.

[0202] "Hybridization" refers to the annealing of complementary nucleic acid molecules. In one embodiment, the complementary nucleic acid molecules include a modified oligonucleotide and a target nucleic acid.

[0203] "Specifically hybridizable" means that there is sufficient complementarity between the modified oligonucleotide and the target nucleic acid to induce the desired effect, and on the other hand, the modified oligonucleotide specifically binds to the desired conditions, i.e., physiological conditions in in vivo assays and therapeutic treatments, and exhibits only minimal or no effect on non-target nucleic acids.

[0204] "Mismatch" or "non-complementary nucleobase" refers to a situation where a nucleobase of a first nucleic acid cannot pair with a corresponding nucleobase of a second nucleic acid or a target nucleic acid.

[0205] "Targeting" or "target" refers to the process of designing and selecting modified oligonucleotides that specifically hybridize to a target nucleic acid and induce a desired effect.

[0206] "Target nucleic acid", "target RNA" and "target RNA transcript" all refer to a nucleic acid that a modified oligonucleotide can target. In certain embodiments, the target nucleic acid comprises a region of a DUX4 nucleic acid.

[0207] "Target fragment" refers to the nucleotide sequence of the target nucleic acid targeted by the modified oligonucleotide. "5' target site" refers to the 5'most nucleotide of the target fragment. "3' target site" refers to the 3'most nucleotide of the target fragment.

[0208] "Active target region" or "target region" refers to a region to which one or more active modified oligonucleotides are targeted. "Active modified oligonucleotide" refers to a modified oligonucleotide that reduces target nucleic acid levels or protein levels.

[0209] "Antisense inhibition" refers to a decrease in target nucleic acid levels or target protein levels in the presence of a modified oligonucleotide that is complementary to the target nucleic acid, compared to the target nucleic acid levels or target protein levels in the absence of the modified oligonucleotide.

[0210] "siRNA" refers to a double-stranded RNA oligonucleotide having a nucleic acid base sequence that can hybridize to a corresponding region or fragment of a target nucleic acid.

[0211] “shRNA” refers to a hairpin-type single-stranded RNA oligonucleotide having a nucleic acid base sequence that can hybridize to a corresponding region or fragment of a target nucleic acid.

[0212] "SnoRNA" refers to a non-coding RNA present in the nucleolus, a chemically modified single-stranded oligonucleotide having a nucleic acid base sequence that can hybridize to a corresponding region or fragment of a target RNA nucleic acid and guide the methylation or pseudouridylation of the target RNA nucleic acid.

[0213] “miRNA” refers to a non-coding RNA that regulates the expression of other genes and is a single-stranded or double-stranded RNA oligonucleotide having a nucleic acid base sequence that can hybridize to a corresponding region or fragment of a target nucleic acid.

[0214] "Cap structure" or "terminal cap moiety" refers to a chemical modification incorporated into either terminus of a modified oligonucleotide.

[0215] A "chemically heterogeneous region" refers to a region within a modified oligonucleotide that is chemically different in some way from other regions within the same modified oligonucleotide. For example, a region having 2'-O-methoxyethyl nucleotides is chemically different from a region having nucleotides that are not modified with 2'-O-methoxyethyl.

[0216] "Chimeric modified oligonucleotide" refers to a modified oligonucleotide having at least two regions of chemical heterogeneity.

[0217] A "motif" refers to a pattern of chemically heterogeneous regions in a modified oligonucleotide.

[0218] "Gapmer" refers to a modified oligonucleotide in which an internal region having multiple nucleosides that support cleavage by RNase H is located between external regions having one or more nucleosides, and the nucleosides constituting the internal region are chemically different from the nucleosides (which may be multiple) constituting the external region. The internal region may be referred to as a "gap segment," and the external region may be referred to as a "wing segment." A wing segment that exists at a position closer to the 5' than the gap segment may be referred to as a "5' wing segment," and a wing segment that exists at a position closer to the 3' than the gap segment may be referred to as a "3' wing segment."

[0219] "Directly adjacent" means that there are no intervening elements between the directly adjacent elements.

[0220] “Nuclear ribonucleases” refer to ribonucleases found in the nucleus. Examples of nuclear ribonucleases include RNase H including RNase H1 and RNase H2, double-stranded RNase Drosha, and other double-stranded RNases, but are not limited to these.

[0221] In a certain embodiment, the gapmer refers to a modified oligonucleotide as follows: it has a 5' wing segment, a 3' wing segment and a gap segment; the number of nucleosides in the 5' wing segment and the 3' wing segment is 1 to 8 respectively; the gap segment is located between the two wing segments and is directly adjacent to them, the number of nucleosides is 6 or more, and the gap segment contains only nucleosides that do not contain modified sugars, or contains 1 or 2 nucleosides that contain modified sugars and other nucleosides that do not contain modified sugars.

[0222] "Agent" refers to an active substance that can confer therapeutic benefit when administered to an animal. "First agent" refers to a therapeutic compound of the present invention. For example, the first agent can also be a modified oligonucleotide that targets DUX4. "Second agent" refers to a second therapeutic compound of the present invention (e.g., a second modified oligonucleotide that targets DUX4) and / or a therapeutic compound that does not target DUX4.

[0223] "Pharmaceutically acceptable salts" refer to physiologically and pharmaceutically acceptable salts of modified oligonucleotides, ie, salts that retain the desired biological activity of the modified oligonucleotide and do not impart undesired toxic effects thereto.

[0224] "Diluent" refers to an ingredient in a composition that is pharmaceutically necessary or desirable, although it lacks pharmacological activity. For example, the diluent in a composition to be injected can be a liquid, such as saline.

[0225] "DUX4-related diseases" refer to diseases caused by abnormal expression of DUX4 mRNA or DUX4 protein, or mRNA or protein of fusion genes caused by translocation of the DUX4 gene. Examples include, but are not limited to, facioscapulohumeral muscular dystrophy, B-cell acute lymphoblastic leukemia, differentiated round cell sarcoma, and embryonic rhabdomyosarcoma.

[0226] "Facioscapulohumeral muscular dystrophy" or "FSHD" refers to a muscular dystrophy caused by decreased muscle strength in the facial, scapular, and humeral muscles. In humans, it is believed that the disease is mainly caused by the shortening of the genomic repeat sequence (D4Z4) near the telomere (chromosome end) of chromosome 4, which results in changes in the genome structure and ectopic expression of the DUX4 gene, which is not originally expressed in muscle (precursor) cells, causing cell death (FSHD1). In addition, in some FSHD patients, gene mutations have been found in SMCHD1, one of the genome structure control factors that inhibit gene expression (FSHD2).

[0227] FSHD is a type of muscular dystrophy associated with progressive muscle weakness and loss of muscle fibers. Unlike Duchenne muscular dystrophy and Becker muscular dystrophy, which mainly affect the lower body, FSHD affects the upper body, mainly in the facial muscles, scapular muscles, and brachial muscles. However, it can also affect the pelvis, waist, and lower limbs. Symptoms of FSHD often appear between the ages of 10 and 26, but it is not uncommon for symptoms to develop much later. Depending on the situation, there are cases where the disease does not develop at all. Symptoms are generally mild and worsen very slowly. Common symptoms include facial muscle weakness, drooping eyelids, inability to whistle, reduced facial expression changes, depressed or angry facial expressions, difficulty in speech articulation, weakness of the scapular muscles (causing deformities such as prominent scapula (winged scapula) and sloping shoulders), weakness of the lower limbs, hearing loss, and concerns about heart disease.

[0228] "Active pharmaceutical agent" refers to a substance (or substances) in a pharmaceutical composition that provides a therapeutic benefit when administered to an animal. For example, in one embodiment, a modified oligonucleotide targeting DUX4 is an active pharmaceutical agent.

[0229] "Concurrent administration" refers to the co-administration of two drugs in any manner in which the pharmacological effects caused by the two drugs are manifested in the patient at the same time. Concurrent administration does not require that the two drugs be in a single pharmaceutical composition, in the same dosage form, or administered by the same route of administration. The pharmacological effects caused by the two drugs do not need to be manifested at the same time. The pharmacological effects only need to be repeated within a certain period of time and do not need to be of the same breadth.

[0230] "Administration" means administering a drug to an animal, and examples thereof include administration by a medical professional and self-administration, but are not limited to these.

[0231] "Improvement" refers to a reduction in at least one indicator, symptom or symptom of the disease, disorder or condition in question. The severity of the indicator can be determined by subjective or objective scales known to those skilled in the art.

[0232] "Animal" refers to humans, or non-human animals including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates including, but not limited to, monkeys and chimpanzees.

[0233] "Co-administration" refers to the administration of two or more drugs to an individual. The two or more drugs may be present in a single pharmaceutical composition or in separate pharmaceutical compositions. The two or more drugs may be administered via the same or different administration routes. Co-administration includes concurrent or sequential administration.

[0234] "Dosage" refers to a specific quantitative amount of a pharmaceutical agent administered in a single dose or within a specific period. In certain embodiments, the dosage can be administered by 1, 2 or more boluses, tablets or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dosage needs to be a volume that is not easily accommodated by a single injection, so in order to achieve the desired dosage, more than 2 injections can be used. In certain embodiments, the pharmaceutical agent is administered long-term or continuously by injection. The dosage can be recorded as the amount of the pharmaceutical agent per hour, day, week or month.

[0235] "Effective amount" or "therapeutically effective amount" refers to the amount of active pharmaceutical agent sufficient to achieve the desired physiological result in an individual in need of the agent. The effective amount may vary from individual to individual depending on the health and physical condition of the individual being treated, the classification group of the individual being treated, the formulation of the composition, the assessment of the individual's medical status, and other related factors.

[0236] An "animal identified as having facioscapulohumeral muscular dystrophy (FSHD)" refers to an animal identified as having a disorder or condition diagnosed as FSHD, or an animal identified as being susceptible to a disorder or condition of FSHD. For example, an individual with a family history may be susceptible to a disorder or condition of FSHD.

[0237] Such determination can be achieved by any method including investigation of individual medical history and standard clinical examination or evaluation. It should be noted that FSHD is known to have FSHD1 and FSHD2 based on the pathogenesis, but FSHD includes both.

[0238] "Subject" refers to a human or non-human animal selected for treatment or therapy.

[0239] "Myotonia" refers to abnormally slow muscle relaxation after voluntary contraction or electrical stimulation.

[0240] "Paranoral administration" refers to administration by injection or infusion. Examples of parenteral administration include subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, such as intramedullary or intraventricular administration. Administration may be continuous or long-term, or short-term or intermittent.

[0241] A "pharmaceutical composition" refers to a mixture of substances suitable for administration to a subject. For example, a pharmaceutical composition may include one or more active agents and a sterile aqueous solution.

[0242] "Prevention" means delaying or preventing the onset or occurrence of a disease, disorder, or undesirable health condition, or one or more symptoms associated with the disease, disorder, or undesirable health condition, for a period of several minutes to indefinitely. Prevention also means reducing the risk of developing a disease, disorder, or undesirable health condition.

[0243] "Treatment" means to alleviate or eliminate a disease, disorder, or undesirable health condition, or one or more symptoms associated with the disease, disorder, or undesirable health condition, or to partially relieve or eradicate one or more causes of the disease, disorder, or undesirable health condition itself.

[0244] "Treatment" includes the above-mentioned prevention or treatment. For example, it also includes administering the pharmaceutical composition of the present invention to change or improve the disease, disorder or undesirable health condition.

[0245] "Prodrug" refers to a therapeutic agent prepared in an inactive form that is converted to an active form in vivo or within a cell thereof by the action of an endogenous enzyme or other chemical substance or condition.

[0246] "Side effects" refer to physiological reactions that may be caused by treatment other than the desired effect. In a certain embodiment, as side effects, injection site reactions, abnormal liver function tests, abnormal renal function, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, myopathy and fatigue can be cited. For example, an increase in the level of alanine aminotransferase (ALT), aspartate aminotransferase (AST) or γ-glutamyl transpeptidase (γ-GTP) in the blood can indicate hepatotoxicity or abnormal liver function. For example, an increase in bilirubin can indicate hepatotoxicity or abnormal liver function. In addition, an increase in urine protein, creatinine in the blood, and an increase in urea nitrogen (UN) can indicate nephrotoxicity or abnormal renal function.

[0247] "Subcutaneous administration" refers to administration just beneath the skin.

[0248] A "therapeutically effective amount" refers to an amount of a drug that confers a therapeutic benefit to a subject.

[0249] (Specific implementation method)

[0250] Implementation

[0251] Certain specific embodiments described below provide compounds for inhibiting DUX4 expression, methods for using the compounds, and pharmaceutical compositions containing the compounds, but are not limited thereto.

[0252] Certain embodiments provide a method for reducing DUX4 expression in an animal, comprising administering to the animal a compound comprising a modified oligonucleotide targeting DUX4.

[0253] One embodiment provides a method for administering a modified oligonucleotide to inhibit the accumulation of a pathogenic DUX4 transcription factor by inhibiting transcription of the DUX4 gene, inhibiting translation of DUX4 mRNA, or inducing cleavage of DUX4 mRNA.

[0254] In one embodiment, a method for treating an animal having facioscapulohumeral muscular dystrophy is provided, comprising the steps of: a) identifying the animal having facioscapulohumeral muscular dystrophy, and b) administering to the animal a therapeutically effective amount of a compound comprising a modified oligonucleotide targeting DUX4.

[0255] In one embodiment, a method for reducing myotonia in a subject in need thereof is provided. The method comprises administering to the subject a modified oligonucleotide complementary to DUX4 mRNA, wherein when the modified oligonucleotide is bound to DUX4 mRNA, ribonucleases or nuclear ribonucleases are activated, thereby reducing myotonia. In one embodiment, the subject has or is suspected of having facioscapulohumeral muscular dystrophy, or has high expression of DUX4 mRNA or a reduced number of repeats of D4Z4 on human chromosome 4, or has or is suspected of having a SMCHD1 (DNA methylase) mutation.

[0256] In certain embodiments, the modified oligonucleotide used in the methods of the present invention is a chimera. In certain embodiments, the modified oligonucleotide used in the methods of the present invention is a gapmer.

[0257] In one embodiment of the method of the present invention described in this specification, the administration is subcutaneous administration. In one embodiment, the administration is intravenous or intramuscular administration.

[0258] In one embodiment, the modified oligonucleotide used in the method of the present invention targets the DUX4 protein coding region, intron, 5'UTR or 3'UTR of DUX4 mRNA. In one embodiment, the modified oligonucleotide used in the method of the present invention targets exon 1, exon 2, exon 3, intron 1, and intron 2 of DUX4 mRNA.

[0259] In one embodiment of the method of the present invention described in the present specification, DUX4 mRNA is cleaved by RNase H1, an intranuclear ribonuclease.

[0260] In one embodiment of the method of the present invention, DUX4 mRNA is reduced in muscle tissue. In one embodiment, splice variants DUX4-FL1 (SEQ ID NO: 1 in the sequence listing) and DUX4-FL2 (SEQ ID NO: 5 in the sequence listing) are preferentially reduced.

[0261] In certain embodiments, DUX4 mRNA has a sequence described in GenBank Accession No. NM_001293798.2 (incorporated into this specification as Sequence No. 1 in the sequence listing). The splice variant of Sequence No. 1 in the sequence listing is also referred to as DUX4-FL1 or mature mRNA of DUX4. In certain embodiments, DUX4 mRNA has a sequence described in GenBank Accession No. NM_001306068.2 (incorporated into this specification as Sequence No. 5 in the sequence listing). The splice variant of Sequence No. 5 in the sequence listing is also referred to as DUX4-FL2. In certain embodiments, DUX4 has a sequence described in GenBank Accession No. NM_001363820.1 (incorporated into this specification as Sequence No. 6 in the sequence listing). The splice variant of Sequence No. 6 in the sequence listing is also referred to as DUX4-s. In certain embodiments, DUX4 mRNA has a SNP of the above-mentioned splice variant.

[0262] In a certain embodiment, the modified oligonucleotide is a modified oligonucleotide composed of 12 to 30 residues and has a nucleic acid base sequence comprising the modified oligonucleotide, which is a nucleic acid base sequence comprising at least 8 consecutive nucleic acid base sequences that are complementary to equal length portions of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list at positions 126 to 147, 232 to 248, 1306 to 1325, or 1472 to 1495 from the 5' end. In a certain embodiment, the modified oligonucleotide is a modified oligonucleotide composed of 12 to 30 residues and has a nucleic acid base sequence comprising the modified oligonucleotide, which is a nucleic acid base sequence comprising at least 9, 10, 11 or 12 consecutive nucleic acid base sequences that are complementary to equal length portions of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list at positions 126 to 147, 232 to 248, 1306 to 1325 or 1472 to 1495 from the 5' end.

[0263] The modified oligonucleotide can be composed of at least 8 consecutive nucleic acid base sequences that are complementary to equal length portions of the nucleic acid bases of the mature mRNA of DUX4 in sequence number 1 of the sequence list at positions 126 to 147, 232 to 248, 1306 to 1325 or 1472 to 1495 from the 5' end, and in addition to the nucleic acid base sequence, it can also have additional sequences on the 5' end side and / or the 3' end side.

[0264] In a certain embodiment, the modified oligonucleotide is a nucleic acid base sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleic acid base sequences that are complementary to an equal length portion of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from the 5' end at positions 126 to 147, and is a modified oligonucleotide within 30 residues.

[0265] In a certain embodiment, the modified oligonucleotide is a nucleic acid base sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 consecutive nucleic acid base sequences that are complementary to the equal length portion of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from positions 232 to 248 from the 5' end, and is a modified oligonucleotide within 30 residues.

[0266] In a certain embodiment, the modified oligonucleotide is a nucleic acid base sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive nucleic acid base sequences that are complementary to an equal length portion of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from positions 1306 to 1325 from the 5' end, and is a modified oligonucleotide within 30 residues.

[0267] In a certain embodiment, the modified oligonucleotide is a nucleic acid base sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 consecutive nucleic acid base sequences that are complementary to an equal length portion of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from positions 1472 to 1495 from the 5' end, and is a modified oligonucleotide within 30 residues.

[0268] In a certain embodiment, the modified oligonucleotide is a modified oligonucleotide composed of 12 to 30 residues, which comprises a nucleic acid base sequence complementary to an equal length portion of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from positions 126 to 147 from the 5' end, and is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to sequence number 1 in the sequence list in the equal length portion. In another embodiment, it is 12 to 29 residues, 12 to 28 residues, 12 to 27 residues, 12 to 26 residues, 12 to 25 residues, 12 to 24 residues, 12 to 23 residues, 12 to 22 residues, 12 to 21 residues, 12 to 20 residues, 12 to 19 residues, 12 to 18 residues, 12 to 17 residues, 12 to 16 residues, 12 to 15 residues, 12 to 14 residues, 13 to 29 residues, 13 to 28 residues, 13 to 27 residues, 13 to 26 residues, 13 to 25 residues, 13 to 24 residues, 13 to 23 residues, 13 to 22 residues, 13-21 residues, 13-20 residues, 13-19 residues, 13-18 residues, 13-17 residues, 13-16 residues, 13-15 residues, 13-14 residues, 14-29 residues, 14-28 residues, 14-27 residues, 14-26 residues, 14-25 residues, 14-24 residues, 14-23 residues, 14-22 residues, 14-21 residues, 14-20 residues, 14-19 residues, 14-18 residues, 14-17 residues, 14-16 residues, 14-15 residues of the above modified oligonucleotides.

[0269] In a certain embodiment, the modified oligonucleotide is a modified oligonucleotide composed of 12 to 30 residues, which comprises a nucleic acid base sequence complementary to an equal length portion of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from positions 232 to 248 from the 5' end, and is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to sequence number 1 in the sequence list in the equal length portion. In another embodiment, it is 12 to 29 residues, 12 to 28 residues, 12 to 27 residues, 12 to 26 residues, 12 to 25 residues, 12 to 24 residues, 12 to 23 residues, 12 to 22 residues, 12 to 21 residues, 12 to 20 residues, 12 to 19 residues, 12 to 18 residues, 12 to 17 residues, 12 to 16 residues, 12 to 15 residues, 12 to 14 residues, 13 to 29 residues, 13 to 28 residues, 13 to 27 residues, 13 to 26 residues, 13 to 25 residues, 13 to 24 residues, 13 to 23 residues, 13 to 22 residues, 13-21 residues, 13-20 residues, 13-19 residues, 13-18 residues, 13-17 residues, 13-16 residues, 13-15 residues, 13-14 residues, 14-29 residues, 14-28 residues, 14-27 residues, 14-26 residues, 14-25 residues, 14-24 residues, 14-23 residues, 14-22 residues, 14-21 residues, 14-20 residues, 14-19 residues, 14-18 residues, 14-17 residues, 14-16 residues, 14-15 residues of the above modified oligonucleotides.

[0270] In a certain embodiment, the modified oligonucleotide is a modified oligonucleotide composed of 12 to 30 residues, which comprises a nucleic acid base sequence complementary to an equal length portion of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from positions 1306 to 1325 from the 5' end, and is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to sequence number 1 in the sequence list in the equal length portion. In another embodiment, it is 12 to 29 residues, 12 to 28 residues, 12 to 27 residues, 12 to 26 residues, 12 to 25 residues, 12 to 24 residues, 12 to 23 residues, 12 to 22 residues, 12 to 21 residues, 12 to 20 residues, 12 to 19 residues, 12 to 18 residues, 12 to 17 residues, 12 to 16 residues, 12 to 15 residues, 12 to 14 residues, 13 to 29 residues, 13 to 28 residues, 13 to 27 residues, 13 to 26 residues, 13 to 25 residues, 13 to 24 residues, 13 to 23 residues, 13 to 22 residues, 13-21 residues, 13-20 residues, 13-19 residues, 13-18 residues, 13-17 residues, 13-16 residues, 13-15 residues, 13-14 residues, 14-29 residues, 14-28 residues, 14-27 residues, 14-26 residues, 14-25 residues, 14-24 residues, 14-23 residues, 14-22 residues, 14-21 residues, 14-20 residues, 14-19 residues, 14-18 residues, 14-17 residues, 14-16 residues, 14-15 residues of the above modified oligonucleotides.

[0271] In a certain embodiment, the modified oligonucleotide is a modified oligonucleotide composed of 12 to 30 residues, which comprises a nucleic acid base sequence complementary to an equal length portion of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from positions 1472 to 1495 from the 5' end, and is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to sequence number 1 in the sequence list in the equal length portion. In another embodiment, the polypeptide comprises 12 to 29 residues, 12 to 28 residues, 12 to 27 residues, 12 to 26 residues, 12 to 25 residues, 12 to 24 residues, 12 to 23 residues, 12 to 22 residues, 12 to 21 residues, 12 to 20 residues, 12 to 19 residues, 12 to 18 residues, 12 to 17 residues, 12 to 16 residues, 12 to 15 residues, 12 to 14 residues, 13 to 29 residues, 13 to 28 residues, 13 to 27 residues, 13 to 26 residues, 13 to 25 residues, 13 to 24 residues, 13 to 23 residues, 13 to The modified oligonucleotides consist of 22 residues, 13-21 residues, 13-20 residues, 13-19 residues, 13-18 residues, 13-17 residues, 13-16 residues, 13-15 residues, 13-14 residues, 14-29 residues, 14-28 residues, 14-27 residues, 14-26 residues, 14-25 residues, 14-24 residues, 14-23 residues, 14-22 residues, 14-21 residues, 14-20 residues, 14-19 residues, 14-18 residues, 14-17 residues, 14-16 residues, and 14-15 residues.

[0272] In a certain embodiment, the modified oligonucleotide is a modified oligonucleotide composed of 12 to 30 residues and has a nucleic acid base sequence comprising the modified oligonucleotide, which is a nucleic acid base sequence comprising at least 8 consecutive nucleic acid base sequences that are complementary to equal length portions of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list at positions 128 to 143, 233 to 248, 1309 to 1323, or 1480 to 1495 from the 5' end. In a certain embodiment, the modified oligonucleotide is a modified oligonucleotide composed of 12 to 30 residues and has a nucleic acid base sequence comprising the modified oligonucleotide, which is a nucleic acid base sequence comprising at least 9, 10, 11, or 12 consecutive nucleic acid base sequences that are complementary to equal length portions of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list at positions 128 to 143, 233 to 248, 1309 to 1323, or 1480 to 1495 from the 5' end.

[0273] In a certain embodiment, the modified oligonucleotide is a nucleic acid base sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15 or 16 consecutive nucleic acid base sequences that are complementary to an equal length portion of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from positions 128 to 143 from the 5' end, and is a modified oligonucleotide within 30 residues.

[0274] In a certain embodiment, the modified oligonucleotide is a nucleic acid base sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15 or 16 consecutive nucleic acid base sequences that are complementary to the equal length portion of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from positions 233 to 248 from the 5' end, and is a modified oligonucleotide within 30 residues.

[0275] In a certain embodiment, the modified oligonucleotide is a nucleic acid base sequence comprising at least 8, 9, 10, 11, 12, 13, 14 or 15 consecutive nucleic acid base sequences that are complementary to an equal length portion of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from positions 1309 to 1323 from the 5' end, and is a modified oligonucleotide within 30 residues.

[0276] In a certain embodiment, the modified oligonucleotide is a sequence of at least 8, 9, 10, 11, 12, 13, 14, 15 or 16 consecutive nucleic acid bases that are complementary to an equal length portion of the nucleic acid bases at positions 1480 to 1495 from the 5' end of the mature mRNA of DUX4 of sequence number 1 in the sequence list, and is a modified oligonucleotide within 30 residues of a nucleic acid base sequence having a complementary base to the base at position 1480 from the 5' end of the nucleic acid base of sequence number 1 at the 3' end.

[0277] In a certain embodiment, the modified oligonucleotide is a modified oligonucleotide composed of 12 to 30 residues, which comprises a nucleic acid base sequence complementary to an equal length portion of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from positions 128 to 143 from the 5' end, and is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to sequence number 1 in the sequence list in the equal length portion. In another embodiment, it is 12 to 29 residues, 12 to 28 residues, 12 to 27 residues, 12 to 26 residues, 12 to 25 residues, 12 to 24 residues, 12 to 23 residues, 12 to 22 residues, 12 to 21 residues, 12 to 20 residues, 12 to 19 residues, 12 to 18 residues, 12 to 17 residues, 12 to 16 residues, 12 to 15 residues, 12 to 14 residues, 13 to 29 residues, 13 to 28 residues, 13 to 27 residues, 13 to 26 residues, 13 to 25 residues, 13 to 24 residues, 13 to 23 residues, 13 to 22 residues, 13-21 residues, 13-20 residues, 13-19 residues, 13-18 residues, 13-17 residues, 13-16 residues, 13-15 residues, 13-14 residues, 14-29 residues, 14-28 residues, 14-27 residues, 14-26 residues, 14-25 residues, 14-24 residues, 14-23 residues, 14-22 residues, 14-21 residues, 14-20 residues, 14-19 residues, 14-18 residues, 14-17 residues, 14-16 residues, 14-15 residues of the above modified oligonucleotides.

