Compound, method and pharmaceutical composition for modulating expression of DUX4

Abstract

To provide a compound, a method and a pharmaceutical composition for normalizing double homeobox 4 (DUX4) of an individual in which the DUX4 gene has abnormally expressed.SOLUTION: Provided is a modified oligonucleotide consisting of 12-30 residues. The modified oligonucleotide includes a nucleobase sequence that includes at least 8 contiguous nucleobase sequences and is complementary to an equal length portion at positions 126-147, 232-248, 1306-1325 or 1472-1495 from a 5' end of a nucleobase of a mature mRNA of DUX4 of SEQ ID NO: 1. The nucleobase sequence of the modified oligonucleotide has at least 90% complementarity to the equal length portion in the nucleobase sequence of the mature mRNA of DUX4 of SEQ ID NO: 1.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a compound for reducing the expression of DUX4 mRNA and protein in animals, a method using the compound, and a pharmaceutical composition containing the compound. The method of the present invention is 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 that occurs at an estimated frequency of 1 in 20,000 worldwide and 1 in 7,500 in Europe. Among muscular dystrophies, it is the third most common disease after Duchenne muscular dystrophy and myotonic dystrophy. The initial symptoms are muscle weakness in the face, upper limbs, scapular region, and upper limb girdle. As the disease progresses, the lower limb girdle and lower limbs are also affected, and about 20% of cases require a wheelchair by the age of 40 (in some cases, there may be only mild facial muscle involvement even in adulthood). Complications such as pain, sensorineural hearing loss, and retinopathy are also common. Approximately 90% of patients develop the disease by the age of 20. Severe patients (about 4%) show muscle weakness from infancy.

[0003] FSHD is classified into two types, FSHD1 and FSHD2, depending on the causative gene. Type 1 FSHD accounts for about 95% of all FSHD patients. In patients with type 1 FSHD, the D4Z4 repeat region on 4q35 is genetically shortened (1 to 10 D4Z4 repeats), which leads to abnormal expression of DUX4 encoded in the D4Z4 repeat region (DUX4 is not expressed in healthy individuals). Type 2 FSHD accounts for about 5% of all FSHD patients, and abnormal expression of DUX4 occurs due to mutations in SMCHD1 (a DNA methyltransferase). DUX4 has a transcription factor-like function and induces the expression of a group of genes that cause apoptosis and muscle atrophy of downstream-encoded muscle cells. Abnormal expression of DUX4 is due to the possession of an allele called 4qA among the two alleles of 4qA and 4qB. The polyadenylation site present in 4qA is necessary for the stabilization of DUX4 mRNA (Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3).

[0004] Antisense technology is emerging as an effective means for regulating the expression of certain gene products and may thus prove uniquely useful for 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), but preparing the adeno-associated virus is complicated, and delivering it to the required whole body muscles is difficult. A method for suppressing the expression of the DUX4 gene using a lentivirus encoding DUX4 shRNA has been reported (Non-Patent Document 2), but preparing the lentivirus is complicated, and delivering it to the required whole body muscles is difficult. Furthermore, the gene suppression effect in vitro in quadriceps and trapezius muscle cells has residual activities of 21% and 44%, respectively, and is not sufficient.

[0006] A method for suppressing the expression of the DUX4 gene using a lentivirus encoding DUX4 shRNA has been reported (Non-Patent Document 2), but preparing the lentivirus is complicated, and delivering it to the required whole body muscles is difficult. Furthermore, the gene suppression effect in vitro in quadriceps and trapezius muscle cells has residual activities of 21% and 44%, respectively, and is not sufficient.

[0007] Compounds in which multiple antisense oligonucleotides against DUX4 are linked have been reported (Patent Document 2), but they are not modified oligonucleotides, and their inhibitory effects are not sufficient.

[0008] Antisense oligonucleotide compounds that bind to the splicing site of DUX4 mRNA have been reported (Patent Document 3). However, since these compounds are selective for pre-mRNA containing introns, their inhibitory effect on mature mRNA is weak, and it is difficult to administer them to a living body due to knockdown by the Lipofection method.

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

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0012] The problems of the present invention are to provide compounds, methods, and pharmaceutical compositions for inhibiting the expression of DUX4 and for treating, i.e., curing, preventing, delaying, or ameliorating DUX4-related diseases and / or their symptoms. The compounds and pharmaceutical compositions disclosed herein also inhibit mutant DUX4 such as SNPs and splicing variants of DUX4.

Means for Solving the Problems

[0013] Certain embodiments provide a method of reducing the expression of DUX4 in an animal (including a human), the method comprising administering to the animal a compound containing a modified oligonucleotide targeting DUX4 as described herein, or a pharmaceutical composition containing the compound.

[0014] Certain embodiments provide a method of knocking down via an endoribonuclease (such as RNase H) by administering to an animal a compound or pharmaceutical composition containing a modified oligonucleotide targeting DUX4. Also provided is a method of inhibiting the transcription of DUX4 mRNA and the translation of DUX4 protein by administering the compound containing the modified oligonucleotide. The modified oligonucleotide is preferably distributed in muscle, particularly preferably in skeletal muscle.

[0015] Certain embodiments provide a method of treating an animal having FSHD. In certain embodiments, 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 herein. In certain embodiments, the method of the present invention comprises identifying an animal having FSHD1 and / or FSHD2.

[0016] Certain embodiments provide a method for treating, i.e., curing, preventing, delaying, or ameliorating muscular atrophy and muscle weakness. This includes alleviating or delaying the progression of poor facial expression / sleeping with eyes open / difficulty in raising the upper limbs / winged scapula caused by muscle weakness in the buccal area, shoulders, and upper arms. Additionally, it is preferable to prevent muscle weakness in the lumbar belt and lower limbs, and further prevent the complication of neural hearing loss and retinopathy.

[0017] In certain embodiments, the DUX4 mRNA has the sequence described in GenBank accession number NM_001293798.2 (incorporated herein as SEQ ID NO: 1 in the Sequence Listing). The splicing variant of the DUX4 mRNA of SEQ ID NO: 1 in the Sequence Listing is also referred to as DUX4-FL1 or the mature mRNA of DUX4. In certain embodiments, the DUX4 mRNA has the sequence described in GenBank accession number NM_001306068.2 (incorporated herein as SEQ ID NO: 5 in the Sequence Listing). The splicing variant of the DUX4 mRNA of SEQ ID NO: 5 in the Sequence Listing is also referred to as DUX4-FL2. In certain embodiments, DUX4 has the sequence described in GenBank accession number NM_001363820.1 (incorporated herein as SEQ ID NO: 6 in the Sequence Listing). The splicing variant of the DUX4 mRNA of SEQ ID NO: 6 in the Sequence Listing is also referred to as DUX4-s. In certain embodiments, DUX4 refers to the SNP of the above splicing variant.

[0018] The present disclosure relates to, but is not limited to, the non-limiting numbered embodiments described in the following items [1] to

[27] .

[0019] Item [1] A modified oligonucleotide consisting of 12 to 30 residues, which is at least 8 consecutive nucleic acid base sequences complementary to the corresponding length portions at positions 126 - 147, 232 - 248, 1306 - 1325, or 1480 - 1495 from the 5'-end of the nucleic acid base sequence of the mature mRNA of DUX4 of SEQ ID NO: 1, The nucleobase sequence of the modified oligonucleotide has at least 90% complementarity to the co-length portion in the nucleobase sequence of the mature mRNA of DUX4 of SEQ ID NO: 1, when the at least 8 consecutive nucleobase sequences include a nucleobase sequence complementary to the co-length portion at positions 1480 to 1495 from the 5'-end of the nucleobase sequence of SEQ ID NO: 1, the modified oligonucleotide consists of a nucleobase sequence having a complementary base of the base at position 1480 from the 5'-end of the nucleobase of SEQ ID NO: 1 at the 3'-end, Modified oligonucleotide.

[0020] Item [2] The modified oligonucleotide according to item [1], wherein one or more modified nucleotides of the modified oligonucleotide contain a modified sugar. Item [3] The modified oligonucleotide according to item [2], wherein the modified sugar is selected from the group consisting of bicyclic sugars, sugars modified with 2'-O-methoxyethyl, and sugars modified with 2'-O-methyl. Item [4] The modified oligonucleotide according to item [3], wherein the bicyclic sugar is selected from the group consisting of LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], and ALNA[Trz].