[0278] In a certain embodiment, the modified oligonucleotide is a modified oligonucleotide composed of 12 to 30 residues, which comprises a nucleic acid base sequence complementary to an equal length portion of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from positions 233 to 248 from the 5' end, and is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to sequence number 1 in the sequence list in the equal length portion. In another embodiment, it is 12 to 29 residues, 12 to 28 residues, 12 to 27 residues, 12 to 26 residues, 12 to 25 residues, 12 to 24 residues, 12 to 23 residues, 12 to 22 residues, 12 to 21 residues, 12 to 20 residues, 12 to 19 residues, 12 to 18 residues, 12 to 17 residues, 12 to 16 residues, 12 to 15 residues, 12 to 14 residues, 13 to 29 residues, 13 to 28 residues, 13 to 27 residues, 13 to 26 residues, 13 to 25 residues, 13 to 24 residues, 13 to 23 residues, 13 to 22 residues, 13-21 residues, 13-20 residues, 13-19 residues, 13-18 residues, 13-17 residues, 13-16 residues, 13-15 residues, 13-14 residues, 14-29 residues, 14-28 residues, 14-27 residues, 14-26 residues, 14-25 residues, 14-24 residues, 14-23 residues, 14-22 residues, 14-21 residues, 14-20 residues, 14-19 residues, 14-18 residues, 14-17 residues, 14-16 residues, 14-15 residues of the above modified oligonucleotides.

[0279] In a certain embodiment, the modified oligonucleotide is a modified oligonucleotide composed of 12 to 30 residues, which comprises a nucleic acid base sequence complementary to an equal length portion of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from positions 1309 to 1323 from the 5' end, and is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to sequence number 1 in the sequence list in the equal length portion. In another embodiment, it is 12 to 29 residues, 12 to 28 residues, 12 to 27 residues, 12 to 26 residues, 12 to 25 residues, 12 to 24 residues, 12 to 23 residues, 12 to 22 residues, 12 to 21 residues, 12 to 20 residues, 12 to 19 residues, 12 to 18 residues, 12 to 17 residues, 12 to 16 residues, 12 to 15 residues, 12 to 14 residues, 13 to 29 residues, 13 to 28 residues, 13 to 27 residues, 13 to 26 residues, 13 to 25 residues, 13 to 24 residues, 13 to 23 residues, 13 to 22 residues, 13-21 residues, 13-20 residues, 13-19 residues, 13-18 residues, 13-17 residues, 13-16 residues, 13-15 residues, 13-14 residues, 14-29 residues, 14-28 residues, 14-27 residues, 14-26 residues, 14-25 residues, 14-24 residues, 14-23 residues, 14-22 residues, 14-21 residues, 14-20 residues, 14-19 residues, 14-18 residues, 14-17 residues, 14-16 residues, 14-15 residues of the above modified oligonucleotides.

[0280] In a certain embodiment, the modified oligonucleotide is a modified oligonucleotide composed of 12 to 30 residues, which is a nucleic acid base sequence complementary to an equal length portion of the 1480th to 1495th positions from the 5' end of the nucleic acid base of the mature mRNA of DUX4 of sequence number 1 in the sequence list, and is composed of a nucleic acid base sequence having a complementary base to the base at the 1480th position from the 5' end of the nucleic acid base of sequence number 1 at the 3' end, and is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to the nucleic acid base sequence of sequence number 1 in the sequence list in the equal length portion. In another embodiment, the polypeptide comprises 12 to 29 residues, 12 to 28 residues, 12 to 27 residues, 12 to 26 residues, 12 to 25 residues, 12 to 24 residues, 12 to 23 residues, 12 to 22 residues, 12 to 21 residues, 12 to 20 residues, 12 to 19 residues, 12 to 18 residues, 12 to 17 residues, 12 to 16 residues, 12 to 15 residues, 12 to 14 residues, 13 to 29 residues, 13 to 28 residues, 13 to 27 residues, 13 to 26 residues, 13 to 25 residues, 13 to 24 residues, 13 to 23 residues, 13 to The modified oligonucleotides consist of 22 residues, 13-21 residues, 13-20 residues, 13-19 residues, 13-18 residues, 13-17 residues, 13-16 residues, 13-15 residues, 13-14 residues, 14-29 residues, 14-28 residues, 14-27 residues, 14-26 residues, 14-25 residues, 14-24 residues, 14-23 residues, 14-22 residues, 14-21 residues, 14-20 residues, 14-19 residues, 14-18 residues, 14-17 residues, 14-16 residues, and 14-15 residues.

[0281] In one embodiment, the modified oligonucleotide provided in the present specification targets any one of the following regions of SEQ ID NO: 1 in the sequence listing: 126 to 141, 126 to 143, 127 to 142, 127 to 143, 127 to 144, 127 to 146, 128 to 143, 128 to 144, 128 to 147, 232 to 245, 232 to 247, 233 to 249 from the 5' end. 6. 233-247, 233-248, 234-247, 234-248, 1304-1323, 1306-1321, 1306-1324, 1307-1323, 1307-1324, 1307-1325, 1307-1326, 1308-1323, 1308-1324, 1308-1322, 1308-1325, 1 309~1323、1309~1324、1309~1325、1309~1322、1310~1323、1310~1324、1472~1485、1472~1486、1472~1487、1472~1488、1473~1487、1473~1488、1473~1489、1474~1488、1474~1 489, 1475-1490, 1476-1490, 1476-1491, 1476-1495, 1477-1495, 1478-1496, 1479-1495, 1479-1496, 1479-1498, 1480-1494, 1480-1495, 1480-1496, 1480-1497 or 1480-1499. In one embodiment, the modified oligonucleotide provided herein targets any one of the following regions of SEQ ID NO: 1 in the sequence listing: 128-143, 232-247, 233-248, 1309-1323 and 1480-1495 from the 5' end.

[0282] In one embodiment, the modified oligonucleotide provided in the present specification has a nucleic acid base sequence, and the nucleic acid base sequence includes a complementary region containing at least 8 consecutive nucleic acid bases that are complementary to the target region. In one embodiment, the modified oligonucleotide provided in the present specification has a nucleic acid base sequence, and the nucleic acid base sequence includes a complementary region containing at least 8 consecutive nucleic acid bases that are complementary to the target region, and the target region is 126 to 141, 126 to 143, 127 to 142, 127 to 143, 127 to 144, 127 to 146, 128 to 143, 128 to 144, 128 from the 5' end of sequence number 1 in the sequence list. ~147、232~245、232~247、233~246、233~247、233~248、234~247、234~248、1304~1323、1306~1321、1306~1324、1307~1323、1307~1324、1307~1325、1307~1326、1308~1323、1308~1324、1308 ~1322、1308~1325、1309~1323、1309~1324、1309~1325、1309~1322、1310~1323、1310~1324、1472~1485、1472~1486、1472~1487、1472~1488、1473~1487、1473~1488、1473~1489、1474~148 8. Position 1474-1489, 1475-1490, 1476-1490, 1476-1491, 1476-1495, 1477-1495, 1478-1496, 1479-1495, 1479-1496, 1479-1498, 1480-1494, 1480-1495, 1480-1496, 1480-1497 or 1480-1499 is the target. In a certain embodiment, the modified oligonucleotide provided in the present specification has a nucleic acid base sequence, which includes a complementary region containing at least 8 consecutive nucleic acid bases that are complementary to the target region, and the target region is targeted at positions 128 to 143, 232 to 247, 233 to 248, 1309 to 1323 or 1480 to 1495 from the 5' end of sequence number 1 in the sequence list.

[0283] In one embodiment, the modified oligonucleotide provided in the present specification has a nucleic acid base sequence, and the nucleic acid base sequence includes a complementary region containing at least 10 consecutive nucleic acid bases that are complementary to the target region. In one embodiment, the modified oligonucleotide provided in the present specification has a nucleic acid base sequence, and the nucleic acid base sequence includes a complementary region containing at least 10 consecutive nucleic acid bases that are complementary to the target region, and the target region is 126 to 141, 126 to 143, 127 to 142, 127 to 143, 127 to 144, 127 to 146, 128 to 143, 128 to 144, 128 to 146, 129 to 150, 129 to 151, 129 to 152, 129 to 153, 129 to 154, 129 to 155, 129 to 156, 129 to 157, 129 to 158, 129 to 159, 129 to 160, 129 to 161, 129 to 162, 129 to 163, 129 to 164, 129 to 165, 129 to 166, 129 to 167, 129 to 168, 129 to 169, 129 to 170, 129 to 171, 129 to 172, 129 to 173, 129 to 174, 129 to 8~147、232~245、232~247、233~246、233~247、233~248、234~247、234~248、1304~1323、1306~1321、1306~1324、1307~1323、1307~1324、1307~1325、1307~1326、1308~1323、1308~1324、130 8~1322、1308~1325、1309~1323、1309~1324、1309~1325、1309~1322、1310~1323、1310~1324、1472~1485、1472~1486、1472~1487、1472~1488、1473~1487、1473~1488、1473~1489、1474~148 8. Position 1474-1489, 1475-1490, 1476-1490, 1476-1491, 1476-1495, 1477-1495, 1478-1496, 1479-1495, 1479-1496, 1479-1498, 1480-1494, 1480-1495, 1480-1496, 1480-1497 or 1480-1499 is the target. In a certain embodiment, the modified oligonucleotide provided in the present specification has a nucleic acid base sequence, which includes a complementary region containing at least 10 consecutive nucleic acid bases that are complementary to the target region, and the target region is targeted at positions 128 to 143, 232 to 247, 233 to 248, 1309 to 1323 or 1480 to 1495 from the 5' end of sequence number 1 in the sequence list.

[0284] In one embodiment, the modified oligonucleotide provided in the present specification has a nucleic acid base sequence, and the nucleic acid base sequence includes a complementary region containing at least 12 consecutive nucleic acid bases that are complementary to the target region. In one embodiment, the modified oligonucleotide provided in the present specification has a nucleic acid base sequence, and the nucleic acid base sequence includes a complementary region containing at least 12 consecutive nucleic acid bases that are complementary to the target region, and the target region is 126 to 141, 126 to 143, 127 to 142, 127 to 143, 127 to 144, 127 to 146, 128 to 143, 128 to 144, 128 to 146, 129 to 150, 129 to 151, 129 to 152, 129 to 153, 129 to 154, 129 to 155, 129 to 156, 129 to 157, 129 to 158, 129 to 159, 129 to 160, 129 to 161, 129 to 162, 129 to 163, 129 to 164, 129 to 165, 129 to 166, 129 to 167, 129 to 168, 129 to 169, 129 to 170, 129 to 171, 129 to 172, 129 to 173, 129 to 174, 129 to 8~147、232~245、232~247、233~246、233~247、233~248、234~247、234~248、1304~1323、1306~1321、1306~1324、1307~1323、1307~1324、1307~1325、1307~1326、1308~1323、1308~1324、130 8~1322、1308~1325、1309~1323、1309~1324、1309~1325、1309~1322、1310~1323、1310~1324、1472~1485、1472~1486、1472~1487、1472~1488、1473~1487、1473~1488、1473~1489、1474~148 8. Position 1474-1489, 1475-1490, 1476-1490, 1476-1491, 1476-1495, 1477-1495, 1478-1496, 1479-1495, 1479-1496, 1479-1498, 1480-1494, 1480-1495, 1480-1496, 1480-1497 or 1480-1499 is the target. In a certain embodiment, the modified oligonucleotide provided in the present specification has a nucleic acid base sequence, which includes a complementary region containing at least 12 consecutive nucleic acid bases that are complementary to the target region, and the target region is targeted at positions 128 to 143, 232 to 247, 233 to 248, 1309 to 1323 or 1480 to 1495 from the 5' end of sequence number 1 in the sequence list.

[0285] In one embodiment, the modified oligonucleotide provided in the present specification has a nucleic acid base sequence, and the nucleic acid base sequence includes a complementary region containing at least 14 consecutive nucleic acid bases that are complementary to the target region. In one embodiment, the modified oligonucleotide provided in the present specification has a nucleic acid base sequence, and the nucleic acid base sequence includes a complementary region containing at least 14 consecutive nucleic acid bases that are complementary to the target region, and the target region is 126 to 141, 126 to 143, 127 to 142, 127 to 143, 127 to 144, 127 to 146, 128 to 143, 128 to 144, 128 to 146, 129 to 150, 129 to 151, 129 to 152, 129 to 153, 129 to 154, 129 to 155, 129 to 156, 129 to 157, 129 to 158, 129 to 159, 129 to 160, 129 to 161, 129 to 162, 129 to 163, 129 to 164, 129 to 165, 129 to 166, 129 to 167, 129 to 168, 129 to 169, 129 to 170, 129 to 171, 129 to 172, 129 to 173, 129 to 174, 129 to 8~147、232~245、232~247、233~246、233~247、233~248、234~247、234~248、1304~1323、1306~1321、1306~1324、1307~1323、1307~1324、1307~1325、1307~1326、1308~1323、1308~1324、130 8~1322、1308~1325、1309~1323、1309~1324、1309~1325、1309~1322、1310~1323、1310~1324、1472~1485、1472~1486、1472~1487、1472~1488、1473~1487、1473~1488、1473~1489、1474~148 8. Position 1474-1489, 1475-1490, 1476-1490, 1476-1491, 1476-1495, 1477-1495, 1478-1496, 1479-1495, 1479-1496, 1479-1498, 1480-1494, 1480-1495, 1480-1496, 1480-1497 or 1480-1499 is the target. In a certain embodiment, the modified oligonucleotide provided in the present specification has a nucleic acid base sequence, which includes a complementary region containing at least 14 consecutive nucleic acid bases that are complementary to the target region, and the target region is targeted at positions 128 to 143, 232 to 247, 233 to 248, 1309 to 1323 or 1480 to 1495 from the 5' end of sequence number 1 in the sequence list.

[0286] In one embodiment, the modified oligonucleotide comprises nucleic acid bases 126 to 141, 126 to 143, 127 to 142, 127 to 143, 127 to 144, 127 to 146, 128 to 143, 128 to 144, 128 to 147, 232 to 245, 232 to 247, 233 to 246, 233 from the 5' end of the mature mRNA of DUX4 of SEQ ID NO: 1 in the sequence listing. ~247、233~248、234~247、234~248、1304~1323、1306~1321、1306~1324、1307~1323、1307~1324、1307~1325、1307~1326、1308~1323、1308~1324、1308~1322、1308~1325、1309~1323、1 309~1324、1309~1325、1309~1322、1310~1323、1310~1324、1472~1485、1472~1486、1472~1487、1472~1488、1473~1487、1473~1488、1473~1489、1474~1488、1474~1489、1475~1490、1 1476-1490, 1476-1491, 1476-1495, 1477-1495, 1478-1496, 1479-1495, 1479-1496, 1479-1498, 1480-1494, 1480-1495, 1480-1496, 1480-1497 or 1480-1499. In one embodiment, the modified oligonucleotide comprises a nucleic acid base sequence complementary to the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the sequence listing at positions 128-143, 232-247, 233-248, 1309-1323, and 1480-1495 from the 5' end.

[0287] In one embodiment, the modified oligonucleotide is a modified oligonucleotide comprising a nucleic acid base sequence described in any one of sequence numbers 2 to 4, 7 to 64, 69 to 97, and 102 to 112 of the sequence listing. In another embodiment, the modified oligonucleotide is a modified oligonucleotide comprising a nucleic acid base sequence described in any one of sequence numbers 2 to 4, 75, and 78 of the sequence listing.

[0288] In one embodiment, the modified oligonucleotide is a modified oligonucleotide consisting of a nucleic acid base sequence recorded in any one of sequence numbers 2 to 4, 7 to 64, 69 to 97, and 102 to 112 of the sequence list. In another embodiment, the modified oligonucleotide is a modified oligonucleotide consisting of a nucleic acid base sequence recorded in any one of sequence numbers 2 to 4, 75, and 78 of the sequence list.

[0289] In one embodiment, the modified oligonucleotide has a nucleic acid base sequence comprising at least 8 consecutive nucleic acid bases of the nucleic acid base sequence described in any one of SEQ ID NOs: 2, 3, 4, 75, or 78 in the sequence listing.

[0290] In one embodiment, the modified oligonucleotide has a nucleic acid base sequence comprising at least 10 consecutive nucleic acid bases of the nucleic acid base sequence described in any one of SEQ ID NOs: 2, 3, 4, 75, or 78 in the sequence listing.

[0291] In one embodiment, the modified oligonucleotide has a nucleic acid base sequence comprising at least 12 consecutive nucleic acid bases of the nucleic acid base sequence described in any one of SEQ ID NOs: 2, 3, 4, 75, or 78 in the sequence listing.

[0292] In one embodiment, the modified oligonucleotide has a nucleic acid base sequence comprising at least 14 consecutive nucleic acid bases of the nucleic acid base sequence described in any one of SEQ ID NOs: 2, 3, 4, 75, or 78 in the sequence listing.

[0293] In one embodiment, the modified oligonucleotide has a nucleic acid base comprising a nucleic acid base sequence consisting of any one of SEQ ID NOs. 2, 3, 4, 75, or 78 in the sequence listing.

[0294] sequence.

[0295] In one embodiment, the modified oligonucleotide has a nucleic acid base sequence consisting of the nucleic acid base sequence described in any one of SEQ ID NOs. 2, 3, 4, 75, or 78 in the sequence listing.

[0296] In one embodiment, the animal is a human.

[0297] In certain embodiments, the administering comprises parenteral administration.

[0298] In certain embodiments, the compound is a single-stranded modified oligonucleotide.

[0299] In a certain embodiment, when the modified oligonucleotide as a whole is measured, the nucleic acid base sequence of the modified oligonucleotide is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to the equal length portion of any one of the above regions of sequence number 1 in the sequence list. In a certain embodiment, when the modified oligonucleotide as a whole is measured, the nucleic acid base sequence of the modified oligonucleotide is 100% complementary to the equal length portion of any one of the above regions of sequence number 1 in the sequence list.

[0300] In one embodiment, at least one internucleoside bond of the modified oligonucleotide is a modified internucleoside bond. In one embodiment, each internucleoside bond is a phosphorothioate internucleoside bond.

[0301] In certain embodiments, at least one nucleoside of the modified oligonucleotide comprises a modified sugar. In certain embodiments, at least one modified sugar is a bicyclic sugar. In certain embodiments, at least one modified sugar comprises 2'-O-methoxyethyl, 2'-O-methyl and / or 4'-(CH2) n -O-2' crosslink (where n is 1 or 2).

[0302] In one embodiment, the sugar moiety of the modified oligonucleotide comprises at least one modified sugar that is a bicyclic sugar. In one embodiment, at least one modified sugar is LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz] and / or ALNA[Trz].

[0303] In one embodiment, at least one nucleoside of the modified oligonucleotide comprises a modified nucleic acid base. In one embodiment, the modified nucleic acid base is 5-methylcytosine.

[0304] In one embodiment, the modified oligonucleotide is a gapmer and comprises the following segments: a) a gap segment consisting of linked deoxynucleosides, b) a 5' wing segment consisting of linked nucleosides, and c) a 3' wing segment consisting of linked nucleosides. The gap segment is located between the 5' wing segment and the 3' wing segment, and each nucleoside of each wing segment comprises a modified sugar such as a 2'-O-methyl modified sugar, a 2'-O-methoxyethyl modified sugar, or a bicyclic sugar.

[0305] In one embodiment, the modified oligonucleotide is a gapmer and comprises the following segments: a) a gap segment comprising a nucleoside containing one or two modified sugars such as 2'-O-methyl modified sugars or 2'-O-methoxyethyl modified sugars and other nucleosides without modified sugars, b) a 5' wing segment comprising linked nucleosides, and c) a 3' wing segment comprising linked nucleosides. The gap segment is located between the 5' wing segment and the 3' wing segment, and each nucleoside of each wing segment contains a modified sugar such as 2'-O-methyl modified sugars, 2'-O-methoxyethyl sugars or bicyclic sugars.

[0306] In a certain embodiment, the modified oligonucleotide is a gapmer and comprises the following fragments: a) a gap fragment having 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 nucleosides, wherein the above nucleosides are exclusively nucleosides without modified sugars, or a gap fragment comprising 1 or 2 nucleosides containing modified sugars such as 2'-O-methyl modified sugars or 2'-O-methoxyethyl modified sugars and other nucleosides without modified sugars, b) a 5' flanking fragment having 2, 3, 4, 5, 6 or 7 nucleosides, and c) a 3' flanking fragment having 2, 3, 4, 5, 6, 7 or 8 nucleosides.

[0307] Here, the gap fragment is located between the 5' flanking fragment and the 3' flanking fragment, each nucleoside of each flanking fragment contains a 2'-O-methyl modified sugar, a 2'-O-methoxyethyl sugar or a bicyclic sugar, each internucleoside bond of the above-mentioned modified oligonucleotide contains a thiophosphate bond, and part or all of the cytosine in the above-mentioned modified oligonucleotide can be 5'-methylcytosine.

[0308] In one embodiment, the modified oligonucleotide is a gapmer, and the number of nucleosides constituting the gapmer is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

[0309] In one embodiment, the modified oligonucleotide is a gapmer, and the sugar portion of the nucleoside contains 2'-O-methyl, 2'-O-methoxyethyl and / or 4'-(CH2) n -O-2' crosslink (where n is 1 or 2).

[0310] In certain embodiments, the modified oligonucleotide is a gapmer and the sugar portion of the nucleoside comprises LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz] and / or ALNA[Trz].

[0311] In a certain embodiment, the modified oligonucleotide is a gapmer, which is a modified oligonucleotide comprising 12 to 30 residues and has a nucleic acid base sequence comprising a modified oligonucleotide, wherein the modified oligonucleotide is a nucleic acid base sequence comprising at least 8 consecutive nucleic acid base sequences that are complementary to equal length portions of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list at positions 126 to 147, 232 to 248, 1306 to 1325, or 1472 to 1495 from the 5' end. In a certain embodiment, the modified oligonucleotide is a modified oligonucleotide composed of 12 to 30 residues and has a nucleic acid base sequence comprising the modified oligonucleotide, which is a nucleic acid base sequence comprising at least 9, 10, 11 or 12 consecutive nucleic acid base sequences that are complementary to equal length portions of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list at positions 126 to 147, 232 to 248, 1306 to 1325 or 1472 to 1495 from the 5' end.

[0312] In a certain embodiment, the modified oligonucleotide is a gapmer, which is a nucleic acid base sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleic acid base sequences that are complementary to an equal length portion of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from the 5' end at positions 126 to 147, and is a modified oligonucleotide within 30 residues.

[0313] In a certain embodiment, the modified oligonucleotide is a gapmer, which is a nucleic acid base sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 consecutive nucleic acid base sequences that are complementary to the equal length portion of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from positions 232 to 248 from the 5' end, and is a modified oligonucleotide within 30 residues.

[0314] In a certain embodiment, the modified oligonucleotide is a gapmer, which is a nucleic acid base sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive nucleic acid base sequences that are complementary to the equal length portion of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from the 5' end at positions 1306 to 1325, and is a modified oligonucleotide within 30 residues.

[0315] In a certain embodiment, the modified oligonucleotide is a gapmer, which is a nucleic acid base sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 consecutive nucleic acid base sequences that are complementary to an equal length portion of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from positions 1472 to 1495 from the 5' end, and is a modified oligonucleotide within 30 residues.

[0316] In a certain embodiment, the modified oligonucleotide is a gapmer of a modified oligonucleotide consisting of 12 to 30 residues, comprising a nucleic acid base sequence complementary to an equal length portion of nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from positions 126 to 147 from the 5' end, and is a modified oligonucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to sequence number 1 in the sequence list in the equal length portion. In another embodiment, it is 12 to 29 residues, 12 to 28 residues, 12 to 27 residues, 12 to 26 residues, 12 to 25 residues, 12 to 24 residues, 12 to 23 residues, 12 to 22 residues, 12 to 21 residues, 12 to 20 residues, 12 to 19 residues, 12 to 18 residues, 12 to 17 residues, 12 to 16 residues, 12 to 15 residues, 12 to 14 residues, 13 to 29 residues, 13 to 28 residues, 13 to 27 residues, 13 to 26 residues, 13 to 25 residues, 13 to 24 residues, 13 to 23 residues, 13 to 22 residues, 13-21 residues, 13-20 residues, 13-19 residues, 13-18 residues, 13-17 residues, 13-16 residues, 13-15 residues, 13-14 residues, 14-29 residues, 14-28 residues, 14-27 residues, 14-26 residues, 14-25 residues, 14-24 residues, 14-23 residues, 14-22 residues, 14-21 residues, 14-20 residues, 14-19 residues, 14-18 residues, 14-17 residues, 14-16 residues, 14-15 residues of the above modified oligonucleotides.

[0317] In a certain embodiment, the modified oligonucleotide is a gapmer of a modified oligonucleotide consisting of 12 to 30 residues, comprising a nucleic acid base sequence complementary to an equal length portion of nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from positions 233 to 248 from the 5' end, and is a modified oligonucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to sequence number 1 in the sequence list in the equal length portion. In another embodiment, it is 12 to 29 residues, 12 to 28 residues, 12 to 27 residues, 12 to 26 residues, 12 to 25 residues, 12 to 24 residues, 12 to 23 residues, 12 to 22 residues, 12 to 21 residues, 12 to 20 residues, 12 to 19 residues, 12 to 18 residues, 12 to 17 residues, 12 to 16 residues, 12 to 15 residues, 12 to 14 residues, 13 to 29 residues, 13 to 28 residues, 13 to 27 residues, 13 to 26 residues, 13 to 25 residues, 13 to 24 residues, 13 to 23 residues, 13 to 22 residues, 13-21 residues, 13-20 residues, 13-19 residues, 13-18 residues, 13-17 residues, 13-16 residues, 13-15 residues, 13-14 residues, 14-29 residues, 14-28 residues, 14-27 residues, 14-26 residues, 14-25 residues, 14-24 residues, 14-23 residues, 14-22 residues, 14-21 residues, 14-20 residues, 14-19 residues, 14-18 residues, 14-17 residues, 14-16 residues, 14-15 residues of the above modified oligonucleotides.

[0318] In a certain embodiment, the modified oligonucleotide is a gapmer of a modified oligonucleotide consisting of 12 to 30 residues, which comprises a nucleic acid base sequence complementary to an equal length portion of nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from positions 1309 to 1323 from the 5' end, and is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to sequence number 1 in the sequence list in the equal length portion. The modified oligonucleotide is a gapmer of a modified oligonucleotide consisting of 12 to 30 residues, which comprises a sequence in the sequence list. In another embodiment, it is 12 to 29 residues, 12 to 28 residues, 12 to 27 residues, 12 to 26 residues, 12 to 25 residues, 12 to 24 residues, 12 to 23 residues, 12 to 22 residues, 12 to 21 residues, 12 to 20 residues, 12 to 19 residues, 12 to 18 residues, 12 to 17 residues, 12 to 16 residues, 12 to 15 residues, 12 to 14 residues, 13 to 29 residues, 13 to 28 residues, 13 to 27 residues, 13 to 26 residues, 13 to 25 residues, 13 to 24 residues, 13 to 23 residues, 13 to 22 residues, 13-21 residues, 13-20 residues, 13-19 residues, 13-18 residues, 13-17 residues, 13-16 residues, 13-15 residues, 13-14 residues, 14-29 residues, 14-28 residues, 14-27 residues, 14-26 residues, 14-25 residues, 14-24 residues, 14-23 residues, 14-22 residues, 14-21 residues, 14-20 residues, 14-19 residues, 14-18 residues, 14-17 residues, 14-16 residues, 14-15 residues of the above modified oligonucleotides.

[0319] In a certain embodiment, the modified oligonucleotide is a gapmer of a modified oligonucleotide consisting of 12 to 30 residues, comprising a nucleic acid base sequence complementary to an equal length portion of nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from positions 1472 to 1495 from the 5' end, and is a modified oligonucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to sequence number 1 in the sequence list in the equal length portion. In another embodiment, it is 12 to 29 residues, 12 to 28 residues, 12 to 27 residues, 12 to 26 residues, 12 to 25 residues, 12 to 24 residues, 12 to 23 residues, 12 to 22 residues, 12 to 21 residues, 12 to 20 residues, 12 to 19 residues, 12 to 18 residues, 12 to 17 residues, 12 to 16 residues, 12 to 15 residues, 12 to 14 residues, 13 to 29 residues, 13 to 28 residues, 13 to 27 residues, 13 to 26 residues, 13 to 25 residues, 13 to 24 residues, 13 to 23 residues, 13 to 22 residues, 13-21 residues, 13-20 residues, 13-19 residues, 13-18 residues, 13-17 residues, 13-16 residues, 13-15 residues, 13-14 residues, 14-29 residues, 14-28 residues, 14-27 residues, 14-26 residues, 14-25 residues, 14-24 residues, 14-23 residues, 14-22 residues, 14-21 residues, 14-20 residues, 14-19 residues, 14-18 residues, 14-17 residues, 14-16 residues, 14-15 residues of the above modified oligonucleotides. At this time, the oligonucleotide contained in the 5' flanking fragment and / or the 3' flanking fragment contains at least one nucleoside, which contains at least one modified sugar selected from GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz] and ALNA[Trz], and may also contain a sugar modified by 2'-O-methoxyethyl and / or a sugar modified by 2'-O-methyl.

[0320] In a certain embodiment, the modified oligonucleotide is a gapmer of a modified oligonucleotide comprising 12 to 30 residues, which is a nucleic acid base sequence complementary to an equal length portion of the nucleic acid bases of the mature mRNA of DUX4 of sequence number 1 in the sequence list from the 1480th to 1495th position from the 5' end, and is composed of a nucleic acid base sequence having a complementary base to the base at the 1480th position from the 5' end of the nucleic acid base of sequence number 1 at the 3' end, and is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to sequence number 1 in the sequence list in the equal length portion. In another embodiment, the polypeptide comprises 12 to 29 residues, 12 to 28 residues, 12 to 27 residues, 12 to 26 residues, 12 to 25 residues, 12 to 24 residues, 12 to 23 residues, 12 to 22 residues, 12 to 21 residues, 12 to 20 residues, 12 to 19 residues, 12 to 18 residues, 12 to 17 residues, 12 to 16 residues, 12 to 15 residues, 12 to 14 residues, 13 to 29 residues, 13 to 28 residues, 13 to 27 residues, 13 to 26 residues, 13 to 25 residues, 13 to 24 residues, 13 to 23 residues, 13 to The modified oligonucleotides consist of 22 residues, 13-21 residues, 13-20 residues, 13-19 residues, 13-18 residues, 13-17 residues, 13-16 residues, 13-15 residues, 13-14 residues, 14-29 residues, 14-28 residues, 14-27 residues, 14-26 residues, 14-25 residues, 14-24 residues, 14-23 residues, 14-22 residues, 14-21 residues, 14-20 residues, 14-19 residues, 14-18 residues, 14-17 residues, 14-16 residues, and 14-15 residues.

[0321] In one embodiment, the modified oligonucleotide provided in the present specification is a gapmer, targeting any one of the following regions of SEQ ID NO: 1 in the sequence listing: 126 to 141, 126 to 143, 127 to 142, 127 to 143, 127 to 144, 127 to 146, 128 to 143, 128 to 144, 128 to 147, 232 to 245, 232 to 247, 23 3-246, 233-247, 233-248, 234-247, 234-248, 1304-1323, 1306-1321, 1306-1324, 1307-1323, 1307-1324, 1307-1325, 1307-1326, 1308-1323, 1308-1324, 1308-1322, 1308-1323 5. 1309-1323, 1309-1324, 1309-1325, 1309-1322, 1310-1323, 1310-1324, 1472-1485, 1472-1486, 1472-1487, 1472-1488, 1473-1487, 1473-1488, 1473-1489, 1474-1488, 1474 1480-1499. In one embodiment, the modified oligonucleotide provided herein is a gapmer, targeting any one of the following regions of SEQ ID NO: 1 in the sequence listing: 128-143, 232-247, 233-248, 1309-1323, and 1480-1495 from the 5' end.

[0322] In certain embodiments, the modified oligonucleotide provided in the present specification is a gapmer having a nucleic acid base sequence, and the nucleic acid base sequence includes a complementary region containing at least 8 consecutive nucleic acid bases complementary to the target region. In certain embodiments, the modified oligonucleotide provided in the present specification has a nucleic acid base sequence, and the nucleic acid base sequence includes a complementary region containing at least 8 consecutive nucleic acid bases complementary to the target region, and the target region is 126 to 141, 126 to 143, 127 to 142, 127 to 143, 127 to 144, 127 to 146, 128 to 143, 128 to 144, 128 from the 5' end of sequence number 1 in the sequence list. ~147、232~245、232~247、233~246、233~247、233~248、234~247、234~248、1304~1323、1306~1321、1306~1324、1307~1323、1307~1324、1307~1325、1307~1326、1308~1323、1308~1324、1308 ~1322、1308~1325、1309~1323、1309~1324、1309~1325、1309~1322、1310~1323、1310~1324、1472~1485、1472~1486、1472~1487、1472~1488、1473~1487、1473~1488、1473~1489、1474~148 8. Position 1474-1489, 1475-1490, 1476-1490, 1476-1491, 1476-1495, 1477-1495, 1478-1496, 1479-1495, 1479-1496, 1479-1498, 1480-1494, 1480-1495, 1480-1496, 1480-1497 or 1480-1499 is the target. In a certain embodiment, the modified oligonucleotide provided in the present specification is a gap body having a nucleic acid base sequence comprising a complementary region containing at least 8 consecutive nucleic acid bases that are complementary to the target region, and the target region is targeted at positions 128 to 143, 232 to 247, 233 to 248, 1309 to 1323 or 1480 to 1495 from the 5' end of sequence number 1 in the sequence list.

[0323] In certain embodiments, the modified oligonucleotide provided in the present specification is a gapmer having a nucleic acid base sequence, and the nucleic acid base sequence includes a complementary region containing at least 10 consecutive nucleic acid bases complementary to the target region. In certain embodiments, the modified oligonucleotide provided in the present specification has a nucleic acid base sequence, and the nucleic acid base sequence includes a complementary region containing at least 10 consecutive nucleic acid bases complementary to the target region, and the target region is 126 to 141, 126 to 143, 127 to 142, 127 to 143, 127 to 144, 127 to 146, 128 to 143, 128 to 144, 128 to 146, 129 to 150, 129 to 151, 129 to 152, 129 to 153, 129 to 154, 129 to 155, 129 to 156, 129 to 157, 129 to 158, 129 to 159, 129 to 160, 129 to 161, 129 to 162, 129 to 163, 129 to 164, 129 to 165, 129 to 166, 129 to 167, 129 to 168, 129 to 169, 129 to 169, 129 to 169, 129 to 169, 129 to 8~147、232~245、232~247、233~246、233~247、233~248、234~247、234~248、1304~1323、1306~1321、1306~1324、1307~1323、1307~1324、1307~1325、1307~1326、1308~1323、1308~1324、130 8~1322、1308~1325、1309~1323、1309~1324、1309~1325、1309~1322、1310~1323、1310~1324、1472~1485、1472~1486、1472~1487、1472~1488、1473~1487、1473~1488、1473~1489、1474~148 8. Position 1474-1489, 1475-1490, 1476-1490, 1476-1491, 1476-1495, 1477-1495, 1478-1496, 1479-1495, 1479-1496, 1479-1498, 1480-1494, 1480-1495, 1480-1496, 1480-1497 or 1480-1499 is the target. In a certain embodiment, the modified oligonucleotide provided in the present specification is a gap body having a nucleic acid base sequence comprising a complementary region containing at least 10 consecutive nucleic acid bases that are complementary to the target region, and the target region is targeted at positions 128 to 143, 232 to 247, 233 to 248, 1309 to 1323 or 1480 to 1495 from the 5' end of sequence number 1 in the sequence list.

[0324] In certain embodiments, the modified oligonucleotide provided in the present specification is a gapmer having a nucleic acid base sequence, and the nucleic acid base sequence comprises a complementary region comprising at least 12 consecutive nucleic acid bases complementary to the target region. In certain embodiments, the modified oligonucleotide provided in the present specification has a nucleic acid base sequence, and the nucleic acid base sequence comprises a complementary region comprising at least 12 consecutive nucleic acid bases complementary to the target region, and the target region is 126 to 141, 126 to 143, 127 to 142, 127 to 143, 127 to 144, 127 to 146, 128 to 143, 128 to 144, 128 to 146, 129 to 150, 129 to 151, 129 to 152, 129 to 153, 129 to 154, 129 to 155, 129 to 156, 129 to 157, 129 to 158, 129 to 159, 129 to 160, 129 to 161, 129 to 162, 129 to 163, 129 to 164, 129 to 165, 129 to 166, 129 to 167, 129 to 168, 129 to 169, 129 to 169, 129 to 169, 129 to 169, 129 to 8~147、232~245、232~247、233~246、233~247、233~248、234~247、234~248、1304~1323、1306~1321、1306~1324、1307~1323、1307~1324、1307~1325、1307~1326、1308~1323、1308~1324、130 8~1322、1308~1325、1309~1323、1309~1324、1309~1325、1309~1322、1310~1323、1310~1324、1472~1485、1472~1486、1472~1487、1472~1488、1473~1487、1473~1488、1473~1489、1474~148 8. Position 1474-1489, 1475-1490, 1476-1490, 1476-1491, 1476-1495, 1477-1495, 1478-1496, 1479-1495, 1479-1496, 1479-1498, 1480-1494, 1480-1495, 1480-1496, 1480-1497 or 1480-1499 is the target. In a certain embodiment, the modified oligonucleotide provided in the present specification is a gap body having a nucleic acid base sequence comprising a complementary region containing at least 12 consecutive nucleic acid bases that are complementary to the target region, and the target region is targeted at positions 128 to 143, 232 to 247, 233 to 248, 1309 to 1323 or 1480 to 1495 from the 5' end of sequence number 1 in the sequence list.

[0325] In certain embodiments, the modified oligonucleotide provided in the present specification is a gapmer having a nucleic acid base sequence, and the nucleic acid base sequence comprises a complementary region comprising at least 14 consecutive nucleic acid bases complementary to the target region. In certain embodiments, the modified oligonucleotide provided in the present specification has a nucleic acid base sequence, and the nucleic acid base sequence comprises a complementary region comprising at least 14 consecutive nucleic acid bases complementary to the target region, and the target region is 126 to 141, 126 to 143, 127 to 142, 127 to 143, 127 to 144, 127 to 146, 128 to 143, 128 to 144, 128 to 146, 129 to 150, 129 to 151, 129 to 152, 129 to 153, 129 to 154, 129 to 155, 129 to 156, 129 to 157, 129 to 158, 129 to 159, 129 to 160, 129 to 161, 129 to 162, 129 to 163, 129 to 164, 129 to 165, 129 to 166, 129 to 167, 129 to 168, 129 to 169, 129 to 169, 129 to 169, 129 to 169, 129 to 8~147、232~245、232~247、233~246、233~247、233~248、234~247、234~248、1304~1323、1306~1321、1306~1324、1307~1323、1307~1324、1307~1325、1307~1326、1308~1323、1308~1324、130 8~1322、1308~1325、1309~1323、1309~1324、1309~1325、1309~1322、1310~1323、1310~1324、1472~1485、1472~1486、1472~1487、1472~1488、1473~1487、1473~1488、1473~1489、1474~148 8. Position 1474-1489, 1475-1490, 1476-1490, 1476-1491, 1476-1495, 1477-1495, 1478-1496, 1479-1495, 1479-1496, 1479-1498, 1480-1494, 1480-1495, 1480-1496, 1480-1497 or 1480-1499 is the target. In a certain embodiment, the modified oligonucleotide provided in the present specification is a gap body having a nucleic acid base sequence comprising a complementary region containing at least 14 consecutive nucleic acid bases that are complementary to the target region, and the target region is targeted at positions 128 to 143, 232 to 247, 233 to 248, 1309 to 1323 or 1480 to 1495 from the 5' end of sequence number 1 in the sequence list.

[0326] In one embodiment, the modified oligonucleotide is a gapmer, which is composed of nucleic acid bases 126 to 141, 126 to 143, 127 to 142, 127 to 143, 127 to 144, 127 to 146, 128 to 143, 128 to 144, 128 to 147, 232 to 245, 232 to 247, 233 to 246, 233-247, 233-248, 234-247, 234-248, 1304-1323, 1306-1321, 1306-1324, 1307-1323, 1307-1324, 1307-1325, 1307-1326, 1308-1323, 1308-1324, 1308-1322, 1308-1325, 1309-1323, 1309-1324, 1309-1325, 1309-1322, 1310-1323, 1310-1324, 1472-1485, 1472-1486, 1472-1487, 1472-1488, 1473-1487, 1473-1488, 1473-1489, 1474-1488, 1474-1489, 1475-1490, 1491-1492 In one embodiment, the modified oligonucleotide is a gapmer, which is a modified oligonucleotide composed of a nucleic acid base sequence complementary to the equal length portion of positions 128 to 143, 232 to 247, 233 to 248, 1309 to 1323, and 1480 to 1495 of the mature mRNA of DUX4 of SEQ ID NO: 1 in the sequence listing.

[0327] In one embodiment, the modified oligonucleotide is a gapmer, which is a modified oligonucleotide consisting of a nucleic acid base sequence recorded in any one of sequence numbers 2 to 4, 7 to 64, 69 to 97, and 102 to 112 of the sequence list. In one embodiment, the modified oligonucleotide is a gapmer, which is a nucleic acid base sequence recorded in any one of sequence numbers 2 to 4, 75, and 78 of the sequence list.

[0328] In one embodiment, the modified oligonucleotide is a gapmer, which is a modified oligonucleotide described in any one of compound numbers 1 to 112, 114 to 132, and 137 to 246. In another embodiment, the modified oligonucleotide is a gapmer, which is a modified oligonucleotide described in any one of compound numbers 1 to 3, 123, 157, 204, 221, and 231.

[0329] In the present specification, in symbols such as "Gls" representing nucleotides, the abbreviations shown on the left side refer to the nucleic acid base portion, the abbreviations shown in the center position refer to the sugar portion, and the abbreviations shown on the right side refer to the pattern of internucleoside bonds.

[0330] In certain embodiments, the modified oligonucleotide is a gapmer having the formula:

[0331] GlsMlsMlsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMlsGlsGl

[0332] The modified oligonucleotide represented by

[0333] In the formula,

[0334] Each nucleic acid base is represented by the following symbols:

[0335] A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine,

[0336] The sugar moieties are represented by the following symbols:

[0337] l=LNA, d=2'-deoxyribose,

[0338] The internucleoside bonds are represented by the following symbols:

[0339] s = phosphorothioate.

[0340] In one embodiment, the modified oligonucleotide is a gapmer.

[0341] is the following formula:

[0342] GmsMmsMmsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMmsGmsGm

[0343] The modified oligonucleotide represented by

[0344] In the formula,

[0345] Each nucleic acid base is represented by the following symbols:

[0346] A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine,

[0347] The sugar moieties are represented by the following symbols:

[0348] m=ALNA[Ms], d=2'-deoxyribose,

[0349] The internucleoside bonds are represented by the following symbols:

[0350] s = phosphorothioate.

[0351] In certain embodiments, the modified oligonucleotide is a gapmer having the formula:

[0352] GmsMmsAmsGdsTdsTdsCdsTdsCdsCdsGdsCdsGmsGmsTm

[0353] The modified oligonucleotide represented by

[0354] In the formula,

[0355] Each nucleic acid base is represented by the following symbols:

[0356] A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine,

[0357] The sugar moieties are represented by the following symbols:

[0358] m=ALNA[Ms], d=2'-deoxyribose,

[0359] The internucleoside bonds are represented by the following symbols:

[0360] s = phosphorothioate.

[0361] In certain embodiments, the modified oligonucleotide is a gapmer having the formula:

[0362] MlsGlsAlsGdsAdsTdsTdsCdsCdsCdsGdsCdsCdsGlsGlsTl

[0363] The modified oligonucleotide represented by

[0364] In the formula,

[0365] Each nucleic acid base is represented by the following symbols:

[0366] A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine,

[0367] The sugar moieties are represented by the following symbols:

[0368] l=LNA, d=2'-deoxyribose,

[0369] The internucleoside bonds are represented by the following symbols:

[0370] s = phosphorothioate.

[0371] Certain embodiments provide a method for reducing the level of DUX4 mRNA and / or DUX4 protein (eg, intramuscular level) in an animal with a DUX4-related disease, comprising administering to the animal an effective amount of a modified oligonucleotide directed against DUX4.

[0372] Certain embodiments provide a method for reducing the level of DUX4 mRNA and / or DUX4 protein (e.g., intramuscular level) in an animal with FSHD, comprising administering to the animal an effective amount of a modified oligonucleotide directed against DUX4. In addition, certain embodiments provide a method for reducing muscle disorders and / or improving motor function in an animal with FSHD, comprising administering to the animal an effective amount of a modified oligonucleotide directed against DUX4.

[0373] In addition, one embodiment provides a method for treating, ie, preventing, ameliorating, or alleviating FSHD, comprising administering to an animal an effective amount of a modified oligonucleotide targeting DUX4.

[0374] A certain embodiment provides a method for preventing, improving, and alleviating various symptoms of FSHD (e.g., facial muscle weakness, ptosis, inability to whistle, reduced facial expression changes, sad or angry facial expressions, difficulty in speech pronunciation, shoulder muscle weakness (deformities such as winged scapula and sloping shoulders), lower limb weakness, hearing loss, and heart disease), comprising administering to an animal an effective amount of a modified oligonucleotide targeting DUX4.

[0375] In one embodiment, a method for reducing the level of mRNA and / or protein produced by fusion of DUX4 gene and IGH gene in an animal with B-cell acute lymphoblastic leukemia or the like (e.g., the level in B cells in the blood) comprises administering to the animal an effective amount of a modified oligonucleotide against DUX4. In addition, in one embodiment, a method for treating, preventing, ameliorating, or alleviating B-cell acute lymphoblastic leukemia or the like is provided, comprising administering to the animal an effective amount of a modified oligonucleotide against DUX4.

[0376] In one embodiment, a method for reducing the level of mRNA and / or protein produced by fusion of the DUX4 gene and the CIC gene (e.g., the level in the sarcoma) in an animal with a differentiated round cell sarcoma or the like is provided, comprising administering to the animal an effective amount of a modified oligonucleotide against DUX4. In addition, in one embodiment, a method for treating, that is, preventing, ameliorating, or alleviating a differentiated round cell sarcoma or the like is provided, comprising administering to the animal an effective amount of a modified oligonucleotide against DUX4.

[0377] In one embodiment, a method for reducing the level of mRNA and / or protein produced by the fusion of the DUX4 gene and the EWSR1 gene (e.g., the level in the sarcoma) in an animal with embryonic rhabdomyosarcoma or the like is provided, comprising administering to the animal a modified oligonucleotide directed against DUX4. In addition, in one embodiment, a method for treating, preventing, ameliorating, or alleviating embryonic rhabdomyosarcoma or the like is provided, comprising administering to the animal an effective amount of a modified oligonucleotide directed against DUX4.

[0378] A certain embodiment provides a method for treating, preventing, improving, and alleviating a DUX4-related disease with few side effects, the method comprising administering a modified oligonucleotide for DUX4 to an animal. In a certain embodiment, as side effects, injection site reactions, abnormal liver function tests, abnormal renal function, hepatotoxicity (abnormal findings in pathological histology: degeneration and necrosis of hepatocytes, hepatocyte hypertrophy, etc.), nephrotoxicity (abnormal findings in pathological histology, etc.), central nervous system abnormalities, myopathy, and fatigue. For example, an increase in the levels of ALT, AST, γ-GTP, GLDH, ALP (alkaline phosphatase), or TBA (total bileacids) in the blood may indicate hepatotoxicity or abnormal liver function. For example, an increase in bilirubin may indicate hepatotoxicity or abnormal liver function. In addition, an increase in urine protein, an increase in creatinine or UN in the blood may indicate renal toxicity or abnormal renal function.

[0379] In one embodiment, there is provided an application of any compound described in this specification or a pharmaceutical composition containing the compound for manufacturing a pharmaceutical for use in any of the treatment methods described in this specification. For example, in one embodiment, there is provided an application of a compound described in this specification or a pharmaceutical composition containing the compound for manufacturing a pharmaceutical for treating, i.e., treating, preventing, delaying or improving FSHD. In one embodiment, there is provided an application of a compound described in this specification or a pharmaceutical composition containing the compound for manufacturing a pharmaceutical for inhibiting DUX4 expression, and for treating, i.e., treating, preventing, delaying or improving DUX4-related diseases and / or their symptoms. In one embodiment, there is provided an application of a compound described in this specification or a pharmaceutical composition containing the compound for manufacturing a pharmaceutical for reducing the expression of DUX4 in animals. In one embodiment, there is provided an application of a compound described in this specification or a pharmaceutical composition containing the compound for manufacturing a pharmaceutical for preferentially reducing the level of DUX4-FLmRNA (e.g., intramuscular level) in animals and reducing myotonia. In one embodiment, there is provided an application of a compound described in this specification or a pharmaceutical composition containing the compound for manufacturing a pharmaceutical for treating animals with FSHD. In one embodiment, there is provided a use of a compound described in the present specification or a pharmaceutical composition containing the compound for manufacturing a pharmaceutical for treating one or more symptoms and consequences associated with the occurrence of FSHD, including muscle stiffness, myotonia, facial muscle weakness, ptosis, inability to whistle, reduced facial expression changes, depressed or angry facial expressions, speech articulation difficulties, shoulder muscle weakness (deformities such as winged scapula and sloping shoulders), lower limb weakness, hearing loss, and heart disease. In one embodiment, there is provided a use of a compound described in the present specification or a pharmaceutical composition containing the compound for manufacturing a pharmaceutical for inhibiting the expression of DUX4 protein by inducing transcription of the DUX4 gene, translation of DUX4 mRNA, or cleavage of DUX4 mRNA.

[0380] In one embodiment, a kit is provided, which is a kit for treating, i.e., treating, preventing or ameliorating FSHD as described in this specification, comprising a) a compound described in this specification and any b) a further agent or therapy described in this specification. The kit may further comprise instructions or a label for using the kit for the purpose of treating, i.e., treating, preventing or ameliorating FSHD.

[0381] In one embodiment, there is provided an application of any compound described in this specification or a pharmaceutical composition containing the compound for manufacturing a pharmaceutical for use in any of the treatment methods described in this specification. For example, in one embodiment, there is provided an application of a compound described in this specification or a pharmaceutical composition containing the compound for manufacturing a pharmaceutical for treating, i.e., treating, improving or preventing FSHD. In one embodiment, there is provided an application of a compound described in this specification or a pharmaceutical composition containing the compound for manufacturing a pharmaceutical for inhibiting DUX4 expression, and treating, i.e., treating, preventing, delaying or improving DUX4-related diseases and / or their symptoms. In one embodiment, there is provided an application of a compound described in this specification or a pharmaceutical composition containing the compound for manufacturing a pharmaceutical for reducing the expression of DUX4 in animals. In one embodiment, there is provided an application of a compound described in this specification or a pharmaceutical composition containing the compound for manufacturing a pharmaceutical for preferentially reducing the level of DUX4-FLmRNA (e.g., intramuscular level) in animals and reducing myotonia. In one embodiment, there is provided an application of a compound described in this specification or a pharmaceutical composition containing the compound for manufacturing a pharmaceutical for treating animals with FSHD. In one embodiment, a compound described in the present specification or a pharmaceutical composition containing the compound is provided for treating one or more symptoms and results related to the occurrence of FSHD, including muscle stiffness, myotonia, facial muscle weakness, ptosis, inability to whistle, reduced facial expression changes, depressed or angry facial expressions, speech articulation difficulties, shoulder muscle weakness (deformities such as winged scapula and sloping shoulders), lower limb weakness, hearing loss and heart disease. In one embodiment, a modified oligonucleotide having a nucleic acid base sequence consisting of the nucleic acid base sequence of sequence numbers 2 to 4, 7 to 64, 69 to 97 or 102 to 112 of the sequence listing, or a compound of compound number 1 to 112, 114 to 132 or 137 to 246 is provided.

[0382] In certain embodiments, when described in the 5' to 3' direction, the modified oligonucleotide has a nucleic acid base sequence of the reverse complementary strand of the target fragment of the target nucleic acid to which it is targeted. In certain such embodiments, when described in the 5' to 3' direction, the modified oligonucleotide has a nucleic acid base sequence of the reverse complementary strand of the target fragment of the target nucleic acid to which it is targeted.

[0383] In one embodiment, the modified oligonucleotide of the present specification targeting DUX4 is 12 to 30 nucleotides in length. In other words, in some embodiments, the modified oligonucleotide is a nucleic acid base composed of 12 to 30 residues linked together. In other embodiments, the modified oligonucleotide comprises 12-29 residues, 12-28 residues, 12-27 residues, 12-26 residues, 12-25 residues, 12-24 residues, 12-23 residues, 12-22 residues, 12-21 residues, 12-20 residues, 12-19 residues, 12-18 residues, 12-17 residues, 12-16 residues, 12-15 residues, 12-14 residues, 13-29 residues, 13-28 residues, 13-27 residues, 13-26 residues, 13-25 residues, 13-24 residues, 13-23 residues, 13-22 A modified oligonucleotide composed of nucleic acid bases of 13 to 21 residues, 13 to 20 residues, 13 to 19 residues, 13 to 18 residues, 13 to 17 residues, 13 to 16 residues, 13 to 15 residues, 13 to 14 residues, 14 to 29 residues, 14 to 28 residues, 14 to 27 residues, 14 to 26 residues, 14 to 25 residues, 14 to 24 residues, 14 to 23 residues, 14 to 22 residues, 14 to 21 residues, 14 to 20 residues, 14 to 19 residues, 14 to 18 residues, 14 to 17 residues, 14 to 16 residues, and 14 to 15 residues. In some such embodiments, the modified oligonucleotide comprises a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 residues connected to form a nucleic acid base, or a modified oligonucleotide consisting of a range determined by any two of the above values. In a certain embodiment, any of these lengths of modified oligonucleotides comprises at least 8 consecutive nucleic acid bases of any one of the nucleic acid base sequences of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 or at least 19 residues (e.g., SEQ ID NOs. 2 to 4, 7 to 64, 69 to 97 or 102 to 112 of the sequence list) of any nucleic acid base sequence described in the present specification.