[0021] Item [5] A modified oligonucleotide consisting of 12 to 30 residues, including at least 8 consecutive nucleobase sequences, which include a nucleobase sequence complementary to the co-length portion at positions 1472 to 1495 from the 5'-end of the nucleobase sequence of the mature mRNA of DUX4 of SEQ ID NO: 1, the nucleobase sequence of the modified oligonucleotide has at least 90% complementarity to the co-length portion in the nucleobase sequence of the mature mRNA of DUX4 of SEQ ID NO: 1, the modified oligonucleotide contains at least one nucleoside containing a modified sugar selected from GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], and ALNA[Trz], Modified oligonucleotide.

[0022] Item [6]. The modified oligonucleotide according to item [5], further comprising a sugar modified with 2'-O-methoxyethyl and / or a sugar modified with 2'-O-methyl.

[0023] Item [7]. The modified oligonucleotide according to any one of claims 1 to 6, wherein at least one modified nucleotide of the modified oligonucleotide contains a modified nucleobase. Item [8]. The modified oligonucleotide according to item [7], wherein the modified nucleobase is 5-methylcytosine.

[0024] Item [9]. The modified oligonucleotide according to any one of items [1] to [8], wherein at least one internucleoside linkage is a modified internucleoside linkage. Item

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

[0025] Item

[11] . The modified oligonucleotide, 1) comprises a gap segment, 2) a 5' wing segment, and 3) a 3' wing segment, wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, and any of the nucleosides of the 5' wing segment and the 3' wing segment contains at least one modified sugar, and the nucleosides of the gap segment are either only nucleosides that do not contain a modified sugar, or contain 1 or 2 nucleosides that contain a modified sugar and the rest are nucleosides that do not contain a modified sugar. The modified oligonucleotide according to any one of items [1] to

[10] .

[0026] Item

[12] . The modified oligonucleotide, of the nucleobase sequence of the mature mRNA of DUX4 of SEQ ID NO: 1 A nucleic acid base sequence from positions 128 to 143 from the 5'-end, a nucleic acid base sequence from positions 232 to 247 from the 5'-end, a nucleic acid base sequence from positions 233 to 248 from the 5'-end, a nucleic acid base sequence from positions 1309 to 1323 from the 5'-end, or a nucleic acid base sequence from positions 1480 to 1495 from the 5'-end, The modified oligonucleotide according to any one of items [1] to

[11] , comprising a nucleic acid base sequence complementary to the above.

[0027] Item

[13] The modified oligonucleotide is 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) The modified oligonucleotide according to any one of items [1] to

[12] , comprising the base sequence of the above.

[0028] Item

[14] The following formula: GlsMlsMlsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMlsGlsGl; The modified oligonucleotide according to item

[13] represented by the above. In the formula, each nucleic acid base is represented by the following symbols: A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine, as shown according to; each sugar moiety is represented by the following symbols: l = LNA, d = 2'-deoxyribose, as shown according to; each internucleoside bond is represented by the following symbol: s = phosphorothioate as shown according to.

[0029] Item

[15] The following formula: GmsMmsMmsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMmsGmsGm; The modified oligonucleotide according to item

[13] represented by In the formula, Each nucleobase is represented by the following symbols: A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine, as shown according to; Each sugar moiety is represented by the following symbols: m = ALNA[Ms], d = 2'-deoxyribose, as shown according to; Each internucleoside linkage is represented by the following symbol: s = phosphorothioate as shown according to.

[0030] Item

[16] The following formula: GmsMmsAmsGdsTdsTdsCdsTdsCdsCdsGdsCdsGmsGmsTm; The modified oligonucleotide according to item

[13] represented by In the formula, Each nucleobase is represented by the following symbols: A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine, as shown according to; Each sugar moiety is represented by the following symbols: m = ALNA[Ms], d = 2'-deoxyribose, as shown according to; Each internucleoside linkage is represented by the following symbol: s = phosphorothioate as shown according to.

[0031] Item

[17] The following formula: MlsGlsAlsGdsAdsTdsTdsCdsCdsCdsGdsCdsCdsGlsGlsTl; The modified oligonucleotide according to item

[13] represented by In the formula, Each nucleobase is represented by the following symbols: A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine, is shown according to; each sugar moiety is represented by the following symbols: l = LNA, d = 2'-deoxyribose, is shown according to; each internucleoside linkage is represented by the following symbols: s = phosphorothioate is shown according to.

[0032] Item

[18] The following formula:

[0033]

Chemical formula

[0034] The modified oligonucleotide or a salt thereof according to item

[14] , represented by.

[0035] Item

[19] The following formula:

[0036]

Chemical formula

[0037] The modified oligonucleotide or a salt thereof according to item

[15] , represented by.

[0038] Item

[20] The following formula:

[0039]

Chemical formula

[0040] The modified oligonucleotide or a salt thereof according to item

[16] , represented by.

[0041] Item

[21] The following formula:

[0042]

Chemical formula

[0043] The modified oligonucleotide according to item

[17] or a salt thereof, as represented by

[0044] 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.

[0045] Item

[23] The pharmaceutical composition according to item

[22] for the treatment, prevention, or delay of the progression of DUX4-related diseases.

[0046] Item

[24] The pharmaceutical composition according to item

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

[0047] Item

[25] A method for treating, preventing, or delaying the progression of DUX4-related diseases in a subject, comprising administering an effective amount of the modified oligonucleotide according to any one of items [1] to

[21] to the subject in need thereof.

[0048] Item

[26] Use of the modified oligonucleotide according to any one of items [1] to

[21] in the manufacture of a medicament for the treatment, prevention, or delay of the progression of DUX4-related diseases.

[0049] Item

[27] Use of the modified oligonucleotide according to any one of items [1] to

[21] for the treatment, prevention, or delay of the progression of DUX4-related diseases.

Advantages of the Invention

[0050] According to the present invention, it is possible to provide a modified oligonucleotide effective for treating diseases such as facioscapulohumeral muscular dystrophy caused by abnormal expression of the DUX4 gene.

Brief Description of the Drawings

[0051]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Mode for Carrying Out the Invention

[0052] It should be understood that both the foregoing summary and the following detailed description are exemplary and explanatory only and do not limit the claimed invention. In this specification, unless otherwise stated, the use of the singular form includes the plural. In this specification, unless otherwise stated, the use of "or" means "and / or". Further, the use of the term "including" and other forms, such as "includes" and "included", is not limiting. Further, unless otherwise stated, terms such as "element" include an element containing one unit and an element containing more than one subunit.

[0053] The headings of the sections used in this specification are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, and papers, are hereby expressly incorporated by reference in their entirety and in part with respect to the portion of the document discussed herein.

[0054] (Definitions) Unless otherwise defined, the nomenclature, procedures, and techniques utilized in connection with analytical chemistry, organic synthetic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and generally used in the art. Standard techniques can be used for chemical synthesis and chemical analysis in this specification. All patents, applications, published applications, and other publications, GenBank accession numbers and related sequence information available through databases such as the National Center for Biotechnology Information (NCBI), and other data referred to throughout the disclosure of this specification, are hereby incorporated by reference in their entirety and in part with respect to the portion of the document discussed herein. Also, this specification is filed with a sequence listing in electronic format, and the information of the sequence listing described in the electronic format is hereby incorporated by reference in its entirety into this specification.

[0055] Unless otherwise indicated, the following terms have the following meanings.

[0056] "Nucleic acid" refers to a molecule composed of monomeric nucleotides. Examples of nucleic acids include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acid (siRNA), and microRNA (miRNA). A nucleic acid can also contain a combination of these elements in a single molecule.

[0057] "Nucleic acid base" means a heterocyclic moiety capable of base pairing with the base of another nucleic acid. Nucleic acid bases include "modified nucleic acid bases" and "unmodified nucleic acid bases".

[0058] "Nucleic acid base sequence" means a sequential order of contiguous nucleic acid bases, independent of any sugar linkage or nucleic acid base modification.

[0059] "Nucleoside" means a nucleic acid base linked to a sugar. In certain embodiments, the nucleoside is linked to a phosphate group.

[0060] "Nucleotide" means a nucleoside having a phosphate group, such as one covalently bonded to the sugar moiety of the nucleoside. Naturally occurring nucleotides have a sugar moiety that is ribose or deoxyribose and are covalently bonded via a phosphodiester bond through the phosphate group.

[0061] "Oligomeric compound" or "oligomer" means a polymer of linked monomer subunits that can hybridize to at least one region of a nucleic acid molecule.

[0062] "Oligonucleotide" means a polymer of linked nucleosides, where each nucleoside and each internucleoside bond may be, independently, either modified or unmodified.

[0063] "Unmodified nucleotide" means a nucleotide consisting of a naturally occurring nucleic acid base, sugar moiety, and internucleoside bond. In certain embodiments, unmodified nucleotides are RNA nucleotides (i.e., β-D-ribonucleosides) or DNA nucleotides (i.e., β-D-deoxyribonucleosides), but are not limited thereto.