[0384] The length of antisense compounds such as antisense oligonucleotides can be increased or decreased and / or mismatches can be introduced without causing the activity to disappear. For example, according to Woolfetal. (Proc. Natl. Acad. Sci. USA 89: 7305-7309, 1992), a series of antisense oligonucleotides of 13 to 25 nucleic acid bases in length were reported to induce the cleavage of target RNA in the oocyte injection model. Although the degree of 25 nucleic acid bases of antisense oligonucleotides with 8 or 11 mismatches near the end of the antisense oligonucleotide was less than that of antisense oligonucleotides without mismatches, they were able to guide the specific cleavage of target mRNA. Similarly, it was reported that antisense oligonucleotides of 13 nucleic acid bases including antisense oligonucleotides with 1 or 3 mismatches can be used to achieve target-specific cleavage.

[0385] According to Gautschi et al. (J. Natl. Cancer Inst. 93: 463-471, March 2001), an oligonucleotide having 100% complementarity to bcl-2 mRNA and 3 mismatches to bcl-xL mRNA was reported to have the ability to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, it was reported that the oligonucleotide exhibited a strong antitumor activity in vivo.

[0386] Target nucleic acids, target regions and nucleotide sequences

[0387] The nucleotide sequence encoding DUX4 is not limited, but the following sequences can be mentioned.

[0388] · The sequence described in GenBank Accession No. NM_001293798.2 (incorporated in the present specification as SEQ ID NO: 1 in the sequence listing). The splice variant of SEQ ID NO: 1 in the sequence listing is also referred to as DUX4-FL1 or mature mRNA of DUX4.

[0389] · The sequence described in GenBank Accession No. NM_001306068.2 (incorporated into the present specification as SEQ ID NO: 5 in the sequence listing). The splice variant of SEQ ID NO: 5 in the sequence listing is also referred to as DUX4-FL2.

[0390] · The sequence described in GenBank Accession No. NM_001363820.1 (incorporated into the present specification as SEQ ID NO: 6 in the sequence listing). The splice variant of SEQ ID NO: 6 in the sequence listing is also referred to as DUX4-s.

[0391] • SNPs for each of the above splice variants.

[0392] The sequences recorded in the sequence numbers of the sequence listings of the embodiments included in this specification have no relation to any modification of the sugar moiety, the internucleoside bond or the nucleic acid base. Therefore, the modified oligonucleotides specified by the sequence numbers of the sequence listings may independently contain one or more modifications of the sugar moiety, the internucleoside bond or the nucleic acid base. The modified oligonucleotides recorded in the compound number represent a combination of a nucleic acid base sequence and a motif.

[0393] In certain embodiments, the target region is a region determined in the structure of the target nucleic acid. For example, the target region may include one or more of a 3'UTR, a 5'UTR, an exon, an intron, an exon / intron junction, a coding region, a translation initiation region, a translation termination region, or other determined nucleic acid regions. The region determined in the structure of DUX4 can be obtained from sequence databases such as NCBI by accession number, and such information is incorporated into this specification as a reference. In certain embodiments, the target region may include a sequence from a 5' target site of a certain target fragment in the target region to a 3' target site of other target fragments in the target region.

[0394] Targeting comprises hybridization of modified oligonucleotides, and the result is the determination of at least one target fragment that produces a desired effect. In certain embodiments, the desired effect is a reduction in the level of mRNA target nucleic acid. In certain embodiments, the desired effect is a reduction in the level of protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid.

[0395] The target region may include one or more target fragments. Multiple target fragments within the target region may be repeated. Or they may not be repeated. In certain embodiments, the target fragments within the target region are separated by about 300 or less nucleotides. In certain embodiments, the target fragments within the target region are separated by 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 nucleotides of the target nucleic acid, or about these numbers of nucleotides, or nucleotides below these numbers, or multiple nucleotides within a range determined by any two of the above values. In certain embodiments, the target fragments within the target region are separated by less than 5 or about less than 5 nucleotides of the target nucleic acid. In certain embodiments, the target fragments are continuous. It is intended to be a target region determined by the range of the starting nucleic acid having either the 5' target site or the 3' target site cited in this specification.

[0396] Suitable target fragments can be found in 5'UTR, coding region, 3'UTR, intron, exon or exon / intron junction. Target fragments containing start codon or stop codon are also suitable target fragments. Suitable target fragments can specifically exclude certain structurally defined regions such as start codon or stop codon.

[0397] The determination of appropriate target fragments can include comparisons of sequences of target nucleic acids with other sequences throughout the genome. For example, the BLAST algorithm can be used to determine similar regions between different nucleic acids. This comparison can prevent selection of modified oligonucleotide sequences that can hybridize with sequences other than the selected target nucleic acid (i.e., non-target sequences or sequences that miss the target) in a non-specific manner.

[0398] The activity of the modified oligonucleotides (e.g., as determined by a percentage reduction in the level of the target nucleic acid) within the active target region may be changed. In certain embodiments, a reduction in the level of DUX4 mRNA is an indicator of inhibition of DUX4 protein expression. A reduction in the level of DUX4 protein is also an indicator of inhibition of target mRNA expression. Furthermore, a change in phenotype, such as a reduction in myotonia or a reduction in muscle disorders, can be an indicator of inhibition of expression of DUX4 mRNA and / or protein.

[0399] Hybridization

[0400] In some embodiments, hybridization occurs between a modified oligonucleotide disclosed in the present specification and a DUX4 nucleic acid. The most common mechanism of hybridization is accompanied by hydrogen bonding (e.g., Watson-Crick, Hoogsteen, or anti-Hoogsteen type hydrogen bonding) between complementary nucleic acid bases of nucleic acid molecules. The strength of hybridization can be represented by the melting temperature Tm. Tm refers to the temperature at which 50% of the hybridized double-stranded nucleic acid dissociates into single-stranded nucleic acids. Although it varies depending on the salt conditions of the solution, the length or base sequence of the nucleic acid, etc., the higher the Tm value, the stronger the hybridization.

[0401] Hybridization occurs under a variety of conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the hybridizing nucleic acid molecules.

[0402] Methods for determining whether a sequence can specifically hybridize to a target nucleic acid are well known in the art (Sambrooke and Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., 2001). In one embodiment, the modified oligonucleotide provided herein can specifically hybridize to a DUX4 nucleic acid.

[0403] Complementarity

[0404] The modified oligonucleotide and target nucleic acid are complementary to each other when a sufficient number of nucleobases of the modified oligonucleotide are capable of forming hydrogen bonds with corresponding nucleobases of the target nucleic acid to produce the desired effect (eg, antisense inhibition of a target nucleic acid such as a DUX4 nucleic acid).

[0405] The modified oligonucleotides can hybridize over one or more segments of a DUX4 nucleic acid in such a way that segments present in between or adjacent to them do not participate in the hybridization phenomenon (eg, loop structures, mismatches, or hairpin structures).

[0406] In certain embodiments, the modified oligonucleotides provided herein or specific portions thereof are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to DUX4 nucleic acids, target regions, target fragments or specific portions thereof. In certain embodiments, the modified oligonucleotide is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to a DUX4 nucleic acid, a target region, a target fragment or a defined portion thereof, and comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 or at least 19 consecutive nucleic acid bases of any nucleic acid base sequence of the exemplary modified oligonucleotides described in the present specification (e.g., at least 8 consecutive nucleic acid bases of a nucleic acid base sequence described in any one of SEQ ID NOs. 2, 3, 4, 7 to 64, 69 to 97 or 102 to 112 of the sequence listing). The percentage of complementarity between a modified oligonucleotide and a target nucleic acid can be determined using conventional methods and can be measured over the entire modified oligonucleotide.

[0407] For example, 18 of the 20 nucleic acid bases of the modified oligonucleotide are complementary to the target region, and therefore, the modified oligonucleotide that performs specific hybridization is considered to be equivalent to 90% complementarity. In this example, the remaining non-complementary nucleic acid bases can be clustered or sandwiched with complementary nucleic acid bases, and do not need to be continuous with each other or with complementary nucleic acid bases. The percentage of complementarity between the modified oligonucleotide and the target nucleic acid region can be routinely determined using the BLAST program (basic local alignment search tools) and the PowerBLAST program (Altschul et al., J. Mol. Biol., 1990, 215, 403410; Zhang and Madden, Genome Res., 1997, 7, 649656) known in the art. The percentage of homology, sequence identity or complementarity can be determined, for example, by the GAP program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using the default settings using the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).

[0408] The position of the non-complementary nucleic acid base can be the 5' end of the modified oligonucleotide, or it can be the 3' end. Alternatively, the non-complementary nucleic acid base (can be multiple) can also be a position inside the modified oligonucleotide. When there are more than two non-complementary nucleic acid bases, they can be continuous (i.e. connected) or non-continuous. In one embodiment, the non-complementary nucleic acid base is located in the flanking fragment of the gap body modified oligonucleotide.

[0409] In one embodiment, a modified oligonucleotide that is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleic acid bases long or at most this length contains less than 3, less than 2 or less than 1 non-complementary nucleic acid base (can be multiple) to a target nucleic acid, such as a DUX4 nucleic acid or a specific portion thereof.

[0410] The modified oligonucleotides provided in this specification also include modified nucleic acid bases that are complementary to a portion of the target nucleic acid. When used in this specification, "portion" refers to a determined number of continuous (i.e., connected) nucleic acid bases in a region or fragment of the target nucleic acid. "Portion" may also refer to a determined number of continuous nucleic acid bases of a modified oligonucleotide. In a certain embodiment, the modified oligonucleotide is partially complementary to at least 8 nucleic acid bases of the target fragment. In a certain embodiment, the modified oligonucleotide is partially complementary to at least 10 nucleic acid bases of the target fragment. In a certain embodiment, the modified oligonucleotide is partially complementary to at least 15 nucleic acid bases of the target fragment. It also refers to a modified oligonucleotide that is complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or more nucleic acid bases of the target fragment, or a range determined by any 2 of these values.

[0411] Identity

[0412] The modified oligonucleotide provided in this specification may also have a determined identity percentage to a specific nucleotide sequence, a compound represented by a sequence number or a specific compound number of a sequence table or a portion thereof. When used in this specification, when the modified oligonucleotide has the same nucleic acid base pairing ability, it is identical to the sequence disclosed in this specification. For example, in an RNA containing uracil instead of the thymine of a disclosed DNA sequence, since both uracil and thymine pair with adenine, it is considered to be identical to the DNA sequence. It also refers to the shortened and extended forms of the modified oligonucleotide of this specification and the compounds having different bases to the modified oligonucleotide provided in this specification. Different bases may be adjacent to each other or dispersed throughout the modified oligonucleotide as a whole. The identity percentage of the modified oligonucleotide is calculated according to the number of bases having the same base pairing to the compared sequence.

[0413] In certain embodiments, the modified oligonucleotide or a portion thereof is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to one or more of the exemplary modified oligonucleotides disclosed in the specification or the sequence number of the sequence listing or these portions.

[0414] Modification

[0415] Nucleoside is a composition of base-sugar. The nucleic acid base (also known as base) part of nucleoside is usually a heterocyclic base part. Nucleotide is a nucleoside further comprising a phosphate group covalently bonded to the sugar part of the nucleoside. For nucleosides comprising pentofuranosyl sugars, the phosphate group can be connected to the 2', 3' or 5' hydroxyl part of the sugar. Oligonucleotides are formed by covalent bonding of adjacent nucleosides to each other, forming a straight-chain polymer oligonucleotide. In the oligonucleotide structure, the phosphate group is generally considered to form the internucleoside bond of the oligonucleotide.

[0416] Modifications in modified oligonucleotides include substitutions or changes to internucleoside bonds, sugar moieties, or nucleic acid bases. Modified oligonucleotides are often preferred over natural types for reasons of desired properties such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity.

[0417] Modified internucleoside bonds

[0418] The naturally occurring internucleoside bond of RNA and DNA is a 3'-5' phosphodiester bond. For reasons of desirable properties such as enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases, modified oligonucleotides having one or more modified, i.e., non-natural, internucleoside bonds are often selected over modified oligonucleotides having naturally occurring internucleoside bonds.

[0419] Oligonucleotides with modified internucleoside bonds include internucleoside bonds that maintain phosphorus atoms and internucleoside bonds that do not have phosphorus atoms. As representative phosphorus-containing internucleoside bonds, more than one of phosphodiester, phosphotriester, methylphosphonate, phosphoramidate and thiophosphate can be enumerated, but are not limited to these. The preparation method of phosphorus-containing and non-phosphorus-containing bonds is well known.

[0420] In certain embodiments, the modified oligonucleotide targeting a DUX4 nucleic acid comprises one or more modified internucleoside bonds. In certain embodiments, the modified internucleoside bonds are phosphorothioate bonds. In certain embodiments, each internucleoside bond of the modified oligonucleotide is a phosphorothioate internucleoside bond.

[0421] Modified sugar moiety

[0422] The modified oligonucleotide of the present invention preferably uses a modified oligonucleotide in which at least one nucleoside constituting the modified oligonucleotide comprises a modified sugar. In the present invention, a modified sugar refers to a sugar in which the sugar moiety is modified, and a modified oligonucleotide comprising one or more of the modified sugars has advantageous features such as enhanced nuclease stability and increased bonding affinity. At least one of the modified sugars preferably has a bicyclic sugar or substituted sugar moiety.

[0423] Examples of nucleosides having modified sugars include, but are not limited to, nucleosides containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, 2'-OCH2CH3, 2'-OCH2CH2F, and 2'-O(CH2)2OCH3 substituents. The substituent at the 2' position may also be selected from allyl, amino, azido, thio, O-allyl, O-C1-C 10 Alkyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n )、O-CH2-C(=O)-N(R m )(R n ) and O-CH2-C(=O)-N(R l )-(CH2)2-N(R m )(R n )(wherein, each R l , R m and R n are independently H or substituted or unsubstituted C1-C 10 alkyl).

[0424] Examples of nucleosides having bicyclic sugars include, but are not limited to, nucleosides containing crosslinks between ribose ring atoms at 4' and 2'. In certain embodiments, the oligonucleotides provided herein contain nucleosides having one or more bicyclic sugars whose crosslinks contain one of the following formulae: 4'-(CH2)-O-2'(LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2'(ENA); 4'-CH(CH3)-O-2' and 4'-CH(CH2OCH3)-O-2' (and their analogs. See U.S. Patent No. 7,399,845); 4 '-C(CH3)(CH3)-O-2' (and their analogs. Refer to WO2009 / 006478); 4'-CH2-N(OCH3)-2' (and their analogs. Refer to WO2008 / 150729); 4'-CH2-ON(CH3)-2' (and their analogs. Refer to US2004-0171570); 4'-CH2-N(R)-O-2' (wherein R is H, C1~C 12 Alkyl or protecting group) (refer to U.S. Patent No. 7427672); 4'-CH2-C(H)(CH3)-2' (and their analogs. Refer to Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and their analogs. Refer to WO2008 / 154401).

[0425] Nucleosides with further bicyclic sugars have been reported in the literature (see, for example, Srivastava et al., J. Am. Chem. Soc., 2007, 129 (26) 8362-8379; Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638; Singh et al., Proc. Natl. Acad. Sci. USA, 2001, 97, 5633-5638). al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al.,; U.S. Patents No. 7,399,845; No. 6,770,748; No. 6,525,191; No. 6,268,490; U.S. Patents No. 2008-0039618; No. 2007-0287831; No. 2004-0171570; No. 2009-0012281; WO 2010 / 036698; WO 2009 / 067647; WO2009 / 067647; WO2007 / 134181; WO2005 / 021570; WO2004 / 106356; WO94 / 14226; WO 2009 / 006478; WO2008 / 154401; and WO2008 / 150729). The nucleosides having the above-mentioned bicyclic sugars can be prepared with one or more stereochemical sugar configurations, for example, including α-L-ribofuranose and β-D-ribofuranose.

[0426] GuNA, a nucleoside having a bicyclic sugar, has been reported as an artificial nucleoside having a guanidine crosslink (see WO2014 / 046212 and WO2017 / 047816). ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Trz] and ALNA[Oxz], bicyclic nucleosides, have been reported as crosslinked artificial nucleic acid amino LNA (ALNA) (see Japanese Patent Application No. 2018-212424).

[0427] In certain embodiments, the nucleoside having a bicyclic sugar comprises a crosslink between the 4' and 2' carbon atoms of the pentofuranosyl sugar moiety, comprising a crosslink comprising -[C(R a )(R b )] n -、-C(R a )=C(R b )-、-C(R a )=N-、-C(=NR a )-, -C(=O)-, -C(=S)-, -N(R a )-、-O-、-Si(R a )2- and -S(=O) x - is independently selected from 1 or 1 to 4 linking groups, but is not limited to these, wherein X is 0, 1 or 2; n is 1, 2, 3 or 4; each R a and R b are independently H, a protecting group, a hydroxyl group, a C1-C 12 Alkyl, substituted C1~C 12 Alkyl, C2~C 12 Alkenyl, substituted C2~C 12 Alkenyl, C2~C 12 Alkynyl, substituted C2~C 12 Alkynyl, aromatic ring group, substituted aromatic ring group, heterocyclic group, substituted heterocyclic group, C5-C7 alicyclic group, substituted C5-C7 alicyclic group, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl group (C(=O)-H), substituted acyl group, CN, sulfonyl group (S(=O)2-J1) or sulfinyl group (S(=O)-J1),

[0428] Each J1 and J2 is independently H, C1~C 12 Alkyl, substituted C1~C 12 Alkyl, C2~C 12 Alkenyl, substituted C2~C 12 Alkenyl, C2~C 12 Alkynyl, substituted C2~C 12Alkynyl, aromatic ring group, substituted aromatic ring group, acyl group (C(=O)-H), substituted acyl group, heterocyclic group, substituted heterocyclic group, C1-C 12 Aminoalkyl, substituted C1~C 12 Aminoalkyl or protecting group.

[0429] In one embodiment, the crosslinking of the bicyclic sugar moiety is -[C(R a )(R b )] n -、-[C(R a )(R b )] n -O-、-C(R a R b )-N(R)-O- or -C(R a R b In one embodiment, the crosslink is 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2' (in this case, the nucleoside with a bicyclic sugar is also referred to as LNA), 4'-(CH2)2-O-2', 4'-CH2-ON(R)-2' and 4'-CH2-N(R)-O-2'-, wherein each R is independently H, a protecting group or a C1~C 12 alkyl.

[0430] In one embodiment, the crosslinking of the bicyclic sugar moiety is 4'-CH2-O-2'-(LNA) or -CH2-N(R)-, wherein each R is independently -SO2-CH3 (ALNA[Ms]), -CO-NH-CH3 (ALNA[mU]), 1,5-dimethyl-1,2,4-triazol-3-yl (ALNA[Trz]), -CO-NH-CH(CH3)2 (ALNA[ipU]), 5-methyl-1,2,4-triazol-3-yl (ALNA[Trz]), 3-oxadiazole (ALNA[Oxz]) (Japanese Patent Application No. 2018-212424).

[0431] In certain embodiments, nucleosides with bicyclic sugars are further defined by isomeric spatial configurations. For example, a nucleoside containing a 4'-(CH2)-O-2' crosslink can exist in either an α-L-steric configuration or a β-D-steric configuration.

[0432] In one embodiment, the nucleoside having a bicyclic sugar includes a nucleoside having a 4'-2' crosslink, and examples of such crosslinks include α-L-4'-(CH2)-O-2', β-D-4'-CH2-O-2', 4'-(CH2)2-O-2', 4'-CH2-ON(R)-2', 4'-CH2-N(R)-O-2', 4'-CH(CH3)-O-2', 4'-CH2-S-2', 4'-CH2-CH(CH3)-2', and 4'-(CH2)3-2' (wherein R is H, a protecting group, a C1 to C 12 Alkyl, or can be C1~C 12 Alkyl-substituted urea or guanidine), but is not limited to these.

[0433] In certain embodiments, the nucleoside having a bicyclic sugar has the formula:

[0434]

[0435] In the formula,

[0436] Bx is a heterocyclic base moiety,

[0437] T a and T b Each independently represents a hydrogen atom, a protective group for a hydroxyl group, a phosphate group which may be substituted, a covalent bond to a phosphorus moiety or a support, etc.,

[0438] Z a It is C1~C6 alkyl, C2~C6 alkenyl, C2~C6 alkynyl, substituted C1~C6 alkyl, substituted C2~C6 alkenyl, substituted C2~C6 alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thiol.

[0439] In one embodiment, the substituents are each independently selected from halogen, oxo, hydroxyl, O c , NJ c J d , SJ c 、N3、OC(=X)J c and N.J. e C(=X)NJ c J d (In the formula, each J c , J d and J e independently H, C1-C6 alkyl or substituted C1-C6 alkyl, X is O or NJ c ) are mono- or poly-substituted by independently selected substituents.

[0440] In certain embodiments, the nucleoside having a bicyclic sugar has the formula:

[0441]

[0442] In the formula,

[0443] Bx is a heterocyclic base moiety,

[0444] T a and T b Each independently represents a hydrogen atom, a protective group for a hydroxyl group, a phosphate group which may be substituted, a covalent bond to a phosphorus portion or a support, etc.,

[0445] Z b It is a C1~C6 alkyl group, a C2~C6 alkenyl group, a C2~C6 alkynyl group, a substituted C1~C6 alkyl group, a substituted C2~C6 alkenyl group, a substituted C2~C6 alkynyl group or a substituted acyl group (C(=O)-).

[0446] In certain embodiments, the nucleoside having a bicyclic sugar has the formula:

[0447]

[0448] In the formula,

[0449] Bx is a heterocyclic base moiety,

[0450] T a and T b Each independently represents a hydrogen atom, a protective group for a hydroxyl group, a phosphate group which may be substituted, a covalent bond to a phosphorus portion or a support, etc.,

[0451] R d is C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl,

[0452] Each q a ,q b ,q c and q d Independently, they are H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, acyl, substituted acyl, C1-C6 aminoalkyl or substituted C1-C6 aminoalkyl.

[0453] In certain embodiments, the nucleoside having a bicyclic sugar has the formula:

[0454]

[0455] In the formula,

[0456] Bx is a heterocyclic base moiety;

[0457] Ta and T b Each independently represents a hydrogen atom, a protective group for a hydroxyl group, a phosphate group which may be substituted, a covalent bond to a phosphorus moiety or a support, etc.,

[0458] q a ,q b ,q e and q f are independently hydrogen, halogen, C1-C 12 Alkyl, substituted C1~C 12 Alkyl, C2~C 12 Alkenyl, substituted C2~C 12 Alkenyl, C2~C 12 Alkynyl, substituted C2~C 12 Alkynyl, C1~C 12 Alkoxy, substituted C1~C 12 Alkoxy, OJ j , SJ j 、SOJ j 、SO2J j , NJ j J k , N3, CN, C(=O)OJj, C(=O)NJ j J k 、C(=O)J j 、OC(=O)NJ j J k 、N(H)C(=NH)NJ j J k 、N(H)C(=O)-NJ j J k or N(H)C(=S)NJ j J k ,

[0459] Or, q e and q f =C(q g )(q h ),

[0460] q g and q h are independently H, halogen, C1-C 12 Alkyl or substituted C1~C 12 alkyl.

[0461] The synthesis and preparation of bicyclic nucleosides (also called LNA) of adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil with 4'-CH2-O-2' crosslinks are described together with their oligomerization and nucleic acid recognition properties (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). The synthesis of nucleosides with bicyclic sugars is also described in WO98 / 39352 and WO99 / 14226.

[0462] Analogs of various bicyclic nucleosides with 4'-2' cross-linking groups such as 4'-CH2-O-2' (the bicyclic nucleoside in this case is also called LNA) and 4'-CH2-S-2' have also been prepared (Kumare al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of oligodeoxyribonucleotide duplexes containing bicyclic nucleosides for use as substrates for nucleic acid polymerases is also described (Wengel et al., WO99 / 14226). Furthermore, the synthesis of 2'-amino-BNA (the bicyclic nucleoside in this case is also called ALNA), i.e., a high-affinity oligonucleotide analog with limited steric structure, has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). Furthermore, 2'-amino- and 2'-methylamino-BNAs have been prepared, and the thermal stability of duplexes with complementary RNA and DNA strands has been reported previously.

[0463] In certain embodiments, the nucleoside having a bicyclic sugar has the formula:

[0464]

[0465] In the formula,

[0466] Bx is a heterocyclic base moiety,

[0467] T a and T b Each independently represents a hydrogen atom, a protective group for a hydroxyl group, a phosphate group which may be substituted, a covalent bond to a phosphorus moiety or a support, etc.,

[0468] Each q i ,q j ,q k and q l are independently H, halogen, C1~C 12 Alkyl, substituted C1~C 12 Alkyl, C2~C 12 Alkenyl, substituted C2~C 12 Alkenyl, C2~C 12 Alkynyl, substituted C2~C 12Alkynyl, C1~C 12 Alkoxy, substituted C1~C 12 Alkoxy, OJ j , SJ j 、SOJ j 、SO2J j , NJ j J k 、N3、CN、C(=O)OJ j 、C(=O)NJ j J k 、C(=O)J j 、OC(=O)NJ j J k 、N(H)C(=NH)NJ j J k 、N(H)C(=O)NJ j J k or N(H)C(=S)NJ j J k ,

[0469] q i and q j or q l and q k =C(q g )(q h ), where q g and q h are independently H, halogen, C1-C 12 Alkyl or substituted C1~C 12 alkyl.

[0470] A carbocyclic bicyclic nucleoside having a 4'-(CH2)3-2' crosslink and an olefinic analog crosslink 4'-CH=CH-CH2-2' has been described (Frier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albake et al., J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides have also been described along with their oligomerization and biochemical studies (Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

[0471] In one embodiment, the nucleoside having a bicyclic sugar includes the following:

[0472] (A) α-L-methyleneoxy (4'-CH2-O-2') BNA,

[0473] (B) β-D-methyleneoxy (4'-CH2-O-2') BNA,

[0474] (C) ethyleneoxy (4'-(CH2)2-O-2') BNA,

[0475] (D) aminooxy (4'-CH2-ON(R)-2') BNA,

[0476] (E) oxyamino (4'-CH2-N(R)-O-2') BNA,

[0477] (F) methyl(methyleneoxy)(4'-CH(CH3)-O-2')BNA (also known as constrained ethyl or cEt),

[0478] (G) Methylene-sulfanyl (4'-CH2-S-2') BNA,

[0479] (H) Methylene-amino (4'-CH2-N(R)-2') BNA,

[0480] (I) methyl carbocyclic (4'-CH2-CH(CH3)-2') BNA,

[0481] (J) propylene carbocyclic (4'-(CH2)3-2') BNA, and

[0482] (K) Vinyl BNA, but not limited to these.