[0064] "Modified nucleotide" means a nucleotide having, independently, a modified sugar moiety, a modified internucleoside bond, or a modified nucleic acid base. "Modified nucleoside" means a nucleoside having, independently, a modified sugar moiety or a modified nucleic acid base.

[0065] "Internucleoside bond" refers to a chemical bond between nucleosides.

[0066] "Linked nucleosides" means adjacent nucleosides that are joined or linked by a nucleoside-nucleoside linkage.

[0067] "Naturally occurring nucleoside-nucleoside linkage" means a 3'-5' phosphodiester linkage.

[0068] "Modified nucleoside-nucleoside linkage" refers to a substitution or any change from a naturally occurring nucleoside-nucleoside linkage (i.e., a phosphodiester nucleoside-nucleoside linkage). For example, but not limited to, phosphorothioate nucleoside-nucleoside linkages.

[0069] "Phosphorothioate nucleoside-nucleoside linkage" means a nucleoside-nucleoside linkage in which a phosphodiester linkage is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is an example of such a modified nucleoside-nucleoside linkage. "Modified nucleobase" refers to any nucleobase other than adenine, cytosine, guanine, thymidine or uracil. For example, but not limited to, 5-methylcytosine. "Unmodified nucleobase" means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

[0070] "Modified oligonucleotide" means an oligonucleotide that contains at least one such modified nucleoside and / or such a modified nucleoside-nucleoside linkage.

[0071] "Salt" is a general term for a compound in which one or more dissociable hydrogen ions contained in an acid are replaced by cations such as metal ions or ammonium ions. Examples of salts of modified oligonucleotides include, but are not limited to, salts (e.g., sodium salts, magnesium salts) formed with inorganic ions (e.g., sodium ions, magnesium ions) on the thio (S) group of a phosphorothioate linkage or a functional group (e.g., amino group) within a modified nucleobase.

[0072] ​ "Sugar" or "sugar moiety" means a natural sugar moiety or a modified sugar moiety.

[0073] "Natural sugar moiety" means the sugar found in DNA (2'-H) or RNA (2'-OH).

[0074] "Modified sugar" refers to a substitution or change from a natural sugar moiety. Examples of modified sugars include substituted sugar moieties and surrogate sugar moieties.

[0075] "Substituted sugar moiety" means a furanosyl other than the natural sugar of RNA or DNA.

[0076] "2'-O-methoxyethyl" (similarly 2'-MOE and 2'-O(CH2)2-OCH3) refers to an O-methoxy-ethyl modification at the 2'-position of the furanosyl ring. A sugar modified with 2'-O-methoxyethyl is a modified sugar.

[0077] "2'-O-methoxyethyl nucleotide" means a nucleotide containing a sugar moiety modified with 2'-O-methoxyethyl.

[0078] "2'-O-methyl" (similarly 2'-OMe and 2'-OCH3) refers to an O-methyl modification at the 2'-position of the furanosyl ring. A sugar modified with 2'-O-methyl is a modified sugar.

[0079] "2'-O-methyl nucleotide" means a nucleotide containing a sugar moiety modified with 2'-O-methyl.

[0080] The "sugar substitute" for the "sugar substitute portion" is intended to represent replacement of only the sugar unit (furanose ring). The sugar substitute can replace the naturally occurring sugar portion of a nucleoside, such that the resulting nucleoside subunit can link to each other and / or link to other nucleosides to form an oligomeric compound that can hybridize with a complementary oligomeric compound. Such structures include rings containing a different number of atoms than furanosyl (e.g., 4-, 6- or 7-membered rings); replacement of the oxygen of furanosyl with a non-oxygen atom (e.g., carbon, sulfur or nitrogen); or both a change in the number of atoms and replacement of oxygen. Such structures can also include substitutions corresponding to those described for the substituted sugar portion (e.g., a 6-membered carbocyclic bicyclic sugar substitute optionally containing additional substituents). The sugar substitute also includes replacement of more complex sugars (e.g., acyclic peptide nucleic acids ). Sugar substitutes include, but are not limited to, morpholino, cyclohexenyl and cyclohexitol.

[0081] "Bicyclic sugar" means a furanosyl ring modified by a bridge between two different carbon atoms present on the same ring. Preferably, "bicyclic sugar" means a modified sugar in which the 2'- and 4'-positions of the furanosyl ring are modified by a bridge. "Bicyclic nucleic acid" refers to a nucleoside or nucleotide in which the furanose portion of the nucleoside or nucleotide contains a "bicyclic sugar".

[0082] "LNA" generally refers to a nucleoside or nucleotide commonly called a 2',4'-locked nucleic acid, for example, of the general formula: wherein

[0083]

Chemical formula

[0084] wherein B is a nucleobase; X and Y are each independently a hydrogen atom, a protecting group for a hydroxyl group, an optionally substituted phosphate group, a phosphorus moiety, or a covalent bond to a support, etc. Examples include nucleosides or nucleotides represented by (see WO98 / 39352). Typical specific examples are the following formula:

[0085]

Chemical formula

[0086] Examples include nucleotides or nucleotides represented by.

[0087] "GuNA" is the following formula:

[0088]

Chemical formula

[0089] [In the formula, B is a nucleobase, and R3, R4, R5, and R6 are each independently a hydrogen atom or a C 1-6 alkyl group optionally substituted with one or more substituents, and R7 and R8 are each independently a hydrogen atom, a protecting group for a hydroxyl group, an optionally substituted phosphate group, a phosphorus moiety, or a covalent bond to a support, etc., and R9, R 10 , R 11 are each independently a hydrogen atom, a C 1-6 alkyl group optionally substituted with one or more substituents, or a protecting group for an amino group. It is a nucleoside or nucleotide represented by. (See, for example, International Publication No. 2014 / 046212 and International Publication No. 2017 / 047816).

[0090] "ALNA[mU]" is the following general formula (I):

[0091]

Chemical formula

[0092] [In the formula, B is a nucleobase; R1, R2, R3 and R4 are each independently a hydrogen atom or a C 1-6 alkyl group which may be substituted with one or more substituents; R5 and R6 are each independently a hydrogen atom, a protecting group for a hydroxyl group, or an optionally substituted phosphate group, a phosphorus moiety or a covalent bond to a support, etc.; m is 1 or 2; X is a group represented by the following formula (II-1):

[0093]

Chemical formula

[0094] and is a group represented by; The symbols described in formula (II-1):

[0095]

Chemical formula

[0096] indicate the bonding point with the 2'-amino group; One of R7 and R8 is a hydrogen atom and the other is a methyl group which may be substituted with one or more substituents.] It is a nucleoside or nucleotide represented by (see, for example, 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.

[0097] "ALNA[ipU]" is a nucleoside or nucleotide represented by the general formula (I) defined in the above "ALNA[mU]", and in the formula, X is a group represented by the following formula (II-1):

[0098]

Chemical formula

[0099] is a group represented by; 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 (see, for example, Japanese Patent Application No. 2018-212424). Typical specific examples are nucleosides or nucleotides in which one of R7 and R8 is a hydrogen atom and the other is an unsubstituted isopropyl group.

[0100] "ALNA[Trz]" is a nucleoside or nucleotide represented by the general formula (I) defined in the above "ALNA[mU]", and in the formula, X is the following formula (II-2):

[0101]

Chemical formula

[0102] is a group represented by; A is a triazolyl group which may be substituted with one or more substituents (see, for example, Japanese Patent Application No. 2018-212424). Typical specific examples are nucleosides or nucleotides in which A is a triazolyl group which may have one or more methyl groups, more specifically a 1,5-dimethyl-1,2,4-triazol- 3-yl group.

[0103] "ALNA[Oxz]" is a nucleoside or nucleotide represented by the general formula (I) defined in the above "ALNA[mU]", and in the formula, X is the following formula (II-2):

[0104]

Chemical formula

[0105] is a group represented by; A is an oxadiazolyl group which may be substituted with one or more substituents (see, for example, Japanese Patent Application No. 2018-212424). Typical specific examples are nucleosides or nucleotides in which A is an oxadiazolyl group which may have one or more methyl groups, and more specifically, a 5-methyl-1,2,4-oxadiazol-3-yl group.

[0106] 「ALNA[Ms]」 is a nucleoside or nucleotide represented by the general formula (I) defined in the above-mentioned 「ALNA[mU]」, and in the formula, X is a group represented by the following formula (II-3):

[0107]

Chemical formula

[0108] and is a group represented by; M is a sulfonyl group substituted with a methyl group which may be substituted with one or more substituents (see, for example, Japanese Patent Application No. 2018-212424). Typical specific examples are nucleosides or nucleotides in which M is a sulfonyl group substituted with an unsubstituted methyl group.