[0483]

[0484] In the formula, Bx is a base moiety, and R is independently a protecting group, a C1-C6 alkyl group or a C1-C6 alkoxy group.

[0485] In one embodiment (LNA), the nucleoside having a bicyclic sugar may be of the following general formula:

[0486]

[0487] [Wherein,

[0488] B is a nucleic acid base,

[0489] X and Y are each independently a hydrogen atom, a protective group for a hydroxyl group, a phosphate group which may be substituted, a covalent bond to a phosphorus part or a support, etc.] and represent a nucleoside (see WO98 / 39352). Typical specific examples include the following formula:

[0490]

[0491] Indicates the nucleotides.

[0492] In one embodiment (GuNA), the nucleoside comprising a bicyclic sugar is of the following general formula:

[0493]

[0494] [wherein, B is a nucleic acid base, R3, R4, R5, and R6 are each independently a hydrogen atom, or a C atom which may be substituted with one or more substituents. 1-6 alkyl, R7 and R8 are each independently a hydrogen atom, a protective group for a hydroxyl group, a phosphate group which may be substituted, a covalent bond to a phosphorus part or a support, etc., R9, R 10 , R 11 Each independently represents a hydrogen atom, which may be substituted with one or more substituents. 1-6 alkyl, or amino protecting group]

[0495] Nucleosides represented by (for example, refer to International Publication No. 2014 / 046212 and International Publication No. 2017 / 047816).

[0496] In one embodiment (ALNA[mU]), the nucleoside comprising a bicyclic sugar is of the following general formula (I):

[0497]

[0498] [Wherein,

[0499] B is a nucleic acid base,

[0500] R1, R2, R3 and R4 are each independently a hydrogen atom, or a C atom which may be substituted with one or more substituents. 1-6 alkyl,

[0501] R5 and R6 are each independently a hydrogen atom, a protective group for a hydroxyl group, a phosphate group which may be substituted, a covalent bond to a phosphorus part or a support, etc.

[0502] m is 1 or 2,

[0503] X is the following formula (II-1):

[0504]

[0505] The group represented by

[0506] Symbols described in formula (II-1):

[0507]

[0508] represents the bonding point to the 2'-amino group,

[0509] One of R7 and R8 is a hydrogen atom, and the other is a methyl group which may be substituted with one or more substituents]

[0510] A nucleoside represented by (for example, see Japanese Patent Application No. 2018-212424). A typical specific example is a nucleoside in which one of R7 and R8 is a hydrogen atom and the other is an unsubstituted methyl group.

[0511] In one embodiment (ALNA[ipU]), the nucleoside comprising a bicyclic sugar is a nucleoside having the general formula (I) defined in ALNA[mU] above, wherein:

[0512] X is the following formula (II-1):

[0513]

[0514] The group represented by

[0515] One of R7 and R8 is a hydrogen atom, and the other is an isopropyl group which may be substituted with one or more substituents (for example, see Japanese Patent Application No. 2018-212424). A typical specific example is a nucleoside in which one of R7 and R8 is a hydrogen atom and the other is an unsubstituted isopropyl group.

[0516] In one embodiment (ALNA[Trz]), the nucleoside comprising a bicyclic sugar is a nucleoside having the general formula (I) above, wherein X is the following formula (II-2):

[0517]

[0518] The group represented by

[0519] A is a triazole group which may be substituted with one or more substituents (see, for example, Japanese Patent Application No. 2018-212424). A typical specific example of ALNA[Trz] is a triazole group which may have one or more methyl groups, more specifically a 1,5-dimethyl-1,2,4-triazol-3-yl nucleoside.

[0520] In one embodiment (ALNA[Oxz]) is a nucleoside having the general formula (I) as defined above for ALNA[mU], wherein:

[0521] X is the following formula (II-2):

[0522]

[0523] The group represented by

[0524] A is substituted by one or more substituents. A is a diazole group (for example, see Japanese Patent Application No. 2018-212424). A typical specific example is A which may have one or more methyl groups. oxadiazolyl, more specifically 5-methyl-1,2,4- Oxazolyl-3-yl nucleosides or nucleotides.

[0525] In one embodiment (ALNA[Ms]), the nucleoside comprising a bicyclic sugar is a nucleoside having the general formula (I) above, wherein X is the following general formula (II-3):

[0526]

[0527] The group represented by

[0528] M is a sulfonyl group which may be substituted with a methyl group, and the methyl group may be substituted with one or more substituents (see, for example, Japanese Patent Application No. 2018-212424). A typical specific example of ALNA[Ms] is a nucleoside in which M is a sulfonyl group substituted with an unsubstituted methyl group.

[0529] In one embodiment, the nucleoside is modified by substitution of a ribosyl ring with a sugar surrogate. Such modifications include substitution of a ribosyl ring with a surrogate ring system (also referred to as a DNA analog), such as a morpholino ring, a cyclohexenyl ring, a cyclohexyl ring or a tetrahydropyranyl ring, such as a ring having one of the following formulas, but are not limited thereto.

[0530]

[0531] In one embodiment, a sugar substitute having the following formula is selected:

[0532]

[0533] In the formula,

[0534] Bx is a heterocyclic base moiety,

[0535] T3 and T4 are each independently an internucleoside linking group obtained by linking a tetrahydropyranoside analog to an oligomer compound, or one of T3 and T4 is an internucleoside linking group obtained by linking a tetrahydropyranoside analog to an oligomer compound or an oligonucleotide, and the other of T3 and T4 is H, a hydroxyl protecting group, a linking conjugation group, or a 5' or 3'-terminal group,

[0536] q1, q2, q3, q4, q5, q6 and q7 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl,

[0537] One of R1 and R2 is hydrogen, and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN (wherein X is O, S or NJ1, and each of J1, J2 and J3 is independently H or C1~C6 alkyl).

[0538] In certain embodiments, Q1, Q2, Q3, Q4, Q5, Q6 and Q7 are each H. In certain embodiments, at least one of Q1, Q2, Q3, Q4, Q5, Q6 and Q7 is not H. In certain embodiments, at least one of Q1, Q2, Q3, Q4, Q5, Q6 and Q7 is methyl. In certain embodiments, a THP nucleoside in which one of R1 and R2 is F can be provided. In certain embodiments, R1 is fluorine and R2 is H; R1 is methoxy and R2 is H; and R1 is methoxyethoxy and R2 is H.

[0539] Examples of such sugar substitutes include those referred to in the art as hexitol nucleic acid (HNA), altritol nucleic acid (ANA), and mannitol nucleic acid (MNA) (see Leumann, CJ, Bioorg. & Med. Chem., 2002, 10, 841-854), but are not limited thereto.

[0540] In certain embodiments, the sugar surrogate comprises a ring having more than 5 atoms and more than 1 heteroatom. For example, nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds have been reported (e.g., see Braaschet al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506).

[0541] As used in this specification, the term "morpholino" refers to a sugar substitute having the following structure:

[0542]

[0543] In one embodiment, for example, the morpholino group can be modified by adding or changing various substituents to the above-mentioned morpholino structure. Such sugar substitutes are referred to as "modified morpholino groups" in this specification.

[0544] In certain embodiments, the oligonucleotide comprises one or more modified cyclohexenyl nucleosides, which are nucleosides having a 6-membered cyclohexenyl group in place of the pentofuranosyl residue of naturally occurring nucleosides. Examples of modified cyclohexenyl nucleosides include modified cyclohexenyl nucleosides described in the art (see, for example, WO2010 / 036696 published on April 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008, 130 (6), 1979-1984; Horvath et al., Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129 (30), 9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005, 24 (5-7), 993-998; Nauwelaerts et al., Nucleic Acids, 2005, 24 (5-7), 993-998; Nauwelaerts et al., Nucleic Acids, 2005, 24 (5-7), 993-998; AcidsResearch, 2005, 33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 2005, F61 (6), 585-586; Guet al., Tetrahedron, 2004, 60 (9), 2111-2123; Guet al., Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem., 2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research,2001,29(24),4941-4947; Wangetal.,J.Org.Chem.,2001,66,8478-82;Wang et al. al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20 (4-7), 785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; WO06 / 047842; and WO01 / 049687; each of which is incorporated herein by reference in its entirety), but is not limited thereto.

[0545] Certain modified cyclohexenyl nucleosides have the formula:

[0546]

[0547] In the formula,

[0548] Bx is a heterocyclic base moiety,

[0549] T3 and T4 are each independently an internucleoside linking group obtained by linking a cyclohexenyl nucleoside analog to an oligonucleotide compound, or one of T3 and T4 is an internucleoside linking group obtained by linking a tetrahydropyranoside analog to an oligonucleotide compound, and the other of T3 and T4 is H, a hydroxyl protecting group, a linking conjugation group or a 5'- or 3'-terminal group,

[0550] q1, q2, q3, q4, q5, q6, q7, q8 and q9 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or other sugar substituents.

[0551] Many other bicyclic and tricyclic sugar surrogate ring systems that can be used to modify nucleosides for incorporation into oligonucleotides are known in the art (see, for example, the general review: Leumann, Christian J., Bioorg. & Med. Chem., 2002, 10, 841-854). Such ring systems can accept various further substitutions in order to enhance activity.

[0552] The preparation method of modified sugar is well known to those skilled in the art. Some representative US patents that teach the preparation of such modified sugars include US: 4981957; 5118800; 5319080; 5359044; 5393878; 5446137; 5466786; 5514785; 5519134; 5567811; 5576427; 5591722; 5597909; 5610300; 5627053; 5639873; 5646265; 5670633; 5700920; 5792847 and 6600032 and WO2005 / 121371, but are not limited to these, and these can be incorporated into the present specification as a reference in their entirety.

[0553] In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified, or a combination thereof) are maintained between hybridizations with the appropriate nucleic acid target.

[0554] Modified Nucleobases

[0555] The modification or substitution of nucleic acid base (or base) can be structurally recognized with naturally occurring or synthetic non-modified nucleic acid base, and further functionally interchangeable with such non-modified nucleic acid base. Both natural and modified nucleic acid bases can be associated with hydrogen bonds. Such nucleic acid base modification can confer nuclease stability, bonding affinity or some other beneficial biological properties to modified oligonucleotides. Modified nucleic acid bases, for example, include synthetic and natural nucleic acid bases such as 5-methylcytosine (5-me-C). Certain nucleic acid base substitutions including 5-methylcytosine substitutions are particularly useful for increasing the bonding affinity of modified oligonucleotides to target nucleic acids. For example, 5-methylcytosine substitutions show that the double-stranded stability of nucleic acids increases by 0.6 to 1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

[0556] As further modified nucleic acid bases, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 5 -uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine.

[0557] The heterocyclic base moiety may also include a heterocyclic base moiety in which a purine or pyrimidine base is substituted with another heterocycle, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. As nucleic acid bases particularly useful for increasing the bonding affinity of modified oligonucleotides, 5-substituted pyrimidine, 6-azapyrimidine and N-2, N-6 and O-6 substituted purines (including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine) can be cited.

[0558] In certain embodiments, the modified oligonucleotide targeting a DUX4 nucleic acid comprises one or more modified nucleic acid bases. In certain embodiments, the modified oligonucleotide targeting a DUX4 nucleic acid comprises one or more modified nucleic acid bases. In certain embodiments, the modified nucleic acid base is 5-methylcytosine. In certain embodiments, each cytosine is 5-methylcytosine.

[0559] Modified oligonucleotide motif

[0560] In a certain embodiment, in order to confer properties such as enhanced inhibitory activity, increased binding affinity for target nucleic acid, or resistance to degradation by nucleases in vivo on the modified oligonucleotide, the modified oligonucleotide targeting DUX4 nucleic acid has a chemically modified subunit configured in a pattern or motif.

[0561] In order to confer increased resistance to degradation by nucleases, increased cellular uptake, increased binding affinity to target nucleic acids and / or increased inhibitory activity, chimeric modified oligonucleotides typically include at least one modified region. The second region of the chimeric modified oligonucleotide can optionally function as a substrate for the intracellular endonuclease RNase H that cuts the RNA strand of the RNA:DNA double strand.

[0562] The modified oligonucleotide with gapmer motif is a chimera modified oligonucleotide. In the gapmer, the inner region with multiple nucleotides that support RNase H to cut off is located between the outer regions, and the outer regions have multiple nucleotides different from the nucleoside chemical properties of the inner region. In the case of the modified oligonucleotide with gapmer motif, the gap fragment generally plays a role as a substrate cut off with endonuclease, but the flanking fragments include modified nucleosides. In certain embodiment, the gapmer region can be distinguished by the type of sugar moieties that each include different regions. In some embodiments, the type of sugar moiety used to distinguish the gap body region may include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2'-modified nucleosides (for such 2'-modified nucleosides, 2'-MOE and 2'-O-CH3 may be included) and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include bicyclic sugar modified nucleosides with LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Trz] and / or ALNA[Oxz]). The wing-gap-wing motif is often recorded as "XYZ," "X" represents the length of the 5' wing region, "Y" represents the length of the gap region, and "Z" represents the length of the 3' wing region. When used in this specification, the gap body recorded as "XYZ" has the following spatial configuration: the gap segment is located directly adjacent to the 5' wing segment and the 3' wing segment, respectively. Thus, there are no intermediate nucleotides between the 5' wing segment and the gap segment, or between the gap segment and the 3' wing segment. Any of the modified oligonucleotides of the present specification may have a gap body motif. In some embodiments, X and Z are the same, and in other embodiments, they are different. In a preferred embodiment, Y is 8 to 16 nucleotides. X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleotides. Therefore, examples of the interposer include 2-10-3, 2-14-2, 2-15-2, 2-16-2, 3-6-7, 3-7-5, 3-8-3, 3-8-4, 3-8-5, 3-9-2, 3-9-3, 3-9-4, 3-9-5, 3-9-8, 3-10-2, 3-10-3, 3-10-4, 3-11-3, 3-12-3, 3-14-3, 4-7-4, 4-7-5, 4-8-3, 4-8-4, 4-9-3, 4-10-3, 5-6-4, 5-6-5, 5-7-3, 5-7-4, 5-8-3, 5

[0563] -8-4 or 7-6-3, but not limited to these.

[0564] In certain embodiments, the modified oligonucleotide targeting DUX4 nucleic acid has a 3-10-3 gap body motif. In certain embodiments, the modified oligonucleotide targeting DUX4 nucleic acid has a 3-9-3 gap body motif. In certain embodiments, the modified oligonucleotide targeting DUX4 nucleic acid has a 3-9-4 gap body motif. In certain embodiments, the modified oligonucleotide targeting DUX4 nucleic acid has a 3-8-5 gap body motif.

[0565] In a certain embodiment, the modified oligonucleotides of these gapmers comprise at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29 or at least 30 consecutive nucleic acid bases of any nucleic acid base sequence of the exemplary modified oligonucleotides described in the present specification (for example, at least 8 consecutive nucleic acid bases of a nucleic acid base sequence described in any one of SEQ ID NOs. 2, 3, 4, 7 to 64, 69 to 97 or 102 to 112 of the sequence listing).

[0566] In certain embodiments, the present invention provides oligomer compounds comprising oligonucleotides. In certain embodiments, such oligonucleotides comprise one or more chemical modifications. In certain embodiments, chemically modified oligonucleotides comprise one or more modified sugars. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleic acid bases. In certain embodiments, chemically modified oligonucleotides comprise one or more modified internucleoside bonds. In certain embodiments, chemical modifications (sugar modifications, nucleic acid base modifications and / or bonding modifications) determine patterns or motifs. In certain embodiments, the patterns of chemical modifications of sugar moieties, internucleoside bonds and nucleic acid bases are each unrelated to each other. Therefore, oligonucleotides can be described by their sugar modification motifs, internucleoside bonding motifs and / or nucleic acid base modification motifs (when used in this specification, nucleic acid base modification motifs have no relationship with the sequence of nucleic acid bases, indicating chemical modifications to nucleic acid bases).

[0567] A sugar moiety

[0568] In certain embodiments, the oligonucleotide comprises one or more types of modified sugar moieties and / or naturally occurring sugar moieties arranged along the oligonucleotide or a region thereof in a defined pattern or sugar modification motif. Such motifs may comprise any of the sugar modifications discussed in this specification and / or other known sugar modifications.

[0569] In a certain embodiment, the oligonucleotide comprises or is composed of a region having a gap body sugar modification motif, which comprises two external regions, namely "flanking segments" and one internal region, namely "gap segments". The three regions of the gap body motif (5' wing segment, gap segment and 3' wing segment) form a continuous sequence of nucleosides, where at least several of the sugar moieties of the nucleosides of each wing segment are different from at least several of the sugar moieties of the nucleosides of the gap segment. In particular, the sugar moieties of the nucleosides of each wing segment closest to the gap segment (the nucleosides on the most 3' side of the 5' wing segment and the nucleosides on the most 5' side of the 3' wing segment) are different from the sugar moieties of the nucleosides of the adjacent gap segments, thereby determining the boundary position between the wing segment and the gap segment. In a certain embodiment, the sugar moieties in the gap segment are identical to each other. In a certain embodiment, the gap segment comprises one or more nucleosides having sugar moieties different from the sugar moieties of one or more other nucleosides of the gap segment. In a certain embodiment, the sugar modification motifs of the two wing segments are identical to each other (symmetrical gap bodies). In certain embodiments, the sugar modification motif of the 5' flanking segment is different from the sugar modification motif of the 3' flanking segment (asymmetric gapmer).

[0570] Certain 5' flanking fragments

[0571] In certain embodiments, the 5' wing segment of the gapmer consists of 1 to 5 linked nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of 3 to 5 linked nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of 4 or 5 linked nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of 1 to 4 linked nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of 1 to 3 linked nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of 1 or 2 linked nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of 2 to 4 linked nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of 2 or 3 linked nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of 3 or 4 linked nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of 1 nucleoside. In certain embodiments, the 5' wing segment of the gapmer consists of 2 linked nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of 3 linked nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of 4 linked nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of 5 linked nucleosides.

[0572] In certain embodiments, the 5' wing segment of the gapmer comprises at least 1 bicyclic nucleoside. In certain embodiments, the 5' wing segment of the gapmer comprises at least 2 bicyclic nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises at least 3 bicyclic nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises at least 4 bicyclic nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises at least 1 constrained ethyl nucleoside. In certain embodiments, the 5' wing segment of the gapmer comprises at least 1 LNA-containing nucleoside, GuNA-containing nucleoside, ALNA[Ms]-containing nucleoside, ALNA[mU]-containing nucleoside, ALNA[ipU]-containing nucleoside, ALNA[Trz]-containing nucleoside and / or ALNA[Oxz]-containing nucleoside. In certain embodiments, each nucleoside of the 5' wing segment of the gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 5' wing segment of the gapmer is a constrained ethyl nucleoside. In one embodiment, each nucleoside of the 5' wing segment of the gapmer is a nucleoside containing LNA, a nucleoside containing GuNA, a nucleoside containing ALNA[Ms], a nucleoside containing ALNA[mU], a nucleoside containing ALNA[ipU], a nucleoside containing ALNA[Trz] and / or a nucleoside containing ALNA[Oxz].

[0573] In certain embodiments, the 5' wing segment of the gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 5' wing segment of the gapmer comprises at least one 2'-substituted nucleoside. In certain embodiments, the 5' wing segment of the gapmer comprises at least one, for example, 3, 4 or 5 2'-MOE nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises at least one 2'-OMe nucleoside. In certain embodiments, each nucleoside of the 5' wing segment of the gapmer is a non-bicyclic modified nucleoside. In certain embodiments, each nucleoside of the 5' wing segment of the gapmer is a 2'-substituted nucleoside. In certain embodiments, each nucleoside of the 5' wing segment of the gapmer is a 2'-MOE nucleoside. In certain embodiments, each nucleoside of the 5' wing segment of the gapmer is a 2'-OMe nucleoside.

[0574] In certain embodiments, the 5' wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5' wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 5' wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 5' wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 5' wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one 2'-deoxynucleoside.

[0575] In certain embodiments, the 5' wing segment of the gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 5' wing segment of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 5' wing segment of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 5' wing segment of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 5' wing segment of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-deoxynucleoside.

[0576] In certain embodiments, the 5'-wing segment of the gapmer comprises at least one modified nucleoside selected from 2'-MOE nucleosides, 2'-OMe nucleosides, LNA-containing nucleosides, GuNA-containing nucleosides, ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, ALNA[ipU]-containing nucleosides, ALNA[Trz]-containing nucleosides, and / or ALNA[Oxz]-containing nucleosides. In certain embodiments, the 5'-wing segment of the gapmer comprises at least two modified nucleosides selected from 2'-MOE nucleosides, 2'-OMe nucleosides, LNA-containing nucleosides, GuNA-containing nucleosides, ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, ALNA[ipU]-containing nucleosides, ALNA[Trz]-containing nucleosides, and / or ALNA[Oxz]-containing nucleosides. In certain embodiments, the 5'-wing segment of the gapmer comprises at least three modified nucleosides selected from 2'-MOE nucleosides, 2'-OMe nucleosides, LNA-containing nucleosides, GuNA-containing nucleosides, ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, ALNA[ipU]-containing nucleosides, ALNA[Trz]-containing nucleosides, and / or ALNA[Oxz]-containing nucleosides. In certain embodiments, the 5'-wing segment of the gapmer comprises at least four modified nucleosides selected from 2'-MOE nucleosides, 2'-OMe nucleosides, LNA-containing nucleosides, GuNA-containing nucleosides, ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, ALNA[ipU]-containing nucleosides, ALNA[Trz]-containing nucleosides, and / or ALNA[Oxz]-containing nucleosides. In certain embodiments, the 5'-wing segment of the gapmer comprises at least 5 modified nucleosides selected from 2'-MOE nucleosides, 2'-OMe nucleosides, LNA-containing nucleosides, GuNA-containing nucleosides, ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, ALNA[ipU]-containing nucleosides, ALNA[Trz]-containing nucleosides and / or ALNA[Oxz]-containing nucleosides.

[0577] In certain embodiments, the 5' wing segment of a gapmer comprises 2 nucleosides containing LNA. In certain embodiments, the 5' wing segment of a gapmer comprises 3 nucleosides containing LNA. In certain embodiments, the 5' wing segment of a gapmer comprises 4 nucleosides containing LNA. In certain embodiments, the 5' wing segment of a gapmer comprises 3 nucleosides containing ALNA[Ms]. In certain embodiments, the 5' wing segment of a gapmer comprises 2 nucleosides containing GuNA. In certain embodiments, the 5' wing segment of a gapmer comprises 3 nucleosides containing GuNA. In certain embodiments, the 5' wing segment of a gapmer comprises 3 nucleosides containing ALNA[mU]. In certain embodiments, the 5' wing segment of a gapmer comprises 3 nucleosides containing ALNA[ipU]. In certain embodiments, the 5' wing segment of a gapmer comprises 2 nucleosides containing LNA and 1 nucleoside containing GuNA. In certain embodiments, the 5' wing segment of a gapmer comprises 3 nucleosides containing ALNA[Trz]. In certain embodiments, the 5'-wing segment of the gapmer comprises 3 nucleosides containing ALNA[Ms] and 1 2'-OMe nucleoside. In certain embodiments, the 5'-wing segment of the gapmer comprises 3 nucleosides containing ALNA[Ms] and 2 2'-OMe nucleosides.

[0578] In certain embodiments, the 5' wing segment of the gapmer comprises 3 constrained ethyl nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises 2 bicyclic nucleosides and 2 non-bicyclic modified nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises 2 constrained ethyl nucleosides and 2 2'-OMe nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises 2 bicyclic nucleosides and 2 non-bicyclic modified nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises 2 constrained ethyl nucleosides and 2 2'-OMe nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises 2 constrained ethyl nucleosides and 3 2'-OMe nucleosides.

[0579] In certain embodiments, the 5' wing segment of the gapmer consists of 1 nucleoside containing ALNA[Ms]. In certain embodiments, the 5' wing segment of the gapmer consists of 2 linked nucleosides containing ALNA[Ms]. In certain embodiments, the 5' wing segment of the gapmer consists of 3 linked nucleosides containing ALNA[Ms]. In certain embodiments, the 5' wing segment of the gapmer consists of 4 linked nucleosides containing ALNA[Ms]. In certain embodiments, the 5' wing segment of the gapmer consists of 5 linked nucleosides containing ALNA[Ms]. In certain embodiments, the 5' wing segment of the gapmer consists of 1, 2 or 3 linked nucleosides containing ALNA[Ms] and 1 2'-OMe nucleoside. In certain embodiments, the 5' wing segment of the gapmer consists of 1, 2 or 3 linked nucleosides containing ALNA[Ms] and 2 2'-OMe nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of 1, 2 or 3 linked nucleosides containing ALNA [Ms] and 3 2'-OMe nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of 3 linked nucleosides containing ALNA [Ms] and 1 2'-MOE nucleoside. In certain embodiments, the 5' wing segment of the gapmer consists of 2 linked nucleosides containing ALNA [Ms] and 2 2'-MOE nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of 3 linked nucleosides containing LNA and 2 2'-MOE nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of 2 linked nucleosides containing LNA and 2 2'-MOE nucleosides.

[0580] In certain embodiments, the 5' wing segment of the gapmer consists of 1, 2 or 3 nucleosides containing ALNA[Ms] linked together and comprises 1 5-methylcytosine. In certain embodiments, the 5' wing segment of the gapmer consists of 1, 2 or 3 nucleosides containing ALNA[Ms] linked together and comprises 2 5-methylcytosines. In certain embodiments, the 5' wing segment of the gapmer consists of 1, 2 or 3 nucleosides containing ALNA[Ms] linked together and comprises 3 5-methylcytosines.

[0581] Certain 3' flanking fragments

[0582] In certain embodiments, the 3' wing segment of the gapmer consists of 1 to 8 linked nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of 2 to 5 linked nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of 3 to 5 linked nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of 4 or 5 linked nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of 1 to 4 linked nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of 1 to 3 linked nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of 1 or 2 linked nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of 2 to 4 linked nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of 2 or 3 linked nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of 3 or 4 linked nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of 1 nucleoside. In certain embodiments, the 3' wing segment of the gapmer consists of 2 linked nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of 3 linked nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of 4 linked nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of 5 linked nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of 6 linked nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of 7 linked nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of 8 linked nucleosides.