[0109] 「5-Methylcytosine」 means cytosine modified with a methyl group bonded to the 5-position. 5-Methylcytosine is a modified nucleobase.

[0110] 「Single-stranded oligonucleotide」 means an oligonucleotide that is not hybridized with a complementary strand.

[0111] 「DUX4」 means a nucleic acid or protein of a transcription factor also called Double homeobox4. Examples of DUX4 include various splicing variants transcribed from the DUX4 gene, or single nucleotide substitution variants (SNPs) thereof, but the variants and / or SNPs may also be used.

[0112] Numerous splicing variants of DUX4 mRNA have been reported. Human DUX4-s (SEQ ID NO: 6 in the Sequence Listing) consists of a short exon 1 (exon 1s), exon 2, and exon 3 due to an atypical splicing donor site within exon 1, and encodes a short, non-toxic DUX4 protein. Human DUX4-FL consists of exon 1, exon 2, and exon 3, and encodes a full-length DUX4 protein. DUX4-FL includes DUX4-FL1 (SEQ ID NO: 1 in the Sequence Listing), a mature mRNA without intron 1, and DUX4-FL2 (SEQ ID NO: 5 in the Sequence Listing), which contains intron 1. Both encode a full-length DUX4 protein, and when expressed in muscle, are considered to be the cause of FSHD (see Non-Patent Document 2).

[0113] DUX4 mRNA is also expressed in normal testes. In addition to DUX4-FL, splicing variants of exon 1, exon 2, exon 6, exon 7, and / or exon 1, exon 2, exon 4, exon 5, exon 6, exon 7 are expressed (see Non-Patent Document 1).

[0114] The DUX4 protein functions as a transcription factor. Examples of genes whose transcription is regulated by DUX4 include MBD3L2, ZSCAN4, TRIM43, DEFB103, and ZNF217 (see Non-Patent Document 2).

[0115] The DUX4 mRNA targeted by the modified oligonucleotide of the present invention is preferably, for example, human DUX4, more preferably DUX4-FL, and even more preferably DUX4-FL1 described in SEQ ID NO: 1 of the Sequence Listing. Further, as the target sites of the modified oligonucleotide against DUX4 of the present invention, exon 1, intron 1, exon 2, intron 2, and exon 3 are preferred.

[0116] ​The DUX4 gene is known to be expressed by fusing with other genes due to chromosomal abnormalities such as translocations. Examples of other genes include IGH (Yasuda et al., Nature Genetics 48(5), 569 (2016)), CIC (Yoshimoto et al., Cancer research 77, 2927 (2017)), EWSR1 (Sirvent et al., Cancer Genetics and Cytogenetics 195, 12 (2009)) have been reported, and are considered to be the causative genes for B-cell acute lymphoblastic leukemia, differentiated round cell sarcoma, fetal rhabdomyosarcoma, etc., respectively. The modified oligonucleotides of the present invention also include compounds that target these fusion genes.

[0117] "Expression of DUX4" means the level of mRNA transcribed from the gene encoding DUX4, or the level of protein translated from that mRNA. The expression of DUX4 can be determined by methods known in the art such as Northern or Western blotting, PCR.

[0118] "DUX4 nucleic acid" means any nucleic acid encoding DUX4. For example, in certain embodiments, the DUX4 nucleic acid includes a DNA sequence encoding DUX4, an RNA sequence transcribed from DNA encoding DUX4 (including genomic DNA containing introns and exons), and an mRNA precursor or spliced mature mRNA encoding DUX4. Also, in certain embodiments, it includes the DNA sequence and RNA sequence of a gene generated by the fusion of the DUX4 gene with other genes.

[0119] "DUX4 mRNA" means mRNA encoding the DUX4 protein.

[0120] "Continuous nucleobases", "adjacent nucleobases" mean nucleobases that are directly adjacent to each other.

[0121] ​"Complementary" means the ability to form base pairs between the nucleobases of a first nucleic acid and a second nucleic acid.

[0122] "Fully complementary (also referred to as complementarity)" or "100% complementary (also referred to as complementarity)" means that every nucleobase of the nucleobase sequence of the first nucleic acid has a complementary nucleobase in the second nucleobase sequence of the second nucleic acid. In certain embodiments, the first nucleic acid is a modified oligonucleotide and the target nucleic acid is the second nucleic acid.

[0123] "Hybridization" means the annealing of complementary nucleic acid molecules. In certain embodiments, the complementary nucleic acid molecules include modified oligonucleotides and target nucleic acids.

[0124] "Specifically hybridizable" has a degree of complementarity between the modified oligonucleotide and the target nucleic acid sufficient to induce the desired effect, while having minimal or no effect on non-target nucleic acids under the conditions where specific binding is desired, i.e., under physiological conditions in in vivo assays and therapeutic treatments.

[0125] "Mismatch" or "non-complementary nucleobase" refers to the case where the nucleobase of the first nucleic acid cannot form a base pair with the corresponding nucleobase of the second nucleic acid or the target nucleic acid.

[0126] "Targeting" or "targeting to" means the process of designing and selecting a modified oligonucleotide that specifically hybridizes to a target nucleic acid and induces the desired effect.

[0127] "Target nucleic acid", "target RNA" and "target RNA transcript" all refer to nucleic acids that can be targeted by a modified oligonucleotide. In certain embodiments, the target nucleic acid includes a region of the DUX4 nucleic acid.

[0128] "Target segment" means the nucleotide sequence of the target nucleic acid that the modified oligonucleotide targets. "5'-target site" refers to the most 5'-terminal nucleotide of the target segment. "3'-target site" refers to the most 3'-terminal nucleotide of the target segment.

[0129] "Active target region" or "target region" means the region targeted by one or more active modified oligonucleotides. "Active modified oligonucleotide" means a modified oligonucleotide that reduces the target nucleic acid level or protein level.

[0130] "Antisense inhibition" means that the target nucleic acid level or target protein level in the presence of a modified oligonucleotide complementary to the target nucleic acid is reduced compared to the target nucleic acid level or target protein level in the absence of the modified oligonucleotide.

[0131] "siRNA" means a double-stranded RNA oligonucleotide having a nucleobase sequence that enables hybridization to a corresponding region or segment of the target nucleic acid.

[0132] "shRNA" means a hairpin-shaped single-stranded RNA oligonucleotide having a nucleobase sequence that enables hybridization to a corresponding region or segment of the target nucleic acid.

[0133] "snoRNA" means a non-coding RNA present in the nucleolus, having a nucleobase sequence that enables hybridization to a corresponding region or segment of the target RNA nucleic acid, and leading to chemical modifications such as methylation or pseudouridylation of the target RNA nucleic acid, and is a single-stranded oligonucleotide.

[0134] "miRNA" means a non-coding RNA that regulates the expression of other genes, and is a single-stranded or double-stranded RNA oligonucleotide having a nucleobase sequence that enables hybridization to a corresponding region or segment of the target nucleic acid.

[0135] The "cap structure" or "terminal cap portion" means a chemical modification incorporated at either end of a modified oligonucleotide.

[0136] The "chemically heterogeneous region" refers to a region within a modified oligonucleotide that is chemically different in some way from another region 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 2'-O-methoxyethyl modified.

[0137] A "chimeric modified oligonucleotide" means a modified oligonucleotide having at least two chemically heterogeneous regions.

[0138] A "motif" means a pattern of chemically heterogeneous regions within a modified oligonucleotide.

[0139] A "gapmer" means a modified oligonucleotide in which an internal region having a plurality of nucleosides that assist in cleavage by RNase H is positioned between external regions having one or more nucleosides, and the nucleosides constituting the internal region are chemically different from the nucleoside(s) constituting the external region(s). The internal region can be referred to as the "gap segment", and the external regions can be referred to as the "wing segments". The wing segment present 5' to the gap segment can be referred to as the "5' wing segment", and the wing segment present 3' to the gap segment can be referred to as the "3' wing segment".

[0140] "Immediately adjacent" means that there is no intervening element between immediately adjacent elements.

[0141] "Nuclear ribonuclease" means a ribonuclease found in the nucleus. Examples of nuclear ribonucleases include, but are not limited to, RNase H including RNase H1 and RNase H2, double-stranded RNase Drosha, and other double-stranded RNases.

[0142] In certain embodiments, a gapmer is a modified oligonucleotide having 5' and 3' wing segments each having from 1 to 8 nucleosides, and a gap segment located between and directly adjacent to both wing segments and having 6 or more nucleosides, the gap segment containing only nucleosides without modified sugars or containing 1 or 2 nucleosides with modified sugars and otherwise nucleosides without modified sugars.

[0143] "Agent" means an active substance that can provide a therapeutic benefit when administered to an animal. "First agent" means the therapeutic compound of the present invention. For example, the first agent may be a modified oligonucleotide that targets DUX4. "Second agent" means the 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.

[0144] "Pharmaceutically acceptable salt" means a physiologically and pharmaceutically acceptable salt of a modified oligonucleotide, i.e., a salt that retains the desired biological activity of the modified oligonucleotide and does not impart undesirable toxic effects thereto.

[0145] "Diluent" means a component in a composition that lacks pharmacological activity but is pharmaceutically necessary or desirable. For example, the diluent in an injectable composition may be a liquid such as saline.

[0146] "DUX4-related diseases" refer to diseases caused by abnormal expression of DUX4 mRNA or DUX4 protein, or mRNA or protein of a fusion gene resulting from translocation of the DUX4 gene. Examples include facioscapulohumeral muscular dystrophy, B-cell acute lymphoblastic leukemia, differentiated round cell sarcoma, and fetal rhabdomyosarcoma, but are not limited thereto. Examples include, but are not limited to, differentiated round cell sarcoma and fetal rhabdomyosarcoma.

[0147] "Facioscapulohumeral muscular dystrophy" or "FSHD" means a muscular dystrophy that develops from muscle weakness in the face, scapula, and upper arm muscles. In humans, it is mainly associated with shortening of the genomic repeat sequence (D4Z4) near the telomere (the end of the chromosome) of chromosome 4, resulting in changes in the genomic structure, and it is thought that the DUX4 gene, which is not normally expressed in muscle (progenitor) cells, is ectopically expressed and causes cell death (FSHD1). In addition, gene mutations have been found in one of the genomic structure regulators, SMCHD1, which suppresses gene expression, in some FSHD patients (FSHD2).

[0148] FSHD is a type of muscular dystrophy associated with progressive muscle weakness and muscle fiber loss. Different from Duchenne muscular dystrophy and Becker muscular dystrophy, which mainly affect the lower body, FSHD develops in the upper body, mainly in the facial muscles, scapular muscles, and upper arm muscles. However, it can also develop in the pelvis, lower back, and lower limbs. The symptoms of FSHD often appear between the ages of 10 and 26, but it is not uncommon for them to develop much later. In some cases, it may not develop at all. The symptoms are usually mild and the rate of deterioration is very slow. Facial muscle weakness is common, and there may be ptosis, inability to whistle, decreased facial expression changes, a depressed or angry facial expression, difficulty pronouncing words, scapular muscle weakness (causing prominence of the scapula (winged scapula) and deformities such as winging of the scapula), lower limb weakness, hearing loss, and the risk of heart disease.

[0149] "Active pharmaceutical agent" means a substance(s) in a pharmaceutical composition that provides a therapeutic benefit when administered to an animal. For example, in certain embodiments, a modified oligonucleotide that targets DUX4 is an active pharmaceutical agent.

[0150] "Administered concomitantly" means that the pharmacological effects resulting from both agents are manifested in the patient simultaneously. Refers to the co - administration of two agents in any manner. Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The pharmacological effects resulting from both agents do not have to be manifested simultaneously. The pharmacological effects only need to overlap within a certain period and do not have to have the same extent.

[0151] "Administering" means giving an agent to an animal, including, but not limited to, administration by a medical professional and self - administration.

[0152] "Improve" means reducing at least one indicator, sign, or symptom of a related disease, disorder, or condition. The severity of the indicator can be determined by subjective or objective scales known to those skilled in the art.

[0153] "Animal" means a human, 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.

[0154] "Co - administration" means the administration of two or more agents to an individual. The two or more agents may be present in a single pharmaceutical composition or in separate pharmaceutical compositions. Each of the two or more agents can be administered via the same or different routes of administration. Co - administration includes concurrent or sequential administration.

[0155] "Dosage" means a specific amount of a pharmaceutical agent administered in a single dose or over a specified period. In certain embodiments, the dosage can be administered as one, two or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dosage requires a volume that cannot easily be contained in a single injection, so two or more injections can be used to achieve the desired dosage. In certain embodiments, the pharmaceutical agent is administered by infusion over a long period or continuously. The dosage can be described as the amount of the pharmaceutical agent per hour, day, week or month.

[0156] "Effective amount" or "therapeutically effective amount" means the amount of an active pharmaceutical agent sufficient to achieve a desired physiological outcome in an individual in need of the agent. The effective amount can vary between individuals depending on the health and physical condition of the treated individual, the taxonomic group of the treated individual, the formulation of the composition, the assessment of the medical condition of the individual and other relevant factors.

[0157] "Identifying an animal having facioscapulohumeral muscular dystrophy (FSHD)" means identifying an animal diagnosed with the disorder or condition of FSHD, or an animal likely to develop the disorder or condition of FSHD. For example, an individual with a family history may be likely to develop the disorder or condition of FSHD. Such identification can be achieved by any method, including examining the individual's medical history and standard clinical tests or evaluations. Although FSHD is known as FSHD1 and FSHD2 according to the pathogenesis, both are included.

[0158] "Individual" means a human or non-human animal selected for treatment or therapy.

[0159] "Myotonia" means an abnormally slow relaxation of muscle after voluntary contraction or electrical stimulation.

[0160] "Parenteral administration" means administration via injection or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intra-arterial administration, intraperitoneal administration or intracranial administration, for example, intrathecal or intraventricular administration. The administration may be continuous or long-term, or short-term or intermittent.

[0161] "Pharmaceutical composition" means a mixture of substances suitable for administration to an individual. For example, a pharmaceutical composition may contain one or more active agents and a sterile aqueous solution.

[0162] "Prevent" 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, over a period ranging from several minutes to an indefinite period. Preventing also means reducing the risk of developing a disease, disorder or undesirable health condition. Preventing also means reducing the risk of developing a disease, disorder or undesirable health condition.

[0163] "Treat" means alleviating or eliminating a disease, disorder or undesirable health condition, or one or more symptoms associated with the disease, disorder or undesirable health condition, or partially resolving or eradicating one or more causes of the disease, disorder or undesirable health condition itself.

[0164] "Treat" is intended to include the above prevention or treatment. For example, it also includes administering the pharmaceutical composition of the present invention to bring about a change or improvement in the disease, disorder or undesirable health condition.

[0165] "Prodrug" means a therapeutic agent prepared in an inactive form that is converted to an active form in the body or within its cells by the action of endogenous enzymes or other chemical substances or conditions.

[0166] "Side effect" means a physiological response that can be caused by a treatment other than the desired effect. In certain embodiments, side effects include injection site reactions, abnormalities in liver function tests, renal function abnormalities, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, myopathy, and fatigue. For example, an increase in the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), or γ-glutamyl transpeptidase (γ-GTP) in the blood may indicate hepatotoxicity or liver function abnormalities. For example, an increase in bilirubin may indicate hepatotoxicity or liver function abnormalities. Also, an increase in urinary protein, an increase in creatinine or urea nitrogen (UN) in the blood may indicate nephrotoxicity or renal function abnormalities.

[0167] "Subcutaneous administration" means administration under the skin.

[0168] "Therapeutically effective amount" means an amount of a drug that confers a therapeutic benefit to an individual.

[0169] (Specific embodiments) Embodiments Certain specific embodiments shown below, but not limited thereto, provide compounds for inhibiting the expression of DUX4, methods using the compounds, and pharmaceutical compositions containing the compounds.

[0170] Certain embodiments provide a method of reducing the expression of DUX4 in an animal, comprising administering to the animal a compound containing a modified oligonucleotide that targets DUX4.

[0171] Certain embodiments provide a method of administering a modified oligonucleotide to prevent the accumulation of pathogenic DUX4 transcription factor by inhibiting the transcription of the DUX4 gene, or inhibiting the translation of DUX4 mRNA, or inducing the cleavage of DUX4 mRNA.

[0172] Certain embodiments provide a method of treating an animal having facioscapulohumeral muscular dystrophy, the method comprising the following steps: a) before having facioscapulohumeral muscular dystrophy identifying the subject animal, and b) administering to the animal a compound comprising a therapeutically effective amount of a modified oligonucleotide that targets DUX4.

[0173] Certain embodiments provide a method of reducing myotonia in a subject in need thereof. The method comprises administering to the subject a modified oligonucleotide complementary to DUX4 mRNA, wherein the modified oligonucleotide, when bound to DUX4 mRNA, activates a ribonuclease or a nuclear ribonuclease, thereby reducing myotonia. In certain embodiments, the subject has, is suspected of having, or highly expresses DUX4 mRNA, has a reduced number of D4Z4 repeats on human chromosome 4, or has or is suspected of having an SMCHD1 (DNA methylase) mutation.