[0583] In certain embodiments, the 3' wing segment of the gapmer comprises at least 1 bicyclic nucleoside. In certain embodiments, the 3' wing segment of the gapmer comprises at least 2 bicyclic nucleosides. In certain embodiments, the 3' wing segment of the gapmer comprises at least 3 bicyclic nucleosides. In certain embodiments, the 3' wing segment of the gapmer comprises at least 4 bicyclic nucleosides. In certain embodiments, the 3' wing segment of the gapmer comprises at least 1 constrained ethyl nucleoside. In certain embodiments, the 3' wing segment of the gapmer comprises at least 1 LNA-containing nucleoside, GuNA-containing nucleoside, ALNA[Ms]-containing nucleoside, ALNA[mU]-containing nucleoside, ALNA[ipU]-containing nucleoside, ALNA[Trz]-containing nucleoside and / or ALNA[Oxz]-containing nucleoside. In certain embodiments, each nucleoside of the 3' wing segment of the gapmer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 3' wing segment of the gapmer is a constrained ethyl nucleoside. In one embodiment, each nucleoside of the 3'-wing segment of the gapmer is a nucleoside containing LNA, a nucleoside containing GuNA, a nucleoside containing ALNA[Ms], a nucleoside containing ALNA[mU], a nucleoside containing ALNA[ipU], a nucleoside containing ALNA[Trz] and / or a nucleoside containing ALNA[Oxz].

[0584] In certain embodiments, the 3' wing segment of the gapmer comprises at least one non-bicyclic modified nucleoside. In certain embodiments, the 3' wing segment of the gapmer comprises at least one 2'-substituted nucleoside. In certain embodiments, the 3' wing segment of the gapmer comprises at least one 2'-MOE nucleoside. In certain embodiments, the 3' wing segment of the gapmer comprises at least one 2'-OMe nucleoside. In certain embodiments, each nucleoside of the 3' wing segment of the gapmer is a non-bicyclic modified nucleoside. In certain embodiments, each nucleoside of the 3' wing segment of the gapmer is a 2'-substituted nucleoside. In certain embodiments, each nucleoside of the 3' wing segment of the gapmer is a 2'-MOE nucleoside. In certain embodiments, each nucleoside of the 3' wing segment of the gapmer is a 2'-OMe nucleoside.

[0585] In certain embodiments, the 3' wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3' wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 3' wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 3' wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 3' wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one 2'-deoxynucleoside.

[0586] In certain embodiments, the 3' wing segment of the gapmer comprises at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3' wing segment of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 3' wing segment of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 3' wing segment of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 3' wing segment of the gapmer comprises at least one constrained ethyl nucleoside and at least one 2'-deoxynucleoside.

[0587] In certain embodiments, the 3'-wing segment of the gapmer comprises at least one modified nucleoside selected from 2'-MOE nucleosides, 2'-OMe nucleosides, LNA-containing nucleosides, GuNA-containing nucleosides, ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, ALNA[ipU]-containing nucleosides, ALNA[Trz]-containing nucleosides, and / or ALNA[Oxz]-containing nucleosides. In certain embodiments, the 3'-wing segment of the gapmer comprises at least two modified nucleosides selected from 2'-MOE nucleosides, 2'-OMe nucleosides, LNA-containing nucleosides, GuNA-containing nucleosides, ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, ALNA[ipU]-containing nucleosides, ALNA[Trz]-containing nucleosides, and / or ALNA[Oxz]-containing nucleosides. In certain embodiments, the 3'-wing segment of the gapmer comprises at least three modified nucleosides selected from 2'-MOE nucleosides, 2'-OMe nucleosides, LNA-containing nucleosides, GuNA-containing nucleosides, ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, ALNA[ipU]-containing nucleosides, ALNA[Trz]-containing nucleosides, and / or ALNA[Oxz]-containing nucleosides. In certain embodiments, the 3'-wing segment of the gapmer comprises at least four modified nucleosides selected from 2'-MOE nucleosides, 2'-OMe nucleosides, LNA-containing nucleosides, GuNA-containing nucleosides, ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, ALNA[ipU]-containing nucleosides, ALNA[Trz]-containing nucleosides, and / or ALNA[Oxz]-containing nucleosides. In certain embodiments, the 3'-wing segment of the gapmer comprises at least 5 modified nucleosides selected from 2'-MOE nucleosides, 2'-OMe nucleosides, LNA-containing nucleosides, GuNA-containing nucleosides, ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, ALNA[ipU]-containing nucleosides, ALNA[Trz]-containing nucleosides, and / or ALNA[Oxz]-containing nucleosides. In certain embodiments, the 3'-wing segment of the gapmer comprises at least 6 modified nucleosides selected from 2'-MOE nucleosides, 2'-OMe nucleosides, LNA-containing nucleosides, GuNA-containing nucleosides, ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, ALNA[ipU]-containing nucleosides, ALNA[Trz]-containing nucleosides, and / or ALNA[Oxz]-containing nucleosides.In certain embodiments, the 3'-wing segment of the gapmer comprises at least 7 modified nucleosides selected from 2'-MOE nucleosides, 2'-OMe nucleosides, LNA-containing nucleosides, GuNA-containing nucleosides, ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, ALNA[ipU]-containing nucleosides, ALNA[Trz]-containing nucleosides, and / or ALNA[Oxz]-containing nucleosides. In certain embodiments, the 3'-wing segment of the gapmer comprises at least 8 modified nucleosides selected from 2'-MOE nucleosides, 2'-OMe nucleosides, LNA-containing nucleosides, GuNA-containing nucleosides, ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, ALNA[ipU]-containing nucleosides, ALNA[Trz]-containing nucleosides, and / or ALNA[Oxz]-containing nucleosides.

[0588] In certain embodiments, the 3' wing segment of a gapmer comprises 2 nucleosides containing LNA. In certain embodiments, the 3' wing segment of a gapmer comprises 3 nucleosides containing LNA. In certain embodiments, the 3' wing segment of a gapmer comprises 4 nucleosides containing LNA. In certain embodiments, the 3' wing segment of a gapmer comprises 3 nucleosides containing ALNA[Ms]. In certain embodiments, the 3' wing segment of a gapmer comprises 2 nucleosides containing GuNA. In certain embodiments, the 3' wing segment of a gapmer comprises 3 nucleosides containing GuNA. In certain embodiments, the 3' wing segment of a gapmer comprises 3 nucleosides containing ALNA[mU]. In certain embodiments, the 3' wing segment of a gapmer comprises 3 nucleosides containing ALNA[ipU]. In certain embodiments, the 3' wing of a gapmer comprises 2 nucleosides containing LNA and 1 nucleoside containing GuNA. In certain embodiments, the 3' wing segment of a gapmer comprises 3 nucleosides containing ALNA[Trz]. In certain embodiments, the 3' wing segment of a gapmer comprises 3 nucleosides containing ALNA[Ms] and 1 2'-OMe nucleoside. In certain embodiments, the 3' wing segment of a gapmer comprises 3 nucleosides containing ALNA[Ms] and 2 nucleosides containing 2'-OMe. In certain embodiments, the 3' wing segment of a gapmer comprises 3 2'-MOE nucleosides. In certain embodiments, the 3' wing segment of a gapmer comprises 4 2'-MOE nucleosides. In certain embodiments, the 3' wing segment of a gapmer comprises 5 2'-MOE. In certain embodiments, the 3' wing segment of a gapmer comprises 3 nucleosides containing ALNA[Ms] and 1 nucleoside containing 2'-MOE. In certain embodiments, the 3' wing segment of a gapmer comprises 3 nucleosides containing ALNA[Ms] and 2 2'-MOE nucleosides. In certain embodiments, the 3' wing segment of a gapmer comprises 2 nucleosides containing ALNA[Ms] and 2 2'-MOE nucleosides. In certain embodiments, the 3' wing segment of a gapmer comprises 2 nucleosides containing ALNA[Ms] and 3 2'-MOE nucleosides. In certain embodiments, the 3' wing segment of a gapmer comprises 3 nucleosides containing ALNA[Ms] and 5 2'-MOE nucleosides. In certain embodiments, the 3' wing segment of a gapmer comprises 1 nucleoside containing ALNA[Ms] and 3 2'-MOE nucleosides. In certain embodiments, the 3' wing segment of a gapmer comprises 2 nucleosides containing LNA and 2 2'-MOE nucleosides.

[0589] In certain embodiments, the 3' wing segment of the gapmer comprises 3 constrained ethyl nucleosides. In certain embodiments, the 3' wing segment of the gapmer comprises 2 bicyclic nucleosides and 2 non-bicyclic modified nucleosides. In certain embodiments, the 3' wing segment of the gapmer comprises 2 constrained ethyl nucleosides and 2 2'-OMe nucleosides. In certain embodiments, the 3' wing segment of the gapmer comprises 2 bicyclic nucleosides and 2 non-bicyclic modified nucleosides. In certain embodiments, the 3' wing segment of the gapmer comprises 2 constrained ethyl nucleosides and 2 2'-OMe nucleosides. In certain embodiments, the 3' wing segment of the gapmer comprises 2 constrained ethyl nucleosides and 3 2'-OMe nucleosides.

[0590] In certain embodiments, the 3' wing segment of the gapmer consists of 1 nucleoside containing ALNA[Ms]. In certain embodiments, the 3' wing segment of the gapmer consists of 2 linked nucleosides containing ALNA[Ms]. In certain embodiments, the 3' wing segment of the gapmer consists of 3 linked nucleosides containing ALNA[Ms]. In certain embodiments, the 3' wing segment of the gapmer consists of 4 linked nucleosides containing ALNA[Ms]. In certain embodiments, the 3' wing segment of the gapmer consists of 5 linked nucleosides containing ALNA[Ms]. In certain embodiments, the 3' wing segment of the gapmer consists of 3, 4 or 5 linked 2'-MOE nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of 1, 2 or 3 linked nucleosides containing ALNA[Ms] and 1 2'-OMe nucleoside. In certain embodiments, the 3' wing segment of the gapmer consists of 1, 2 or 3 linked nucleosides containing ALNA[Ms] and 2 2'-OMe nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of 1, 2 or 3 ALNA[Ms]-containing nucleosides and 3 2'-OMe nucleosides linked. In certain embodiments, the 3' wing segment of the gapmer consists of 1, 2 or 3 ALNA[Ms]-containing nucleosides and 1 2'-MOE nucleoside linked. In certain embodiments, the 3' wing segment of the gapmer consists of 1, 2 or 3 ALNA[Ms]-containing nucleosides and 2 2'-MOE nucleosides linked. In certain embodiments, the 3' wing segment of the gapmer consists of 1, 2 or 3 ALNA[Ms]-containing nucleosides and 3 2'-MOE nucleosides linked. In certain embodiments, the 3' wing segment of the gapmer consists of 1, 2 or 3 ALNA[Ms]-containing nucleosides and 5 2'-MOE nucleosides linked. In certain embodiments, the 3' wing segment of the gapmer consists of 2 LNA-containing nucleosides and 2 2'-MOE nucleosides linked.

[0591] In certain embodiments, the 3' wing segment of the gapmer consists of 1, 2 or 3 nucleosides containing ALNA[Ms] linked together and comprises 1 5-methylcytosine. In certain embodiments, the 3' wing segment of the gapmer consists of 1, 2 or 3 nucleosides containing ALNA[Ms] linked together and comprises 2 5-methylcytosines. In certain embodiments, the 3' wing segment of the gapmer consists of 1, 2 or 3 nucleosides containing ALNA[Ms] linked together and comprises 3 5-methylcytosines. In certain embodiments, the 3' wing segment of the gapmer consists of 1, 2, 3, 4 or 5 2'-MOE nucleosides linked together and comprises 1 5-methylcytosine.

[0592] In certain embodiments, the 5' wing segment of the gapmer consists of 1 nucleoside containing ALNA[Ms], and the 3' wing segment consists of 1 nucleoside containing ALNA[Ms]. In certain embodiments, the 5' wing segment of the gapmer consists of 2 linked nucleosides containing ALNA[Ms], and the 3' wing segment consists of 2 linked nucleosides containing ALNA[Ms]. In certain embodiments, the 5' wing segment of the gapmer consists of 3 linked nucleosides containing ALNA[Ms], and the 3' wing segment consists of 3 linked nucleosides containing ALNA[Ms]. In certain embodiments, the 5' wing segment of the gapmer consists of 4 linked nucleosides containing ALNA[Ms], and the 3' wing segment consists of 4 linked nucleosides containing ALNA[Ms]. In certain embodiments, the 5' wing segment of the gapmer consists of 5 linked nucleosides containing ALNA[Ms], and the 3' wing segment consists of 5 linked nucleosides containing ALNA[Ms].

[0593] In certain embodiments, the 5' wing segment of the gapmer consists of 1, 2 or 3 ALNA[Ms]-containing nucleosides and 1 2'-OMe nucleoside linked, and the 3' wing segment consists of 1, 2 or 3 ALNA[Ms]-containing nucleosides and 1 2'-OMe nucleoside linked. In certain embodiments, the 5' wing segment of the gapmer consists of 1, 2 or 3 ALNA[Ms]-containing nucleosides and 2 2'-OMe nucleosides linked, and the 3' wing segment consists of 1, 2 or 3 ALNA[Ms]-containing nucleosides and 2 2'-OMe nucleosides linked. In certain embodiments, the 5' wing segment of the gapmer consists of 1, 2 or 3 ALNA[Ms]-containing nucleosides and 3 2'-OMe nucleosides linked, and the 3' wing segment consists of 1, 2 or 3 ALNA[Ms]-containing nucleosides and 3 2'-OMe nucleosides linked.

[0594] In certain embodiments, the 5' wing segment of the gapmer consists of 1, 2 or 3 nucleosides containing ALNA [Ms] and 1 or 2 2'-MOE nucleosides linked, and the 3' wing segment consists of 1, 2 or 3 nucleosides containing ALNA [Ms] and 1 or 2 2'MOE nucleosides linked. In certain embodiments, the 5' wing segment of the gapmer consists of 1, 2 or 3 nucleosides containing ALNA [Ms] linked, and the 3' wing segment consists of 1, 2 or 3 nucleosides containing ALNA [Ms] and 1, 2 or 3 2'-MOE nucleosides linked. In certain embodiments, the 5' wing segment of the gapmer consists of 1, 2 or 3 nucleosides containing ALNA [Ms] linked, and the 3' wing segment consists of 1, 2, 3, 4 or 5 2'-MOE nucleosides linked. In certain embodiments, the 5' wing segment of the gapmer consists of 1, 2, 3, 4 or 5 linked 2'-MOE nucleosides, and the 3' wing segment consists of 1, 2 or 3 linked ALNA[Ms].

[0595] In certain embodiments, the 5' wing segment of the gapmer consists of 1, 2 or 3 nucleosides containing ALNA[Ms] linked together and contains 1 5-methylcytosine, and further, the 3' wing segment of the gapmer consists of 1, 2 or 3 nucleosides containing ALNA[Ms] linked together and contains 1 5-methylcytosine. In certain embodiments, the 5' wing segment of the gapmer consists of 1, 2 or 3 nucleosides containing ALNA[Ms] linked together and contains 2 5-methylcytosines, and further, the 3' wing segment of the gapmer consists of 1, 2 or 3 nucleosides containing ALNA[Ms] linked together and contains 1 5-methylcytosine. In certain embodiments, the 5' wing segment of the gapmer consists of 1, 2 or 3 nucleosides containing ALNA[Ms] linked together and contains 2 5-methylcytosines, and further, the 3' wing segment of the gapmer consists of 1, 2 or 3 nucleosides containing ALNA[Ms] linked together and contains 2 5-methylcytosines. In certain embodiments, the 5' wing segment of the gapmer consists of 1, 2 or 3 linked nucleosides containing ALNA[Ms] and contains 3 5-methylcytosines, and further, the 3' wing segment of the gapmer consists of 1, 2 or 3 linked nucleosides containing ALNA[Ms] and contains 3 5-methylcytosines. In certain embodiments, the 5' wing segment of the gapmer consists of 1, 2 or 3 linked nucleosides containing ALNA[Ms] and contains 1 5-methylcytosine, and further, the 3' wing segment of the gapmer consists of 1, 2, 3, 4 or 5 linked 2'-MOE nucleosides and contains 1 5-methylcytosine. In one embodiment, the 5' wing segment of the gapmer consists of 1, 2 or 3 linked nucleosides containing ALNA[Ms], and the 3' wing segment of the gapmer consists of 1, 2 or 3 linked nucleosides containing ALNA[Ms] and 1, 2 or 3 2'-MOE nucleosides, and contains 1 5-methylcytosine.

[0596] In one embodiment, the gap segment of the gapmer comprises 10 consecutive nucleosides, any one of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th or 10th nucleosides comprises a 2'-OMe nucleoside, and the remaining nucleosides are deoxynucleosides.

[0597] Compositions and methods for formulating pharmaceutical compositions

[0598] In order to prepare a pharmaceutical composition or preparation, the modified oligonucleotide can be mixed with one or more pharmaceutically acceptable active or inactive substances. The composition and method for preparing a pharmaceutical composition are determined based on some criteria including the route of administration, the severity of the disease or the amount of administration, but are not limited to these.

[0599] By combining an appropriate pharmaceutically acceptable diluent or carrier with the modified oligonucleotide, the modified oligonucleotide targeting DUX4 nucleic acid can be utilized in a pharmaceutical composition. As a pharmaceutically acceptable diluent, phosphate buffered saline (PBS) can be cited. PBS is a diluent suitable for use in compositions for parenteral delivery. Therefore, in one embodiment, a pharmaceutical composition comprising a modified oligonucleotide targeting DUX4 nucleic acid and a pharmaceutically acceptable diluent can be used in the method described in this specification. In a certain embodiment, the pharmaceutically acceptable diluent is PBS.

[0600] When administered to an animal including a human, a pharmaceutical composition comprising a modified oligonucleotide comprises any pharmaceutically acceptable salt, ester, or salt of such an ester or any other oligonucleotide that can (directly or indirectly) provide a biologically active metabolite or residue thereof. Thus, for example, the present disclosure also relates to pharmaceutically acceptable salts, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other biological equivalents of modified oligonucleotides. As suitable pharmaceutically acceptable salts, sodium salts and potassium salts can be cited, but are not limited to these.

[0601] In order to form an active modified oligonucleotide, the prodrug may comprise incorporating additional nucleosides at one or both ends of the modified oligonucleotide, which is cleaved by endogenous nucleases in vivo.

[0602] Conjugated oligonucleotides

[0603] The modified oligonucleotide can be covalently bonded to one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the resulting modified oligonucleotide. Typical conjugated groups include cholesterol moieties and lipid moieties. Further conjugated groups include carbohydrates, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin and pigments.

[0604] For example, in order to enhance properties such as nuclease stability, the modified oligonucleotide can also be modified in a manner having one or more stabilizing groups generally bonded to one or both ends of the modified oligonucleotide. Stabilizing groups include cap structures. These terminal modifications protect the modified oligonucleotides with terminal nucleic acids from exonuclease decomposition, which can contribute to intracellular delivery and / or positioning. The cap can be present at the 5' end (5' cap) or the 3' end (3' cap), or at both ends. The cap structure is well known in the art, for example, an inverted deoxy abasic cap can be cited. As further 3' and 5' stabilizing groups that can be used to cap one or both ends of the modified oligonucleotide to impart nuclease stability, the stabilizing groups disclosed in WO03 / 004602 can be cited.

[0605] Cell culture and modified oligonucleotide treatment

[0606] The effect of modified oligonucleotides on the level, activity or expression of DUX4 nucleic acid can be tested in vitro in various cell types. The cell types used in such analysis can be obtained from commercial suppliers (e.g., American Cell Culture System Preservation Agency, Manassus, VA; Zen-Bio, Inc., Research Triangle Park, NC; Clonetics Corporation, Walkersville, MD), and the cells are cultured using commercially available reagents (e.g., Invitrogen Life Technologies, Carlsbad, CA) according to the supplier's instructions. As exemplary cell types, C2C12 cells, HepG2 cells, Hep3B cells, primary hepatocytes, A549 cells, GM04281 fibroblasts, and LLC-MK2 cells can be cited, but are not limited to these. These cells can be transfected with vectors expressing human DUX4 mRNA for use. As a vector, it is preferably expressed as a fusion protein with a reporter gene such as luciferase or GFP, for example, psiCHECK-2 vector (Promega) can be cited.

[0607] In vitro testing of modified oligonucleotides

[0608] The method of treating cells with modified oligonucleotides is described in the present specification, and this can be appropriately changed depending on the type of modified oligonucleotides.

[0609] Typically, cells are treated with modified oligonucleotides when they reach about 60-80% confluence in culture.

[0610] The modified oligonucleotides are introduced into cells, for example, by lipofection.

[0611] As a reagent commonly used to introduce modified oligonucleotides into cultured cells, LIPOFECTIN (registered trademark) (Invitrogen, Carlsbad, CA), a cationic lipid transfection reagent, can be cited. In OPTI-MEM (registered trademark) 1 (Invitrogen, Carlsbad, CA), the modified oligonucleotide and LIPOFECTIN (registered trademark) are mixed to obtain the desired final concentration of the modified oligonucleotide and typically a LIPOFECTIN (registered trademark) concentration of 2 to 12 μg / mL per 100 nM of the modified oligonucleotide.

[0612] As other reagents for introducing modified oligonucleotides into cultured cells, LIPOFECTAMINE 2000 (registered trademark) (Invitrogen, Carlsbad, CA) can be cited. In OPTI-MEM (registered trademark) 1 serum-reduced medium (Invitrogen, Carlsbad, CA), the modified oligonucleotide is mixed with LIPOFECTAMINE 2000 (registered trademark) to obtain the desired concentration of the modified oligonucleotide and typically a LIPOFECTIN (registered trademark) concentration of 2 to 12 μg / mL per 100 nM of the modified oligonucleotide.

[0613] As other reagents for introducing modified oligonucleotides into cultured cells, Cytofectin (registered trademark) (Invitrogen, Carlsbad, CA) can be cited. The modified oligonucleotides are mixed with Cytofectin (registered trademark) in OPTI-MEM (registered trademark) 1 serum-reduced medium (Invitrogen, Carlsbad, CA) to obtain the desired concentration of the modified oligonucleotide and typically a Cytofectin (registered trademark) concentration of 2 to 12 μg / mL per 100 nM modified oligonucleotide.

[0614] Another method for introducing modified oligonucleotides into cultured cells is electroporation.

[0615] The modified oligonucleotide can be introduced into cells even without using LIPOFECTIN etc. The method of suppressing the expression of the target gene using the modified oligonucleotide in this case is called the Gymnosis method.

[0616] Cells are treated with modified oligonucleotides by conventional methods. Typically, cells are collected 16 to 48 hours after modified oligonucleotide treatment, at which point RNA or protein levels of the target nucleic acid are measured by methods known in the art and described in this specification. Typically, when treatment is repeated multiple times, data are displayed as the average of the repeated treatments.

[0617] The concentration of the modified oligonucleotide used varies depending on the cell system. Methods for determining the most suitable modified oligonucleotide concentration for a specific cell system are well known in the art. When LIPOFECTAMINE 2000 (registered trademark), LIPOFECTIN or Cytofectin is used for transfection, typically, the modified oligonucleotide is used at a concentration of 1 nM to 300 nM. When electroporation is used for transfection, the modified oligonucleotide is used at a higher concentration of 625 to 20000 nM. The modified oligonucleotide concentration IC50 that inhibits 50% of gene expression can be calculated from the inhibition rate of gene expression at each concentration.

[0618] RNA Isolation

[0619] RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. Methods for RNA isolation are well known in the art. For RNA, methods known in the art are used, such as TRIZOL (registered trademark) reagent (Invitrogen, Carlsbad, CA) are used to prepare according to the manufacturer's recommended protocol.

[0620] Analysis of inhibition of target levels or expression

[0621] Inhibition of DUX4 nucleic acid levels or expression can be analyzed by various methods known in the art. For example, target nucleic acid levels can be quantified, for example, by Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitative real-time PCR. RNA analysis can be performed on total cellular RNA or poly (A) + mRNA. Methods for RNA isolation are well known in the art. Northern blot analysis is also customary in the art. Quantitative real-time PCR can be conveniently implemented using a commercially available ABI PRISM (registered trademark) 7600, 7700, or 7900 sequence detection system used in accordance with the manufacturer's instructions available from PE-Applied Biosystems, Foster City, CA.

[0622] Quantitative real-time PCR analysis of target RNA levels

[0623] Quantification of target RNA levels can be achieved by quantitative real-time PCR using the ABI PRISM (registered trademark) 7600, 7700 or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Quantitative real-time PCR methods are well known in the art.

[0624] Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction, thereby generating complementary DNA (cDNA) that is subsequently used as a substrate for real-time PCR amplification. RT and real-time PCR reactions are performed sequentially in the same sample well. RT and real-time PCR reagents can be obtained from Invitrogen (Carlsbad, CA). RT and real-time PCR reactions are performed by methods known to those skilled in the art.

[0625] The amount of gene (or RNA) target obtained by real-time PCR is standardized by using the expression level of a gene with constant expression such as cyclophilin A, or by quantifying total RNA using RIBOGREEN (registered trademark) (Invitrogen, Inc. Carlsbad, CA). The expression of cyclophilin A is quantified by real-time PCR multiple times or individually with the target. Total RNA is quantified using RIBOGREEN (registered trademark) RNA quantification reagent (Invitrogen, Inc. Eugene, OR). The quantification method of RNA using RIBOGREEN (registered trademark) is taught in Jones, LJ, et al, (Analytical Biochemistry, 1998, 265, 368-374). RIBOGREEN (registered trademark) fluorescence is measured using CYTOFLUOR (registered trademark) 4000 equipment (PE Applied Biosystems).

[0626] Probes and primers are designed to hybridize to DUX4 nucleic acid. Methods for designing probes and primers for real-time PCR are well known in the art and may include the use of software such as PRIMER EXPRESS (registered trademark) software (Applied Biosystems, Foster City, CA).

[0627] Protein level analysis

[0628] Antisense inhibition of DUX4 nucleic acid is evaluated by measuring the level of DUX4 protein. The level of DUX4 protein can be investigated or quantified by various methods known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein analysis, protein activity analysis (e.g., caspase activity analysis), immunohistochemistry, immunocytochemistry, or fluorescence activated cell sorting (FACS). Antibodies against the target can be determined and obtained from various supply sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or can be prepared by conventional monoclonal or polyclonal antibody production methods known in the art.

[0629] Analysis of gene expression

[0630] The level of DUX4 gene expression can also be measured using a reporter gene such as luciferase. For example, when using psiCHECK-2 vector (Promega), the expression of the DUX4 gene can be measured by the luminescence of Renilla luciferase, which is a fusion protein with DUX4, and by correcting with the luminescence of firefly luciferase present on the same vector, the effects of nonspecific cell death can be eliminated.

[0631] In vivo testing of modified oligonucleotides

[0632] In order to evaluate the ability of modified oligonucleotides to inhibit DUX4 expression and cause phenotypic changes, experiments are performed in animals. The experiments can be performed in normal animals or experimental disease models, such as DUX4 transgenic mouse models (Jones, T. et al. PLoS One. 2018; 13 (2), Article number e0192657), DUX4 gene expression mice using genetically recombinant AAV viruses (Wallace, LM et al. Mol Ther. 2012; 20 (7): 1417, Wallace, LM et al. Ann Neurol. 2011; 69 (3): 540).