[0174] In certain embodiments, the modified oligonucleotide used in the methods of the invention is chimeric. In certain embodiments, the modified oligonucleotide of the methods used in the invention is a gapmer.

[0175] In certain embodiments of the methods of the invention described herein, the administration is subcutaneous administration. In certain embodiments, the administration is intravenous or intramuscular administration.

[0176] In certain embodiments, the modified oligonucleotide used in the methods of the invention targets the DUX4 protein coding region, intron, 5'UTR or 3'UTR of DUX4 mRNA. In certain embodiments, the modified oligonucleotide used in the methods of the invention targets exon 1, exon 2, exon 3, intron 1, intron 2 of DUX4 mRNA.

[0177] In certain embodiments of the methods of the invention described herein, DUX4 mRNA is cleaved by the nuclear ribonuclease RNase H1.

[0178] In certain embodiments of the method of the present invention, DUX4 mRNA is reduced in muscle tissue. In certain embodiments, splicing 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.

[0179] In certain embodiments, DUX4 mRNA has the sequence described in GenBank accession number NM_001293798.2 (incorporated herein as SEQ ID NO: 1 in the Sequence Listing). The splicing variant of SEQ ID NO: 1 in the Sequence Listing is also referred to as DUX4-FL1 or the mature mRNA of DUX4. In certain embodiments, DUX4 mRNA has the sequence described in GenBank accession number NM_001306068.2 (incorporated herein as SEQ ID NO: 5 in the Sequence Listing). The splicing variant of SEQ ID NO: 5 in the Sequence Listing is also referred to as DUX4-FL2. In certain embodiments, DUX4 has the sequence described in GenBank accession number NM_001363820.1 (incorporated herein as SEQ ID NO: 6 in the Sequence Listing). The splicing variant of SEQ ID NO: 6 in the Sequence Listing is also referred to as DUX4-s. In certain embodiments, DUX4 mRNA has SNPs of the above splicing variants.

[0180] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues and contains a nucleotide sequence containing at least 8 consecutive nucleotide sequences complementary to an isologous portion at positions 126 to 147, 232 to 248, 1306 to 1325, or 1472 to 1495 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing. In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues and having a nucleotide sequence comprising a modified oligonucleotide which is a nucleotide sequence comprising at least 9, 10, 11 or 12 consecutive nucleotide sequences complementary to an isologous portion at positions 126 to 147, 232 to 248, 1306 to 1325 or 1472 to 1495 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing. The modified oligonucleotide may consist of at least 8 consecutive nucleotide sequences complementary to an isologous portion at positions 126 to 147, 232 to 248, 1306 to 1325 or 1472 to 1495 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, or may have additional sequences on the 5'-end side and / or 3'-end side in addition to the nucleotide sequence.

[0181] In certain embodiments, the modified oligonucleotide is a nucleotide sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotide sequences complementary to an isologous portion at positions 126 to 147 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and is a modified oligonucleotide within 30 residues.

[0182] In certain embodiments, the modified oligonucleotide is a nucleotide sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 consecutive nucleotide sequences complementary to an isologous portion at positions 232 to 248 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and is a modified oligonucleotide within 30 residues.

[0183] In certain embodiments, the modified oligonucleotide is a nucleotide sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 consecutive nucleotide sequences complementary to an isologous portion at positions 1306 to 1325 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and is a modified oligonucleotide within 30 residues.

[0184] In certain embodiments, 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 complementary to an isometric portion at positions 1472 to 1495 from the 5'-end of the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and is a modified oligonucleotide within 30 residues.

[0185] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues, and comprises a nucleic acid base sequence complementary to an isometric portion at positions 126 to 147 from the 5'-end of the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and is a modified oligonucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to SEQ ID NO: 1 in the Sequence Listing in the isometric portion. Also, in certain embodiments, 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 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, 14 to 15 residues of the above-mentioned modified oligonucleotide.

[0186] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues, and contains a nucleobase sequence complementary to the 232nd to 248th position of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and is a modified oligonucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to SEQ ID NO: 1 in the Sequence Listing in the equivalent-length portion. Also, in certain embodiments, the modified oligonucleotide 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 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, 14 to 15 residues of the above-mentioned modified oligonucleotide.

[0187] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues and contains a nucleobase sequence complementary to the 1306-1325 position of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and is a modified oligonucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to SEQ ID NO: 1 in the Sequence Listing at the equal-length portion. Also, in certain embodiments, the modified oligonucleotide 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 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, 14 to 15 residues of the above-mentioned modified oligonucleotide.

[0188] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues, and contains a nucleobase sequence complementary to the equi-length portion at positions 1472 to 1495 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and is a modified oligonucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to SEQ ID NO: 1 in the Sequence Listing in the equi-length portion. Also, in certain embodiments, 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 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 1 The modified oligonucleotide consisting of 9 residues, 14 to 18 residues, 14 to 17 residues, 14 to 16 residues, 14 to 15 residues.

[0189] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues and having a nucleotide sequence comprising at least 8 consecutive nucleotide sequences complementary to an isologous portion at positions 128 to 143, 233 to 248, 1309 to 1323, or 1480 to 1495 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing. In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues and having a nucleotide sequence comprising at least 9, 10, 11, or 12 consecutive nucleotide sequences complementary to an isologous portion at positions 128 to 143, 233 to 248, 1309 to 1323, or 1480 to 1495 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing.

[0190] In certain embodiments, the modified oligonucleotide is a nucleotide sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive nucleotide sequences complementary to an isologous portion at positions 128 to 143 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and is a modified oligonucleotide within 30 residues.

[0191] In certain embodiments, the modified oligonucleotide is a nucleotide sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive nucleotide sequences complementary to an isologous portion at positions 233 to 248 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and is a modified oligonucleotide within 30 residues.

[0192] In certain embodiments, the modified oligonucleotide is a nucleotide sequence comprising at least 8, 9, 10, 11, 12, 13, 14, or 15 consecutive nucleotide sequences complementary to an isometric portion at positions 1309 to 1323 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and is a modified oligonucleotide within 30 residues.

[0193] In certain embodiments, the modified oligonucleotide is a nucleotide sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive nucleotide sequences complementary to an isometric portion at positions 1480 to 1495 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and consists of a nucleotide sequence having a complementary base of the base at position 1480 from the 5'-end of the nucleobases of SEQ ID NO: 1 at the 3'-end, and is a modified oligonucleotide within 30 residues.

[0194] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues, and comprises a nucleotide sequence complementary to an isometric portion at positions 128 to 143 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to SEQ ID NO: 1 in the isometric portion. Also, in certain embodiments, 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 to 21 residues, 13 to 20 residues, 13 to 19 residues, 13 to 18 residues It is the modified oligonucleotide of a base, 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, 14 to 15 residues.

[0195] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues, and contains a nucleobase sequence complementary to the equi-length portion at positions 233 to 248 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and is a modified oligonucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to SEQ ID NO: 1 in the Sequence Listing in the equi-length portion. Also, in certain embodiments, it is the modified oligonucleotide of 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 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, 14 to 15 residues.

[0196] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues and contains a nucleobase sequence complementary to the 1309-1323 position isologous portion of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and is a modified oligonucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to SEQ ID NO: 1 in the Sequence Listing in the isologous portion. Also, in certain embodiments, the modified oligonucleotide 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 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, 14 to 15 residues of the above-mentioned modified oligonucleotide.

[0197] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues and is a nucleobase sequence complementary to an isologous portion at positions 1480 to 1495 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, consisting of a nucleobase sequence having the complementary base of the base at position 1480 from the 5'-end of the nucleobases of SEQ ID NO: 1 at the 3'-end, and is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to SEQ ID NO: 1 in the Sequence Listing in the isologous portion. Also, in certain embodiments, 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, 1 The modified oligonucleotide consisting of 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 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, 14 to 15 residues.

[0198] In certain embodiments, the modified oligonucleotides provided herein target any one of the following regions of SEQ ID NO:1 in the Sequence Listing: positions 126-141, 126-143, 127-142, 127-143, 127-144, 127-146, 128-143, 128-144, 128-147, 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, 1474-1488, 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, counting from the 5' end. In certain embodiments, the modified oligonucleotides provided herein target any one of the following regions of SEQ ID NO:1 in the Sequence Listing: positions 128-143, 232-247, 233-248, 1309-1323 and 1480-1495, counting from the 5' end.

[0199] In certain embodiments, the modified oligonucleotides provided herein have a nucleobase sequence comprising a complementary region containing at least 8 consecutive nucleobases complementary to a target region. In certain embodiments, the modified oligonucleotides provided herein have a nucleobase sequence comprising a complementary region containing at least 8 consecutive nucleobases complementary to a target region, and the target region is positions 126-141, 126-143, 127-142, 127-143, 127-144, 127-146, 128-143, 128-144, 128-147, 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, 1474-1488, 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 counted from the 5'-end of SEQ ID NO: 1 in the Sequence Listing. In certain embodiments, the modified oligonucleotides provided herein have a nucleobase sequence comprising a complementary region containing at least 8 consecutive nucleobases complementary to a target region, and the target region is positions 128-143, 232-247, 233-248, 1309-1323 or 1480-1495 counted from the 5'-end of SEQ ID NO: 1 in the Sequence Listing.

[0200] In certain embodiments, the modified oligonucleotides provided herein have a nucleobase sequence comprising a complementary region containing at least 10 consecutive nucleobases complementary to a target region. In certain embodiments, the modified oligonucleotides provided herein have a nucleobase sequence comprising a complementary region containing at least 10 consecutive nucleobases complementary to a target region, and the target region is positions 126-141, 126-143, 127-142, 127-143, 127-144, 127-146, 128-143, 128-144, 128-147, 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, 1474-1488, 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 counted from the 5'-end of SEQ ID NO: 1 in the Sequence Listing. In certain embodiments, the modified oligonucleotides provided herein have a nucleobase sequence comprising a complementary region containing at least 10 consecutive nucleobases complementary to a target region, and the target region is positions 128-143, 232-247, 233-248, 1309-1323 or 1480-1495 counted from the 5'-end of SEQ ID NO: 1 in the Sequence Listing.

[0201] In certain embodiments, the modified oligonucleotides provided herein have a nucleobase sequence comprising a complementary region containing at least 12 consecutive nucleobases complementary to a target region. In certain embodiments, the modified oligonucleotides provided herein have a nucleobase sequence comprising a complementary region containing at least 12 consecutive nucleobases complementary to a target region, and the target region is positions 126-141, 126-143, 127-142, 127-143, 127-144, 127-146, 128-143, 128-144, 128-147, 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, 1474-1488, 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 counting from the 5'-end of SEQ ID NO:1 in the Sequence Listing. In certain embodiments, the modified oligonucleotides provided herein have a nucleobase sequence comprising a complementary region containing at least 12 consecutive nucleobases complementary to a target region, and the target region is positions 128-143, 232-247, 233-248, 1309-1323 or 1480-1495 counting from the 5'-end of SEQ ID NO:1 in the Sequence Listing.

[0202] In certain embodiments, the modified oligonucleotides provided herein are directed to a target region It has a nucleobase sequence comprising a complementary region containing at least 14 consecutive nucleobases that are complementary to a target region. In certain embodiments, the modified oligonucleotides provided herein have a nucleobase sequence comprising a complementary region containing at least 14 consecutive nucleobases that are complementary to a target region, and the target region is positions 126-141, 126-143, 127-142, 127-143, 127-144, 127-146, 128-143, 128-144, 128-147, 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, 1474-1488, 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 counted from the 5'-end of SEQ ID NO:1 in the Sequence Listing. In certain embodiments, the modified oligonucleotides provided herein have a nucleobase sequence comprising a complementary region containing at least 14 consecutive nucleobases that are complementary to a target region, and the target region is positions 128-143, 232-247, 233-248, 1309-1323 or 1480-1495 counted from the 5'-end of SEQ ID NO:1 in the Sequence Listing.

[0203] In certain embodiments, the modified oligonucleotide comprises a nucleotide sequence complementary to an isometric portion at positions 126-141, 126-143, 127-142, 127-143, 127-144, 127-146, 128-143, 128-144, 128-147, 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, 1474-1488, 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 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing. Further, in certain embodiments, the modified oligonucleotide is a modified oligonucleotide comprising a nucleotide sequence complementary to an isometric portion at positions 128-143, 232-247, 233-248, 1309-1323, 1480-1495 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing.

[0204] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 2-4, 7-64, 69-97, 102-112 in the Sequence Listing. Further, in certain embodiments, the modified oligonucleotide is a modified oligonucleotide comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 2-4, 75, 78 in the Sequence Listing.

[0205] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of the nucleobase sequence set forth in any one of SEQ ID NOs: 2-4, 7-64, 69-97, 102-112 in the Sequence Listing. Also, in certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of the nucleobase sequence set forth in any one of SEQ ID NOs: 2-4, 75, 78 in the Sequence Listing.

[0206] In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 8 consecutive nucleobases of the nucleobase sequence set forth in any one of SEQ ID NOs: 2, 3, 4, 75, or 78 in the Sequence Listing.

[0207] In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 10 consecutive nucleobases of the nucleobase sequence set forth in any one of SEQ ID NOs: 2, 3, 4, 75, or 78 in the Sequence Listing.

[0208] In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 12 consecutive nucleobases of the nucleobase sequence set forth in any one of SEQ ID NOs: 2, 3, 4, 75, or 78 in the Sequence Listing.

[0209] In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 14 consecutive nucleobases of the nucleobase sequence set forth in any one of SEQ ID NOs: 2, 3, 4, 75, or 78 in the Sequence Listing.

[0210] In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising a nucleobase of the nucleobase sequence set forth in any one of SEQ ID NOs: 2, 3, 4, 75, or 78 in the Sequence Listing.

[0211] In certain embodiments, the modified oligonucleotide has a nucleobase sequence consisting of the nucleobase sequence set forth in any one of SEQ ID NOs: 2, 3, 4, 75, or 78 in the Sequence Listing.

[0212] In certain embodiments, the animal is a human.

[0213] In certain embodiments, administration includes parenteral administration.

[0214] In certain embodiments, the compound is a single-stranded modified oligonucleotide.

[0215] In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to an equi-length portion of any one of the regions of SEQ ID NO: 1 in the Sequence Listing when measured over the entire length of the modified oligonucleotide. In certain embodiments, the nucleobase sequence of the modified oligonucleotide is 100% complementary to an equi-length portion of any one of the regions of SEQ ID NO: 1 in the Sequence Listing when measured over the entire length of the modified oligonucleotide.

[0216] In certain embodiments, at least one internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage. In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage.

[0217] 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 includes 2'-O-methoxyethyl, 2'-O-methyl and / or 4'-(CH2) n -O-2' bridge (wherein n is 1 or 2).

[0218] In certain embodiments, the sugar moiety of the modified oligonucleotide comprises a modified sugar that is at least one bicyclic sugar. In certain embodiments, at least one modified sugar is LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], and / or ALNA[Trz].

[0219] In certain embodiments, at least one nucleoside of the modified oligonucleotide comprises a modified nucleobase. In certain embodiments, the modified nucleobase is 5-methylcytosine.

[0220] In certain embodiments, the modified oligonucleotide is a gapmer and comprises: 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.

[0221] In certain embodiments, the modified oligonucleotide is a gapmer and comprises: a) a gap segment that includes nucleosides that include modified sugars such as one or two 2'-O-methyl modified sugars or 2'-O-methoxyethyl modified sugars and other nucleosides that do not include modified sugars; 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 sugar, or a bicyclic sugar.

[0222] In certain embodiments, the modified oligonucleotide is a gapmer and comprises the following: a) a gap segment having 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 nucleosides, wherein the nucleosides are either only nucleosides without modified sugars or contain 1 or 2 nucleosides with modified sugars such as 2'-O-methyl modified sugars or 2'-O-methoxyethyl modified sugars and the rest are nucleosides without modified sugars; b) a 5' wing segment having 2, 3, 4, 5, 6 or 7 nucleosides; and c) a 3' wing segment having 2, 3, 4, 5, 6, 7 or 8 nucleosides. Here, the gap segment is located between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment contains a 2'-O-methyl modified sugar, a 2'-O-methoxyethyl sugar or a bicyclic sugar, each internucleoside linkage of the modified oligonucleotide contains a phosphorothioate linkage, and some or all of the cytosines in the modified oligonucleotide may be 5'-methylcytosine.

[0223] In certain embodiments, 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.

[0224] In certain embodiments, the modified oligonucleotide is a gapmer and in the sugar moiety of the nucleoside, it contains 2'-O-methyl, 2'-O-methoxyethyl and / or 4'-(CH2) n -O-2' crosslinking (wherein n is 1 or 2).

[0225] In certain embodiments, the modified oligonucleotide is a gapmer and in the sugar moiety of the nucleoside, it contains LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], and / or ALNA[Trz].