[0633] For administration to animals, the modified oligonucleotide is formulated in a pharmaceutically acceptable diluent, such as phosphate buffered saline. Administration includes parenteral administration routes. After the treatment period with the modified oligonucleotide, RNA is isolated from the tissue and changes in DUX4 nucleic acid expression are measured. Changes in the level of DUX4 protein are also measured.

[0634] Some kind of antisense mechanism

[0635] FSHD is caused by abnormal expression of the DUX4 gene (particularly a splice variant of DUX4-FL) in muscle. On the other hand, DUX4 is also expressed in healthy people, for example, in the testis. Among certain splice variants of DUX4 expressed in the testis, in addition to DUX4-FL, splice variants of exon 1, exon 2, exon 6, and exon 7, and splice variants of exon 1, exon 2, exon 4, exon 5, exon 6, and exon 7 are expressed (Non-Patent Document 1 above).

[0636] A biomarker

[0637] The expression of genes such as MBD3L2, ZSCAN4, TRIM43, DEFB103, and ZNF217 is regulated at least in part by the accumulation level of DUX4 protein (Non-Patent Document 2). In addition, creatine kinase in blood can be measured as a marker for muscle disorders.

[0638] Certain indications

[0639] In certain embodiments, the present disclosure provides a method of treating an individual, comprising administering one or more pharmaceutical compositions described in the present disclosure. In certain embodiments, the individual has FSHD.

[0640] Therefore, the present specification provides a method for improving symptoms associated with FSHD in a subject in need of improving symptoms associated with FSHD. In a certain embodiment, a method for reducing the incidence of one or more symptoms associated with FSHD is provided. In a certain embodiment, a method for reducing the severity of symptoms associated with FSHD is provided. In a certain embodiment, symptoms associated with FSHD include muscle stiffness, myotonia, facial muscle weakness, ptosis, inability to whistle, reduced facial expression changes, depressed or angry facial expressions, dysphonia, scapular muscle weakness (deformities such as winged scapula and oblique shoulders), lower limb weakness, hearing loss and heart disease.

[0641] In certain embodiments, the methods of the invention comprise administering to a subject in need thereof a therapeutically effective amount of a compound that targets a DUX4 nucleic acid.

[0642] In certain embodiments, administration of a modified oligonucleotide targeting a DUX4 nucleic acid results in a decrease in the expression of DUX4 of at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%, or a range determined by any two of these values.

[0643] In certain embodiments, a pharmaceutical composition comprising a modified oligonucleotide targeting DUX4 can be used to prepare a medicament for treating a patient suffering from or susceptible to a DUX4-related disease such as FSHD.

[0644] In one embodiment, the method described in the present specification includes administering a compound comprising a modified oligonucleotide having a sequence described in SEQ ID NOs. 2, 3, 4, 7 to 64, 69 to 97 or 102 to 112 of the sequence listing and a continuous nucleic acid base portion described in the present specification.

[0645] Drug administration

[0646] In one embodiment, the compound and pharmaceutical composition described in the present specification are administered parenterally.

[0647] In certain embodiments, the parenteral administration is performed by injection. The injection can be continuous or long-term, or short-term or intermittent. In certain embodiments, the injected pharmaceutical agent is delivered using a pump. In certain embodiments, the parenteral administration is performed by injection (e.g., bolus injection). The injection can be delivered using a syringe.

[0648] Examples of parenteral administration include subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, such as intramedullary or intraventricular administration. Administration may be continuous or long-term, short-term or intermittent.

[0649] In certain embodiments, the delivery of a compound of a pharmaceutical composition described in this specification results in at least 70% downregulation of the level of a target mRNA and / or a target protein. In certain embodiments, the delivery of a compound or composition described in this specification lasts for at least 1 day, at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 20 days, at least 21 days, at least 28 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 55 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 76 days, at least 77 days, at least 78 days. The invention also provides for 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% downregulation of the levels of target mRNA and / or target protein for at least 1 day, at least 79 days, at least 80 days, at least 85 days, at least 90 days, at least 95 days, at least 100 days, at least 105 days, at least 110 days, at least 115 days, at least 120 days, or at least 1 year.

[0650] In certain embodiments, the modified oligonucleotide is delivered by injection or infusion once a day, once every three days, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, once every six months, twice a year, or once a year.

[0651] Certain combined therapies

[0652] In certain embodiments, a first agent comprising a modified oligonucleotide of the present invention is co-administered with one or more second agents. In certain embodiments, such a second agent is designed to treat the same FSHD as the first agent described in this specification. In certain embodiments, such a second agent is designed to treat a disease, disorder or state different from the first agent described in this specification. In certain embodiments, such a second agent is designed to treat an undesirable side effect of one or more pharmaceutical compositions described in this specification. In certain embodiments, the second agent is co-administered with the first agent in order to treat the undesirable effects of the first agent. In certain embodiments, the second agent is co-administered with the first agent in order to bring about a combined effect. In certain embodiments, the second agent is co-administered with the first agent in order to bring about a synergistic effect.

[0653] In certain embodiments, the first agent and one or more second agents are administered simultaneously. In certain embodiments, the first agent and one or more second agents are administered at different times. In certain embodiments, the first agent and one or more second agents are prepared together in a single pharmaceutical preparation. In certain embodiments, the first agent and one or more second agents are prepared separately.

[0654] Certain compounds

[0655] In one embodiment, the compounds disclosed in this specification can be synthesized into oligomers by the phosphoramidite method using commercially available amidites for DNA and RNA synthesis (including LNA). The artificial nucleic acid GuNA can be synthesized into oligomers by the method described in WO2014 / 046212 and WO2017 / 047816. The artificial nucleic acids ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Trz] and ALNA[Oxz] can be synthesized into oligomers by the method described in Japanese Patent Application No. 2018-212424.

[0656] In certain embodiments, the compounds disclosed herein benefit from improved one or more in vitro and / or in vivo properties compared to appropriate comparative compounds.

[0657] In a certain embodiment, it is a compound No. 1 having a sequence of (5' to 3')ngagattcccgccggt (n is 5-methylcytosine, incorporated in this specification as Sequence No. 2), that is, a compound in which the 5' flank and the 3' flank are each composed of a gap body consisting of 3 LNA-containing nucleosides, and the bonds between each nucleoside are phosphorothioate bonds.

[0658] In a certain embodiment, it is a compound numbered 2 having a sequence of (5' to 3')gnagttctccgcggt (n is 5-methylcytosine, incorporated in this specification as sequence number 3 in the sequence list), that is, a compound in which the 5' flank and the 3' flank are each composed of a gap body consisting of 3 nucleosides containing ALNA[Ms], and the bonds between each nucleoside are phosphorothioate bonds.

[0659] In a certain embodiment, it is a compound No. 3 having a sequence of (5' to 3')gnntagacagcgtngg (n is 5-methylcytosine, incorporated in this specification as sequence No. 4 in the sequence list), that is, a compound in which the 5' side and the 3' side are gap bodies consisting of 3 LNA-containing nucleosides each, and the bonds between each nucleoside are phosphorothioate bonds.

[0660] In a certain embodiment, it is a compound No. 123 having a sequence of (5' to 3')gnntagacagcgtngg (n is 5-methylcytosine, incorporated in this specification as sequence No. 4 in the sequence list), that is, a compound in which the 5' flank and the 3' flank are each composed of 3 nucleosides containing ALNA[Ms], and the bonds between each nucleoside are phosphorothioate bonds.

[0661] Non-Limiting Disclosure and Incorporation by Reference

[0662] Certain compounds, compositions and methods described in this specification are specifically described according to certain embodiments, but the following examples only serve to illustrate the compounds described in this specification and are not intended to limit the present invention. References, GENBANK accession numbers, etc. described in this application are incorporated into this specification in their entirety by reference.

[0663] The sequence table attached to the application specifies each sequence in the form of any one of "RNA" or "DNA" as needed, but in fact these sequences can be modified by any combination of chemical modifications. It can be easily understood by those skilled in the art that the designation of "RNA" or "DNA" for illustrating modified oligonucleotides is arbitrary according to the circumstances. For example, an oligonucleotide comprising a nucleoside containing a 2'-OH sugar moiety and a thymine base can be described as a DNA with a modified sugar (2'-OH relative to the natural 2'-H of DNA), or an RNA with a modified base (thymine (methylated uracil) relative to the natural uracil of RNA).

[0664] Therefore, the nucleic acid sequences provided in this specification including the nucleic acid sequences in the sequence table are not limited to these, and are intended to include nucleic acids containing natural or modified RNA and / or DNA in any combination including nucleic acids having modified nucleic acid bases, but are not limited to these. Although not limited, as a further example, the oligomer compound having the nucleic acid base sequence "ATCGATCG" may be modified or unmodified, including any oligomer compound having such a nucleic acid base sequence, including such compounds containing RNA bases, such as compounds having the sequence "AUCGAUCG", and compounds having some DNA bases and some RNA bases such as "AUCGATCG", and oligomer compounds having other modified or naturally occurring bases such as "ATmeCGAUCG" (here, meC represents a cytosine base containing a methyl group at position 5), but are not limited to these.

[0665] Example

[0666] Non-Limiting Disclosure and Incorporation by Reference

[0667] Certain compounds, compositions and methods described in this specification are specifically described according to certain embodiments, but the following examples only serve to illustrate the compounds described in this specification and are not intended to limit the present invention. The references described in this application are incorporated into this specification in their entirety by reference.

[0668] The structures of the artificial nucleic acids used in this specification are shown in the following structural formulas together with their respective abbreviations.

[0669] The structures and abbreviations of artificial nucleic acids

[0670]

[0671] Example 1

[0672] Synthesis and purification of modified oligonucleotide compounds for in vitro evaluation

[0673] Using various amidites (LNA amidite was purchased from Chem Genes and Hongene Biotechnology Limited, 2'-OMe amidite was purchased from Sigma-Aldrich, GuNA was synthesized by the method described in WO2014 / 046212 and WO2017 / 047816, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Trz] and ALNA[Oxz] were synthesized by the method described in Japanese Patent Application No. 2018-212424), modified oligonucleotide compounds were synthesized using CPG or polystyrene carriers on a 0.2 or 1.0 μmol scale using a DNA / RNA oligonucleotide automatic synthesizer nS-8II (GeneDesign Co., Ltd.). All amidites were adjusted to 0.1M acetonitrile solution, the coupling time in non-natural nucleosides was 10 minutes, and the other steps were performed under the standard conditions of nS-8II. Activator 42 (Sigma-Aldrich) was used as the activator, and Sulfurizing Reagent II (Gren Research Corporation) was used for sulfurization. The synthesized oligonucleotide was added with 28% ammonia solution and reacted at 60-65°C for 8 hours to perform excision from the carrier and deprotection of the base. After the ammonia was concentrated and distilled off, it was purified by reverse phase HPLC.

[0674] Example 2

[0675] Synthesis and purification of modified oligonucleotide compounds for in vivo evaluation

[0676] Using various amidites, using the DNA / RNA oligonucleotide automatic synthesizer AKTA oligopilot plus 10 (manufactured by GE Healthcare Japan Co., Ltd.), modified oligonucleotide compounds were synthesized using a polystyrene carrier at a scale of 20 to 50 μmol. DNA amidite was adjusted to 0.1M, non-natural amidite was adjusted to 0.05 to 0.1M acetonitrile solution, the coupling recycle time in non-natural nucleosides was 20 minutes, and when the first base was introduced into the universal carrier (universal carrier), the coupling, sulfurization, and capping steps were performed twice in succession. The other steps were carried out under the standard conditions of AKTA oligopilot plus 10. Activator 42 (Sigma-Aldrich) was used as the activator, and Sulfurizing Reagent II (Gren Research Corporation) was used for sulfurization. The synthesized oligonucleotide is decyanoethylated on a solid phase using 20% ​​diethylamine / acetonitrile or 50% triethylamine / acetonitrile, and a 28% aqueous ammonia solution is added and reacted at 60-65°C for 8-24 hours to perform excision from the carrier and deprotection of the base portion. After the ammonia is concentrated and distilled off, it is purified using an anion exchange column. The remaining salt contained after the anion exchange is removed using a desalting column.

[0677] Example 3

[0678] Purity confirmation of modified oligonucleotide compounds

[0679] The purification and purity of the synthesized modified oligonucleotide compounds were confirmed by reverse phase HPLC under the following conditions. The purity of all the compounds was 85% or more.

[0680] Reverse phase HPLC (Purification)

[0681] Mobile Phase:

[0682] Solution A: 400mM hexafluoroisopropanol, 15mM triethylamine

[0683] Liquid B: Methanol

[0684] Gradient: A:B = 85:15 → 70:30 (10 min)

[0685] Use column:

[0686] AssignWaters XBridge@Oligonucleotide BEH C18 OBDTMPrep Column, 2.5μm,10mm*50mm

[0687] Flow Rate:

[0688] Dispensing 5mL / min

[0689] Column temperature: 60℃.

[0690] Detection: UV (260nm)

[0691] Reverse phase HPLC (purity confirmation)

[0692] Mobile Phase:

[0693] Solution A: 400mM hexafluoroisopropanol, 15mM triethylamine aqueous solution Solution B: methanol

[0694] Gradient: A:B = 80:20 → 70:30 (6.5 min)

[0695] Use column:

[0696] AnalyzeWaters ACQUITY UPLC@Oligonucleotide BEH C18Column, 1.7μm,2.1mm*50mm

[0697] Flow rate: 0.2mL / min

[0698] Column temperature: 60℃.

[0699] Detection: UV (260nm)

[0700] Anion exchange purification

[0701] Mobile Phase:

[0702] Solution A: 1mM NaOH 20% acetonitrile aqueous solution

[0703] Solution B: 1 mM NaOH, 1.5 M NaCl / 20% acetonitrile aqueous solution

[0704] Column used: TSKgel SuperQ-5PW(13)

[0705] Flow rate: 7mL / min

[0706] Column temperature: room temperature

[0707] Detection: UV (260nm)

[0708] Desalting columns

[0709] Mobile Phase:

[0710] Solution A: 20% acetonitrile aqueous solution

[0711] Solution B: 20% acetonitrile in water

[0712] Use column:

[0713] GE HiPrep 26 / 10 Desalting * 4 in series

[0714] Flow rate: 12mL / min

[0715] Column temperature: room temperature

[0716] Example 4

[0717] Molecular weight determination of modified oligonucleotide compounds

[0718] The molecular weight of the synthesized modified oligonucleotide compound was measured using Waters ZQ under the following conditions.

[0719] Mobile Phase:

[0720] Solution A: 400mM hexafluoroisopropanol, 15mM triethylamine aqueous solution

[0721] Liquid B: Methanol

[0722] Gradient: A:B = 80:20 → 70:30 (6.5 min)

[0723] Use column:

[0724] Waters ACQUITY UPLC@Oligonucleotide BEH C18 Column, 1.7μm,2.1mm*50mm

[0725] Flow rate: 0.2mL / min

[0726] Column temperature: 60℃.

[0727] Detection: UV (260nm)

[0728] Example 5

[0729] Molecular weight of synthetic modified oligonucleotide compounds

[0730] The synthesized modified oligonucleotide compounds are shown in the following Table 1. In the description of the compounds, each nucleotide is represented by three letters. Among them, the nucleotide at the 3' end has no internucleoside bond and is therefore represented by two letters.

[0731] 1) The first letter is capitalized, indicating the following nucleic acid bases:

[0732] A = adenine, T = thymine, G = guanine, C = cytosine, U = uracil, M = 5-methylcytosine,

[0733] 2) The second letter represents the following sugar moieties:

[0734] l=LNA, g=GuNA, m=ALNA[Ms], u=ALNA[mU], p=ALNA[ipU], t=ALNA[Trz], e=2′-MOE, o=2′-OMe, d=2′-deoxyribose,

[0735] 3) The third letter represents the following internucleoside bond:

[0736] s = phosphorothioate, p = phosphodiester.

[0737] As the target position, the 5' target site of DUX4 mature mRNA of the modified oligonucleotide (the position corresponding to SEQ ID NO: 1 in the sequence listing at the 3' end of the modified oligonucleotide) is shown.

[0738] [Table 1-1]

[0739]

[0740] [Table 1-2]

[0741] (Continued Table 1)

[0742]

[0743] [Table 1-3]

[0744] (Continued Table 1)

[0745]

[0746] [Table 1-4]

[0747] (Continued Table 1)

[0748]

[0749] [Table 1-5]

[0750] (Continued Table 1)

[0751]

[0752] [Table 1-6]

[0753] (Continued Table 1)

[0754]

[0755] Example 6

[0756] In vitro DUX4 knockdown activity assay (lipid transfection method)

[0757] On a transfection reagent mixed with DUX4 modified oligonucleotide and lipofectamine RNAi reagent, 1.25 × 104 Cells / cm 2 C2C12 cells were inoculated and cultured in a CO2 incubator overnight. On the next day, the cells were transfected with a reporter plasmid in which the DUX4 sequence was cloned in the multiple cloning site of the psiCHECK-2 vector (Promega) using the Lipofectamine 2000 reagent, and cultured in a CO2 incubator for about 24 hours. Then, the Dual-Glo Luciferase Assay System was used to detect the luminescence values ​​of firefly luciferase and Renilla luciferase in the cells using a microplate reader. In order to correct the effects of transfection efficiency and cell number using the luminescence value generated by Renilla luciferase activity, the ratio to the luminescence value generated by firefly luciferase activity was calculated. The inhibition rate was calculated based on the reduction rate of Renilla / firefly when the modified oligonucleotide was added, with 100 points, and the IC was calculated from the concentrations at 2 points before and after 50% and the inhibition rate at this time. 50 Compared with the compounds complementary to positions 232 to 248, 1306 to 1325, or 1472 to 1495 of DUX4 mature mRNA (compound numbers 1 to 132), it can be seen that the inhibition rates of compounds No. 133 (complementary to positions 214 to 227 of sequence number 1 of the sequence list), 134 (complementary to positions 1323 to 1336 of sequence number 1 of the sequence list), 135 (complementary to positions 1458 to 1471 of sequence number 1 of the sequence list), and 136 (complementary to positions 1495 to 1508 of sequence number 1 of the sequence list) are significantly weaker.

[0758] Example 7

[0759] In vitro DUX4 knockdown activity assay (Gymnosis method)

[0760] In the DUX4 modified oligonucleotide solution, 6 × 10 3 Cells / cm 2 C2C12 cells were inoculated and cultured in a CO2 incubator for 2 nights. After 2 days, the culture medium containing the DUX4 modified oligonucleotide solution was removed from the cells and washed with new culture medium. Then, the cells were transfected with a reporter plasmid in which the DUX4 sequence was cloned in the multiple cloning site of the psiCHECK-2 vector (Promega) using the Lipofectamine 2000 reagent and cultured in a CO2 incubator for about 24 hours. Then, the luminescence values ​​of firefly luciferase and Renilla luciferase in the cells were detected using a microplate reader using the Dual-Glo Luciferase Assay System. In order to correct the effects of transfection efficiency and cell number using the luminescence value generated by Renilla luciferase activity, the ratio to the luminescence value generated by firefly luciferase activity was calculated. The inhibition rate was calculated based on the reduction rate of Renilla / firefly when the modified oligonucleotide was added, with 100 points, and the IC was calculated from the concentrations at 2 points before and after 50% and the inhibition rate at this time.50 The results are shown in Table 2 below. Compared with the compounds containing nucleic acid base sequences complementary to equal length portions in the region of positions 232 to 248, 1306 to 1325, or 1472 to 1495 of DUX4 mature mRNA (compound numbers 1 to 132), it is known that the inhibition rates of compound numbers 133 (complementary to positions 214 to 227 of sequence number 1 of the sequence list), 134 (complementary to positions 1323 to 1336 of sequence number 1 of the sequence list), 135 (complementary to positions 1458 to 1471 of sequence number 1 of the sequence list), and 136 (complementary to positions 1495 to 1508 of sequence number 1 of the sequence list) are significantly weaker.

[0761] [Table 2-1]

[0762]

[0763] [Table 2-2]

[0764] (Continued Table 2)

[0765] 35 0.009 0.105 36 0.006 0.390 37 0.003 0.133 38 0.005 0.125 39 0.006 0.143 40 0.002 0.051 41 0.007 0.184 42 0.008 0.166 43 0.008 0.148 44 0.009 0.174 45 0.023 0.699 46 0.003 0.109 47 0.005 0.181 48 0.005 0.166 49 0.009 0.261 50 0.003 0.206 51 0.005 0.419 52 0.006 0.414 53 0.016 0.164 54 0.010 0.402 55 0.006 0.052 56 <0.03 0.577 57 0.012 0.435 58 0.002 0.058 59 0.002 0.052 60 0.005 0.100 61 0.002 0.048 62 0.003 0.038 63 0.004 0.090 64 0.019 0.426 65 0.002 0.128 66 0.002 0.066 67 0.003 0.188 68 0.013 0.850 69 0.002 0.083 70 0.005 0.111

[0766] [Table 2-3]

[0767] (Continued Table 2)

[0768] 71 0.002 0.037 72 0.002 0.072 73 0.005 0.292 74 0.002 0.079 75 0.005 0.237 76 0.003 0.073 77 0.004 0.091 78 0.010 0.132 79 0.010 0.099 80 0.009 0.373 81 0.003 0.115 82 0.011 0.529 83 0.003 0.113 84 0.007 0.207 85 0.017 0.400 86 0.005 0.106 87 0.005 0.097 88 0.007 0.154 89 0.019 0.434 90 0.008 0.077 91 0.009 0.148 92 0.015 0.763 93 0.007 0.135 94 <0.03 0.793 95 0.021 0.496 96 0.036 0.306 97 0.009 0.546 98 0.010 0.363 99 0.008 0.624 100 0.014 0.669 101 0.029 0.443 102 0.014 0.657 103 0.011 0.307 104 0.007 0.255 105 0.005 0.122 106 0.006 0.291

[0769] [Table 2-4]

[0770] (Continued Table 2)

[0771] 107 0.004 0.321 108 0.076 0.877 109 0.075 0.730 110 0.020 0.266 111 0.017 0.223 112 0.050 0.616 113 0.016 0.569 114 0.013 0.259 115 0.011 0.531 116 0.003 0.112 117 0.003 0.092 118 0.015 0.461 119 0.004 0.467 120 0.004 0.456 121 0.008 0.239 122 0.006 0.289 123 0.013 0.154 124 0.003 0.338 125 0.010 0.272 126 0.004 0.159 127 0.003 0.093 128 0.006 0.256 129 0.008 0.088 130 0.003 0.079 131 0.005 0.245 132 0.006 0.294 133 >0.3 >3 134 >0.3 >3 135 0.140 >3 136 >0.3 >3

[0772] Example 8

[0773] Synthesis of modified oligonucleotide compounds and in vitro DUX4 knockdown activity assay (Gymnosis method)

[0774] Table 3 shows the newly synthesized modified oligonucleotide compounds and the results of the in vitro DUX4 knockdown activity test performed in the same manner as in Example 7 on the compounds.

[0775] In addition, in the description of the compounds in Table 3, each nucleotide is represented by three letters. Among them, the nucleotide at the 3' end has no internucleoside bond and is therefore represented by two letters.

[0776] 1) The first letter is capitalized, indicating the following nucleic acid bases:

[0777] A = adenine, T = thymine, G = guanine, C = cytosine, U = uracil, M = 5-methylcytosine,

[0778] 2) The second letter represents the following sugar moieties:

[0779] l=LNA, m=ALNA[Ms], e=2'-MOE, o=2'-OMe, d=2'-deoxyribose,

[0780] 3) The third letter represents the following internucleoside bond:

[0781] s = phosphorothioate, p = phosphodiester.

[0782] As the target position, the 5' target site of DUX4 mature mRNA of the modified oligonucleotide (the position corresponding to SEQ ID NO: 1 in the sequence listing at the 3' end of the modified oligonucleotide) is shown.

[0783] Compared with the compounds containing nucleic acid base sequences complementary to equal-length portions within the region of positions 126 to 147, 232 to 248, 1306 to 1325 or 1472 to 1495 of DUX4 mature mRNA (compound numbers 137 to 247), it can be seen that the inhibition rates of compounds numbered 248 (complementary to positions 112 to 127 of sequence number 1 in the sequence listing), 249 (complementary to positions 162 to 177 of sequence number 1 in the sequence listing), 250 (complementary to positions 264 to 279 of sequence number 1 in the sequence listing), and 251 (complementary to positions 1273 to 1288 of sequence number 1 in the sequence listing) are significantly weaker.

[0784] [Table 3]

[0785] [Table 3-1]

[0786]

[0787] Table 3

[0788] [Table 3-2]

[0789]

[0790] Example 9

[0791] In vivo DUX4 knockdown activity assay

[0792] Adeno-associated virus vector AAV-DUX4 (SignaGen Laboratories, Cat. #S L100862) expressing DUX4 mature mRNA of sequence number 1 in the sequence list was prepared. AAV-DUX4 was intramuscularly administered to the tibialis anterior muscle of 8-week-old C57BL / 6J mice (male, Charles River, Japan) at 1E+10 VG / 50 μL under isoflurane (Pfizer) anesthesia. Three days later, modified oligonucleotides targeting DUX4 were prepared in physiological saline at 1, 3, 10, and 50 mg / 5 mL / kg, and intravenously administered to 8-week-old C57BL / 6J mice (male, Charles River, Japan). After 72 hours, whole blood was collected from the abdominal vena cava under cervical dislocation or isoflurane (Pfizer) anesthesia, and the mice were killed. After death, the tibialis anterior muscle was collected, immersed in RNAlater Soln. (invitrogen), and frozen at -80°C. The homogenization buffer of Maxwell RSC simplyRNA Tissue Kit (Promega) was added to the tissue, and it was disrupted using a multi-bead oscillator, and the RNA was purified according to the protocol described in the kit. 400 ng of RNA was reverse transcribed, and the obtained cDNA was used for quantitative PCR. The knockdown activity of the modified oligonucleotide is expressed as a relative value relative to the vector group to express the quantitative ratio of 18S rRNA relative to DUX4. The results at 1, 3, 10 and 50 mg / 5 mL / kg are shown in Figures 1 to 4Compound No. 1 (sequence complementary to positions 233 to 248 of DUX4 mature mRNA), Compound No. 2 (sequence complementary to positions 1309 to 1323 of DUX4 mature mRNA), Compound No. 3 (sequence complementary to positions 1480 to 1495 of DUX4 mature mRNA), Compound No. 13 (sequence complementary to positions 234 to 247 of DUX4 mature mRNA), Compound No. 41 (sequence complementary to positions 1308 to 1323 of DUX4 mature mRNA), Compound No. 54 (sequence complementary to positions 1309 to 1323 of DUX4 mature mRNA), Compound No. 57 (sequence complementary to positions 1480 to 1495 of DUX4 mature mRNA), Compound No. 13 (sequence complementary to positions 234 to 247 of DUX4 mature mRNA), Compound No. 41 (sequence complementary to positions 1308 to 1323 of DUX4 mature mRNA), Compound No. 57 (sequence complementary to positions 1480 to 1495 of DUX4 mature mRNA), Compound No. 13 ... The following compounds: compound No. 68 (a sequence complementary to positions 1309 to 1323 of DUX4 mature mRNA), compound No. 78 (a sequence complementary to positions 1309 to 1324 of DUX4 mature mRNA), compound No. 88 (a sequence complementary to positions 1310 to 1323 of DUX4 mature mRNA), compound No. 104 (a sequence complementary to positions 1473 to 1488 of DUX4 mature mRNA), and compound No. 122 (a sequence complementary to positions 1480 to 1495 of DUX4 mature mRNA) can inhibit the expression of the DUX4 gene in the muscle even when administered to a living body.

[0793] Example 10

[0794] Safety of modified oligonucleotides

[0795] Compounds No. 3, 42, and 123 were intravenously administered to 6-week-old ICR mice (male, Charles River, Japan) at a maximum dose of 100 mg / kg. No hepatotoxicity (increase in ALT and AST in the blood and abnormal pathological findings), nephrotoxicity (increase in UN and creatinine in the blood and abnormal pathological findings), general symptom changes, or death was observed.

[0796] Embodiment 11

[0797] In vivo Tg mouse DUX4 knockdown activity assay

[0798] 9-week-old male FLExDUX4-heteto / HSA-MCM-hetero:TG (DUX4-Tg) and FLExDUX4-wild / HSA-MCM-hetero:TG (MCM, control) (male, introduced from The Jackson Laboratory to Charles River Co., Ltd., Japan) were used. Modified oligonucleotides targeting DUX4 were prepared using physiological saline in such a way that the dosage of each dosage solution became 5 mL / kg, and the modified oligonucleotides were administered intravenously into the tail vein once a week. One week after the end of the 4-week administration, whole blood was collected from the abdominal vena cava under deep anesthesia with isoflurane, and the animals were euthanized. Plasma was separated with EDTA, and creatine kinase (CK) was measured. Muscles of the lower limbs were collected, the wet weight was measured, and gene expression analysis was performed.

[0799] like Figure 5 and Figure 6 As shown, compound No. 3 and compound No. 123 inhibited the mRNA expression of DUX4. In addition, the blood level of CK, a muscle disorder marker, was reduced. On the other hand, no clear effect on DUX4 mRNA expression and blood CK level was confirmed for compound No. 113 and compound No. 247.

[0800] Example 12

[0801] Continuous drug administration toxicity test in mice

[0802] Using physiological saline, a modified oligonucleotide targeting DUX4 was prepared in a manner of 100 mg / 5 mL / kg, and 6-week-old ICR mice (male, Charles River, Japan) were intravenously administered in the tail vein for 4 consecutive days. After the final administration for 72 hours, blood was collected from the posterior great vein under isoflurane anesthesia, and clinical biochemical examination was performed. In addition, after bleeding to death, an autopsy was performed, and a pathological histological examination of the liver and kidney was performed. In compound No. 123, death and general state, food intake and body weight changes were not confirmed after administration, and no hepatotoxicity (the rise of ALT and AST in serum and abnormal findings in pathological histology) and nephrotoxicity (the rise of UN and creatinine in serum and abnormal findings in pathological histology) were observed. On the other hand, for Compound No. 113 and Compound No. 247, although no death or changes in general state, food intake, or body weight were observed after administration, clear hepatotoxicity (increases in serum ALT, AST, GLDH, ALP, bilirubin, and bile acid, abnormal pathological histological findings: degeneration and necrosis of hepatocytes, hepatocyte hypertrophy) and renal toxicity (increase in serum creatinine) were observed for both. In addition, the liver and kidney concentrations of Compound No. 123 under the test conditions were 323 and 251 μg / g, respectively, which were comparable to or higher than the liver and kidney concentrations of Compound No. 247 (111 and 185 μg / g) and the liver and kidney concentrations of Compound No. 113 (39.3 and 235 μg / g), but no liver and kidney toxicity was observed.

[0803] [Table 4]

[0804] Table 4. Blood biochemical examination of mice in continuous drug administration experiment

[0805] Inspection items Control group Compound No. 123 Control group Compound No. 247 Compound No. 113 AST(U / L) 53.8 60.2 38.0 1187.0 1355.8 ALT(U / L) 30.4 38.6 17.6 2300.2 1282.8 GLDH(U / L) 16.0 36.0 7.0 813.2 768.8 ALP(U / L) 340.2 345.2 204.2 1063.8 640.6 T-Bil (mg / dL) 0.086 0.052 0.106 1.208 0.298 D-Bil(mg / dL) 0.024 0.012 0.044 1.086 0.238 I-Bil (mg / dL) 0.062 0.040 0.062 0.122 0.060 TBA (μmol / L) 0.80 1.60 0.90 28.02 63.32 UN(mg / dL) 20.34 18.62 18.32 23.02 23.20 Cre (mg / dL) 0.056 0.054 0.062 0.126 0.152

[0806] [Table 5]

[0807] Table 5. Pathological findings in the continuous drug administration experiment in mice

[0808] Pathological findings Control group Compound No. 123 Control group Compound No. 247 Compound No. 113 Degeneration and necrosis of liver cells 0 / 5 0 / 5 0 / 5 3 / 5 4 / 5 Hepatocyte hypertrophy 0 / 5 0 / 5 0 / 5 4 / 5 5 / 5

[0809] Reference example

[0810] The synthesis routes of nucleotides containing ALNA[Ms], nucleotides containing ALNA[mU], nucleotides containing ALNA[ipU], nucleotides containing ALNA[Trz], and nucleotides containing ALNA[Oxz] are shown below. It should be noted that the starting compounds 1a, 1d, and 1g can be synthesized by the method described in International Publication No. 2017 / 047816.

[0811] Synthesis of ALNA[Ms]-T

[0812]

[0813] Synthesis of ALNA[Ms]-mC

[0814]

[0815] Synthesis of ALNA[Ms]-G

[0816]

[0817] Synthesis of ALNA[Ms]-A

[0818]

[0819] Synthesis of ALNA[mU]-T

[0820]

[0821] Synthesis of ALNA[mU]-mC

[0822]

[0823] Synthesis of ALNA[mU]-G

[0824]

[0825] Synthesis of ALNA[mU]-A

[0826]

[0827] Synthesis of ALNA[ipU]-T

[0828]

[0829] Synthesis of ALNA[ipU]-mC

[0830]

[0831] Synthesis of ALNA[ipU]-G

[0832]

[0833] Synthesis of ALNA[ipU]-A

[0834]

[0835] Synthesis of ALNA[Trz]-T

[0836]

[0837] Synthesis of ALNA[Trz]-mC

[0838]

[0839] Synthesis of ALNA[Trz]-G

[0840]

[0841] Synthesis of ALNA[Trz]-A

[0842]

[0843] Synthesis of ALNA[Oxz]-T

[0844]

[0845] Synthesis of ALNA[Oxz]-mC

[0846]

[0847] Industrial Applicability

[0848] The modified oligonucleotide of the present invention can be used as a compound useful for treating, preventing or delaying the progression of a DUX4-related disease.

[0849] [Sequence Listing Free Text]

[0850] Sequence No. 1 in the sequence listing represents the base sequence of DUX4 mature mRNA.

[0851] Sequence numbers 2 to 4 and 7 to 112 in the sequence listing represent base sequences of modified oligonucleotides.

[0852] SEQ ID NOs. 5 to 6 in the sequence listing show the base sequences of DUX4-FL2 and DUX4-s, which are splice variants of SEQ ID NO. 1, respectively. Sequence Listing <110> Mitsubishi Tanabe Pharma Corporation <120> Compounds, methods and pharmaceutical compositions for regulating the expression of DUX4 <130> A19030-19510 <150> JP2019-67914 <151> 2019-03-29 <160> 109 <170> PatentIn version 3.5 <210> 1 <211> 1574 <212> DNA <213> Homo sapiens <400> 1 atggccctcc cgacaccctc ggacagcacc ctccccgcgg aagcccgggg acgaggacgg 60 cgacggagac tcgtttggac cccgagccaa agcgaggccc tgcgagcctg ctttgagcgg 120 aacccgtacc cgggcatcgc caccagagaa cggctggccc aggccatcgg cattccggag 180 cccagggtcc agatttggtt tcagaatgag aggtcacgcc agctgaggca gcaccggcgg 240 gaatctcggc cctggcccgg gagacgcggc ccgccagaag gccggcgaaa gcggaccgcc 300 gtcaccggat cccagaccgc cctgctcctc cgagcctttg agaaggatcg ctttccaggc 360 atcgccgccc gggaggagct ggccagagag acgggcctcc cggagtccag gattcagatc 420 tggtttcaga atcgaagggc caggcacccg ggacagggtg gcagggcgcc cgcgcaggca 480 ggcggcctgt gcagcgcggc ccccggcggg ggtcaccctg ctccctcgtg ggtcgccttc 540 gcccacaccg gcgcgtgggg aacggggctt cccgcacccc acgtgccctg cgcgcctggg 600 gctctcccac agggggcttt cgtgagccag gcagcgaggg ccgcccccgc gctgcagccc 660 agccaggccg cgccggcaga ggggatctcc caacctgccc cggcgcgcgg ggatttcgcc 720 tacgccgccc cggctcctcc ggacggggcg ctctcccacc ctcaggctcc tcgctggcct 780 ccgcacccgg gcaaaagccg ggaggaccgg gacccgcagc gcgacggcct gccgggcccc 840 tgcgcggtgg cacagcctgg gcccgctcaa gcggggccgc agggccaagg ggtgcttgcg 900 ccacccacgt cccaggggag tccgtggtgg ggctggggcc ggggtcccca ggtcgccggg 960 gcggcgtggg aaccccaagc cggggcagct ccacctcccc agcccgcgcc cccggacgcc 1020 tccgcctccg cgcggcaggg gcagatgcaa ggcatcccgg cgccctccca ggcgctccag 1080 gagccggcgc cctggtctgc actcccctgc ggcctgctgc tggatgagct cctggcgagc 1140 ccggagtttc tgcagcaggc gcaacctctc ctagaaacgg aggccccggg ggagctggag 1200 gcctcggaag aggccgcctc gctggaagca cccctcagcg aggaagaata ccgggctctg 1260 ctggaggagc tttaggacgc ggggtctagg cccggtgaga gactccacac cgcggagaac 1320 tgccattctt tcctgggcat cccggggatc ccagagccgg cccaggtacc agcagacctg 1380 cgcgcagtgc gcaccccggc tgacgtgcaa gggagctcgc tggcctctct gtgcccttgt 1440 tcttccgtga aattctggct gaatgtctcc ccccaccttc cgacgctgtc taggcaaacc 1500 tggattagag ttacatctcc tggatgatta gttcagagat atattaaaat gccccctccc 1560 tgtggatcct atag 1574 <210> 2 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (1) <223> m5c <400> 2 cgagattccc gccggt 16 <210> 3 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2) <223> m5c <400> 3 gcagttctcc gcggt 15 <210> 4 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(3), (14)..(14) <223> m5c <400> 4 gcctagacag cgtcgg 16 <210> 5 <211> 1710 <212> DNA <213> Homo sapiens <400> 5 atggccctcc cgacaccctc ggacagcacc ctccccgcgg aagcccgggg acgaggacgg 60 cgacggagac tcgtttggac cccgagccaa agcgaggccc tgcgagcctg ctttgagcgg 120 aacccgtacc cgggcatcgc caccagagaa cggctggccc aggccatcgg cattccggag 180 cccagggtcc agatttggtt tcagaatgag aggtcacgcc agctgaggca gcaccggcgg 240 gaatctcggc cctggcccgg gagacgcggc ccgccagaag gccggcgaaa gcggaccgcc 300 gtcaccggat cccagaccgc cctgctcctc cgagcctttg agaaggatcg ctttccaggc 360 atcgccgccc gggaggagct ggccagagag acgggcctcc cggagtccag gattcagatc 420 tggtttcaga atcgaagggc caggcacccg ggacagggtg gcagggcgcc cgcgcaggca 480 ggcggcctgt gcagcgcggc ccccggcggg ggtcaccctg ctccctcgtg ggtcgccttc 540 gcccacaccg gcgcgtgggg aacggggctt cccgcacccc acgtgccctg cgcgcctggg 600 gctctcccac agggggcttt cgtgagccag gcagcgaggg ccgcccccgc gctgcagccc 660 agccaggccg cgccggcaga ggggatctcc caacctgccc cggcgcgcgg ggatttcgcc 720 tacgccgccc cggctcctcc ggacggggcg ctctcccacc ctcaggctcc tcgctggcct 780 ccgcacccgg gcaaaagccg ggaggaccgg gacccgcagc gcgacggcct gccgggcccc 840 tgcgcggtgg cacagcctgg gcccgctcaa gcggggccgc agggccaagg ggtgcttgcg 900 ccacccacgt cccaggggag tccgtggtgg ggctggggcc ggggtcccca ggtcgccggg 960 gcggcgtggg aaccccaagc cggggcagct ccacctcccc agcccgcgcc cccggacgcc 1020 tccgcctccg cgcggcaggg gcagatgcaa ggcatcccgg cgccctccca ggcgctccag 1080 gagccggcgc cctggtctgc actcccctgc ggcctgctgc tggatgagct cctggcgagc 1140 ccggagtttc tgcagcaggc gcaacctctc ctagaaacgg aggccccggg ggagctggag 1200 gcctcggaag aggccgcctc gctggaagca cccctcagcg aggaagaata ccgggctctg 1260 ctggaggagc tttaggacgc ggggttggga cggggtcggg tggttcgggg cagggcggtg 1320 gcctctcttt cgcggggaac acctggctgg ctacggaggg gcgtgtctcc gccccgcccc 1380 ctccaccggg ctgaccggcc tgggattcct gccttctagg tctaggcccg gtgagagact 1440 ccacaccgcg gagaactgcc attctttcct gggcatcccg gggatcccag agccggccca 1500 ggtaccagca gacctgcgcg cagtgcgcac cccggctgac gtgcaaggga gctcgctggc 1560 ctctctgtgc ccttgttctt ccgtgaaatt ctggctgaat gtctcccccc accttccgac 1620 gctgtctagg caaacctgga ttagagttac atctcctgga tgattagttc agagatatat 1680 taaaatgccc cctccctgtg gatcctatag 1710 <210> 6 <211> 768 <212> DNA <213> Homo sapiens <220> <223> Description of artificial sequence: Oligonucleotide <400> 6 atggccctcc cgacaccctc ggacagcacc ctccccgcgg aagcccgggg acgaggacgg 60 cgacggagac tcgtttggac cccgagccaa agcgaggccc tgcgagcctg ctttgagcgg 120 aacccgtacc cgggcatcgc caccagagaa cggctggccc aggccatcgg cattccggag 180 cccagggtcc agatttggtt tcagaatgag aggtcacgcc agctgaggca gcaccggcgg 240 gaatctcggc cctggcccgg gagacgcggc ccgccagaag gccggcgaaa gcggaccgcc 300 gtcaccggat cccagaccgc cctgctcctc cgagcctttg agaaggatcg ctttccaggc 360 atcgccgccc gggaggagct ggccagagag acgggcctcc cggagtccag gattcagatc 420 tggtttcaga atcgaagggc caggcacccg ggacagggtg gcagggcgcc cgcgcaggtc 480 taggcccggt gagagactcc acaccgcgga gaactgccat tctttcctgg gcatcccggg 540 gatcccagag ccggcccagg taccagcaga cctgcgcgca gtgcgcaccc cggctgacgt 600 gcaagggagc tcgctggcct ctctgtgccc ttgttcttcc gtgaaattct ggctgaatgt 660 ctccccccac cttccgacgc tgtctaggca aacctggatt agagttacat ctcctggatg 720 attagttcag agatatatta aaatgccccc tccctgtgga tcctatag 768 <210> 7 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotides <400> 7 gattcccgcc ggtg 14 <210> 8 <211> 14 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 8 agattcccgc cggt 14 <210> 9 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 9 gagattcccg ccggt 15 <210> 10 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (1)..(1), (8)..(10), (12)..(13) <223> m5c <400> 10 cgagattccc gccggt 16 <210> 11 <211> 14 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (12)..(12) <223> m5c <400> 11 gagattcccg ccgg 14 <210> 12 <211> 14 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (7)..(9), (11)..(12) <223> m5c <400> 12 gagattcccg ccgg 14 <210> 13 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (1)..(1), (8)..(10), (12)..(13), <223> m5c <400> 13 cgagattccc gccgg 15 <210> 14 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (1)..(1), (13)..(13) <223> m5c <400> 14 cgagattccc gccgg 15 <210> 15 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 15 agttctccgc ggtgtg 16 <210> 16 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2) <223> m5c <400> 16 gcagttctcc gcggtgt 17 <210> 17 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (3) <223> m5c <400> 17 ggcagttctc cgcggtgt 18 <210> 18 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (1) <223> m5c <400> 18 cagttctccg cggtg 15 <210> 19 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (1)..(1), (6)..(6), (8)..(9), (11)..(11) <223> m5c <400> 19 cagttctccg cggtg 15 <210> 20 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 20 gcagttctcc gcggtg 16 <210> twenty one <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2) <223> m5c <400> twenty one gcagttctcc gcggtg 16 <210> twenty two <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(2), (7)..(7), (9)..(10), (12)..(12) <223> m5c <400> twenty two gcagttctcc gcggtg 16 <210> twenty three <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (3) <223> m5c <400> twenty three ggcagttctc cgcggtg 17 <210> twenty four <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (3)..(3), (8)..(8), (10)..(11), (13)..(13) <223> m5c <400> twenty four ggcagttctc cgcggtg 17 <210> 25 <211> 14 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (1) <223> m5c <400> 25 cagttctccg cggt 14 <210> 26 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (12)..(12) <223> m5c <400> 26 gcagttctcc gcggt 15 <210> 27 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(2), (12)..(12) <223> m5c <400> 27 gcagttctcc gcggt 15 <210> 28 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(2), (7)..(7), (9)..(10), (12)..(12) <223> m5c <400> 28 gcagttctcc gcggt 15 <210> 29 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(2), (7)..(7), (9)..(10) <223> m5c <400> 29 gcagttctcc gcggt 15 <210> 30 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(2), (7)..(7), (9)..(10), (12)..(12) <223> m5c <400> 30 gcagttctcc gcggt 15 <210> 31 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(2), (7)..(7), (9)..(10) <223> m5c <400> 31 gcagutctcc gcggt 15 <210> 32 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(2), (7)..(7), (9)..(10), (12)..(12) <223> m5c <400> 32 gcagutctcc gcggt 15 <210> 33 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 33 ggcagttctc cgcggt 16 <210> 34 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (3) <223> m5c <400> 34 ggcagttctc cgcggt 16 <210> 35 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (3)..(3), (8)..(8), (10)..(11), (13)..(13) <223> m5c <400> 35 ggcagttctc cgcggt 16 <210> 36 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 36 tggcagttct ccgcggt 17 <210> 37 <211> 14 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2) <223> m5c <400> 37 gcagttctcc gcgg 14 <210> 38 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (13) <223> m5c <400> 38 ggcagttctc cgcgg 15 <210> 39 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (3) <223> m5c <400> 39 ggcagttctc cgcgg 15 <210> 40 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (3)..(3), (13)..(13) <223> m5c <400> 40 ggcagttctc cgcgg 15 <210> 41 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (3)..(3), (8)..(8), (10)..(11), (13)..(13) <223> m5c <400> 41 ggcagttctc cgcgg 15 <210> 42 <211> 14 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (1) <223> m5c <400> 42 cgtcggaagg tggg 14 <210> 43 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2) <223> m5c <400> 43 gcgtcggaag gtggg 15 <210> 44 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (3) <223> m5c <400> 44 agcgtcggaa ggtggg 16 <210> 45 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (1) <223> m5c <400> 45 cagcgtcgga aggtggg 17 <210> 46 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (3) <223> m5c <400> 46 agcgtcggaa ggtgg 15 <210> 47 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (3)..(3), (6)..(6) <223> m5c <400> 47 agcgtcggaa ggtgg 15 <210> 48 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (1) <223> m5c <400> 48 cagcgtcgga aggtgg 16 <210> 49 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (1)..(1), (4)..(4), (7)..(7) <223> m5c <400> 49 cagcgtcgga aggtgg 16 <210> 50 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2) <223> m5c <400> 50 acagcgtcgg aaggtgg 17 <210> 51 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (1) <223> m5c <400> 51 cagcgtcgga aggtg 15 <210> 52 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(2) <223> m5c <400> 52 acagcgtcgg aaggtg 16 <210> 53 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (3) <223> m5c <400> 53 gacagcgtcg gaaggt 16 <210> 54 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (3) <223> m5c <400> 54 gacagcgtcg gaagg 15 <210> 55 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 55 agacagcgtc ggaagg 16 <210> 56 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (1)..(2), (13)..(13) <223> m5c <400> 56 cctagacagc gtcgg 15 <210> 57 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (3)..(3), (14)..(14) <223> m5c <400> 57 gcctagacag cgtcgg 16 <210> 58 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2) <223> m5c <400> 58 gcctagacag cgtcgg 16 <210> 59 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(2), (14)..(14) <223> m5c <400> 59 gcctagacag cgtcgg 16 <210> 60 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(3) <223> m5c <400> 60 gcctagacag cgtcgg 16 <210> 61 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(3), (8)..(8), (11)..(11), (14)..(14) <223> m5c <400> 61 gcctagacag cgtcgg 16 <210> 62 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(3), (8)..(8), (11)..(11), (14)..(14) <223> m5c <400> 62 gcctagacag cgucgg 16 <210> 63 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(3), (8)..(8), (11)..(11), (14)..(14) <223> m5c <400> 63 gccuagacag cgtcgg 16 <210> 64 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(3), (8)..(8), (11)..(11), (14)..(14) <223> m5c <400> 64 gccuagacag cgucgg 16 <210> 65 <211> 14 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (1)..(1), (3)..(3) <223> m5c <400> 65 ctcagctggc gtga 14 <210> 66 <211> 14 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (1)..(2) <223> m5c <400> 66 ccaggaaaga atgg 14 <210> 67 <211> 14 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (14)..(14) <223> m5c <400> 67 gggagacatt cagc 14 <210> 68 <211> 14 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (1) <223> m5c <400> 68 ctaatccagg tttg 14 <210> 69 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (3)..(3),(16)..(16) <223> m5c <400> 69 ggcgatgccc gggtac 16 <210> 70 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (18) <223> m5c <400> 70 gtggcgatgc ccgggtac 18 <210> 71 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 71 ggtggcgatg cccgggta 18 <210> 72 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 72 gtggcgatgc ccgggta 17 <210> 73 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 73 tggcgatgcc cgggta 16 <210> 74 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (1) <223> m5c <400> 74 ctggtggcga tgcccgggta 20 <210> 75 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 75 gtggcgatgc ccgggt 16 <210> 76 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 76 ggtggcgatg cccgggt 17 <210> 77 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2) <223> m5c <400> 77 tctggtggcg atgcccgggt 20 <210> 78 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (12)..(12) <223> m5c <400> 78 gagattcccg ccggtg 16 <210> 79 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2) <223> m5c <400> 79 gcagttctcc gcggtgtgga 20 <210> 80 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 80 ggcagttctc cgcggtgtg 19 <210> 81 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 81 atggcagttc tccgcggtgt 20 <210> 82 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 82 tggcagttct ccgcggtgt 19 <210> 83 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 83 tggcagttct ccgcggtg 18 <210> 84 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(2) <223> m5c <400> 84 gcctagacag cgtcggaagg 20 <210> 85 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(2) <223> m5c <400> 85 gcctagacag cgtcggaag 19 <210> 86 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 86 tgcctagaca gcgtcggaa 19 <210> 87 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 87 tttgcctaga cagcgtcgga 20 <210> 88 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 88 tgcctagaca gcgtcgga 18 <210> 89 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(3) <223> m5c <400> 89 gcctagacag cgtcgga 17 <210> 90 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 90 gtttgcctag acagcgtcgg 20 <210> 91 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 91 ttgcctagac agcgtcgg 18 <210> 92 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (3)..(3), (15)..(15) <223> m5c <400> 92 tgcctagaca gcgtcgg 17 <210> 93 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(3), (14)..(14) <223> m5c <400> 93 gcctagacag cgtcgg 16 <210> 94 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(2), (12)..(12) <223> m5c <400> 94 gcagttctcc gcggtg 16 <210> 95 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 95 ggcagttctc cgcggtgt 18 <210> 96 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (12)..(12) <223> m5c <400> 96 gtggcgatgc ccgggt 16 <210> 97 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (12)..(12) <223> m5c <400> 97 gcagttctcc gcggtg 16 <210> 98 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2) <223> m5c <400> 98 acgggttccg ctcaaa 16 <210> 99 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (1)..(1), (15)..(16) <223> m5c <400> 99 cggaatgccg atggcc 16 <210> 100 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (3)..(3), (14)..(14) <223> m5c <400> 100 ttctggcggg ccgcgt 16 <210> 101 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (14)..(14) <223> m5c <400> 101 tagaccccgc gtccta 16 <210> 102 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(3), (14)..(14) <223> m5c <400> 102 gccuagacag cgtcgg 16 <210> 103 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(3), (14)..(14) <223> m5c <400> 103 gcctagacag cgucgg 16 <210> 104 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(3), (14)..(14) <223> m5c <400> 104 gccuagacag cgtcgg 16 <210> 105 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2)..(3), (14)..(14) <223> m5c <400> 105 gccuagacag cgucgg 16 <210> 106 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <400> 106 gtggcgaugc ccgggt 16 <210> 107 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2) <223> m5c <400> 107 gcagutctcc gcggt 15 <210> 108 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2) <223> m5c <400> 108 gcagtuctcc gcggt 15 <210> 109 <211> 15 <212> DNA <213> Artificial sequence <220> <223> Synthetic oligonucleotides <220> <221> Modified bases <222> (2) <223> m5c <400> 109 gcagttcucc gcggt 15

Claims

1. A modified oligonucleotide represented by the following formula: GmsMmsMmsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMmsGmsGm, In the formula, Each nucleic acid base is represented by the following symbols: A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine, Each sugar moiety is represented by the following symbols: m = ALNA [Ms], d = 2'-deoxyribose, The ALNA[Ms] is represented by the following structural formula: In the formula, Base represents each nucleic acid base, The internucleoside bonds are represented by the following symbols: s = phosphorothioate.

2. A modified oligonucleotide or a salt thereof, represented by the following formula: 。 3. A pharmaceutical composition comprising the modified oligonucleotide according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, which is used for the treatment, prevention or delay of progression of facioscapulohumeral muscular dystrophy, B-cell acute lymphoblastic leukemia, differentiated round cell sarcoma or embryonal rhabdomyosarcoma.

5. The pharmaceutical composition according to claim 4, which is used for treating, preventing or delaying the progression of facioscapulohumeral muscular dystrophy.

6. Use of the modified oligonucleotide according to claim 1 or 2 in the manufacture of a pharmaceutical for treating, preventing or delaying the progression of facioscapulohumeral muscular dystrophy, B-cell acute lymphoblastic leukemia, differentiated round cell sarcoma or embryonal rhabdomyosarcoma.

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