[0226] In certain embodiments, the modified oligonucleotide is a gapmer, which is a modified oligonucleotide consisting of 12 to 30 residues and having a nucleotide sequence comprising at least 8 consecutive nucleotide sequences complementary to an isologous portion at positions 126 to 147, 232 to 248, 1306 to 1325, or 1472 to 1495 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO:1 in the Sequence Listing. In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues and having a nucleotide sequence comprising at least 9, 10, 11, or 12 consecutive nucleotide sequences complementary to an isologous portion at positions 126 to 147, 232 to 248, 1306 to 1325, or 1472 to 1495 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO:1 in the Sequence Listing.

[0227] In certain embodiments, the modified oligonucleotide is a gapmer and is a nucleotide sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotide sequences complementary to an isologous portion at positions 126 to 147 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO:1 in the Sequence Listing, and is a modified oligonucleotide within 30 residues.

[0228] In certain embodiments, the modified oligonucleotide is a gapmer and is a nucleotide sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 consecutive nucleotide sequences complementary to an isologous portion at positions 232 to 248 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO:1 in the Sequence Listing, and is a modified oligonucleotide within 30 residues.

[0229] In certain embodiments, the modified oligonucleotide is a gapmer and is a nucleic acid sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleic acid base sequences complementary to an equal-length portion at positions 1306 to 1325 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO:1 in the Sequence Listing, and is a modified oligonucleotide within 30 residues.

[0230] In certain embodiments, the modified oligonucleotide is a gapmer and is a nucleic acid 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 complementary to an equal-length portion at positions 1472 to 1495 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO:1 in the Sequence Listing, and is a modified oligonucleotide within 30 residues.

[0231] In certain embodiments, the modified oligonucleotide is a gapmer of a modified oligonucleotide consisting of 12 to 30 residues and comprises a nucleic acid sequence complementary to an equal-length portion at positions 126 to 147 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO:1 in the Sequence Listing, and is a modified oligonucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to SEQ ID NO:1 in the Sequence Listing in the equal-length portion. Also, in certain embodiments, 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 The modified oligonucleotide is one of 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 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.

[0232] In certain embodiments, the modified oligonucleotide is a gapmer of a modified oligonucleotide consisting of 12-30 residues, and contains a nucleobase sequence complementary to an equi-length portion at positions 233-248 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and is a modified oligonucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to SEQ ID NO: 1 in the Sequence Listing in the equi-length portion. Also, in certain embodiments, the modified oligonucleotide is one of 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 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.

[0233] In certain embodiments, the modified oligonucleotide is a gapmer of a modified oligonucleotide consisting of 12 to 30 residues, and contains a nucleobase sequence complementary to an equal-length portion at positions 1309 to 1323 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and is a modified oligonucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to SEQ ID NO: 1 in the Sequence Listing in the equal-length portion. Also, in certain embodiments, it is the above-mentioned modified oligonucleotide consisting of 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 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, 14 to 15 residues.

[0234] In certain embodiments, the modified oligonucleotide is a gapmer of a modified oligonucleotide consisting of 12 to 30 residues, and contains a nucleobase sequence complementary to an equal-length portion at positions 1472 to 1495 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and is a modified oligonucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to SEQ ID NO: 1 in the Sequence Listing in the equal-length portion. Also, in certain embodiments, 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 The modified oligonucleotide is one selected from 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 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 14 to 15. In this case, the oligonucleotide contained in the 5' wing segment and / or the 3' wing segment contains at least one nucleoside containing a modified sugar selected from at least one of GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz] and ALNA[Trz], and may further contain a sugar modified with 2'-O-methoxyethyl and / or a sugar modified with 2'-O-methyl.

[0235] In certain embodiments, the modified oligonucleotide is a gapmer of a modified oligonucleotide consisting of 12 to 30 residues, and is a nucleobase sequence complementary to an equi-length portion at positions 1480 to 1495 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, consisting of a nucleobase sequence having a complementary base of the base at position 1480 from the 5'-end of the nucleobases of SEQ ID NO: 1 at the 3'-end, and is a modified oligonucleotide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to SEQ ID NO: 1 in the Sequence Listing in the equi-length portion. Also, in certain embodiments, the modified oligonucleotide consists of 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 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, 14 to 15 residues.

[0236] In certain embodiments, the modified oligonucleotides provided herein are gapmers and target any one of the following regions of SEQ ID NO:1 in the Sequence Listing: positions 126-141, 126-143, 127-142, 127-143, 127-144, 127-146, 128-143, 128-144, 128-147, 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, 1474-1488, 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, counted from the 5' end. In certain embodiments, the modified oligonucleotides provided herein are gapmers and target any one of the following regions of SEQ ID NO:1 in the Sequence Listing: positions 128-143, 232-247, 233-248, 1309-1323 and 1480-1495, counted from the 5' end.

[0237] In certain embodiments, the modified oligonucleotides provided herein are gapmers and have a nucleobase sequence that includes a complementary region containing at least 8 consecutive nucleobases complementary to a target region. In certain embodiments, the modified oligonucleotides provided herein have a nucleobase sequence that includes a complementary region containing at least 8 consecutive nucleobases complementary to a target region, and the target region is positions 126-141, 126-143, 127-142, 127-143, 127-144, 127-146, 128-143, 128-144, 128-147, 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, 1474-1488, 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 from the 5'-end of SEQ ID NO: 1 in the Sequence Listing. In certain embodiments, the modified oligonucleotides provided herein are gapmers and have a nucleobase sequence that includes a complementary region containing at least 8 consecutive nucleobases complementary to a target region, and the target region is positions 128-143, 232-247, 233-248, 1309-1323 or 1480-1495 from the 5'-end of SEQ ID NO: 1 in the Sequence Listing.

[0238] In certain embodiments, the modified oligonucleotides provided herein are gapmers and have a nucleobase sequence that includes a complementary region containing at least 10 consecutive nucleobases complementary to a target region. In certain embodiments, the modified oligonucleotides provided herein have a nucleobase sequence that includes a complementary region containing at least 10 consecutive nucleobases complementary to a target region, and the target region is positions 126-141, 126-143, 127-142, 127-143, 127-144, 127-146, 128-143, 128-144, 128-147, 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, 1474-1488, 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 counted from the 5'-end of SEQ ID NO:1 in the Sequence Listing. In certain embodiments, the modified oligonucleotides provided herein are gapmers and have a nucleobase sequence that includes a complementary region containing at least 10 consecutive nucleobases complementary to a target region, and the target region is positions 128-143, 232-247, 233-248, 1309-1323 or 1480-1495 counted from the 5'-end of SEQ ID NO:1 in the Sequence Listing.

[0239] In certain embodiments, the modified oligonucleotides provided herein are gapmers and have a nucleobase sequence comprising a complementary region containing at least 12 consecutive nucleobases complementary to a target region. In certain embodiments, the modified oligonucleotides provided herein have a nucleobase sequence comprising a complementary region containing at least 12 consecutive nucleobases complementary to a target region, and the target region is positions 126-141, 126-143, 127-142, 127-143, 127-144, 127-146, 128-143, 128-144, 128-147, 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, 1474-1488, 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 from the 5'-end of SEQ ID NO: 1 in the Sequence Listing. In certain embodiments, the modified oligonucleotides provided herein are gapmers and have a nucleobase sequence comprising a complementary region containing at least 12 consecutive nucleobases complementary to a target region, and the target region is positions 128-143, 232-247, 233-248, 1309-1323 or 1480-1495 from the 5'-end of SEQ ID NO: 1 in the Sequence Listing.

[0240] In certain embodiments, the modified oligonucleotides provided herein are gapmers and have a nucleobase sequence that includes a complementary region containing at least 14 consecutive nucleobases complementary to a target region. In certain embodiments, the modified oligonucleotides provided herein have a nucleobase sequence that includes a complementary region containing at least 14 consecutive nucleobases complementary to a target region, and the target region is positions 126-141, 126-143, 127-142, 127-143, 127-144, 127-146, 128-143, 128-144, 128-147, 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, 1474-1488, 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 from the 5'-end of SEQ ID NO: 1 in the Sequence Listing. In certain embodiments, the modified oligonucleotides provided herein are gapmers and have a nucleobase sequence that includes a complementary region containing at least 14 consecutive nucleobases complementary to a target region, and the target region is positions 128-143, 232-247, 233-248, 1309-1323 or 1480-1495 from the 5'-end of SEQ ID NO: 1 in the Sequence Listing.

[0241] In certain embodiments, the modified oligonucleotide is a gapmer and the sequence of the Sequence Listing A modified oligonucleotide consisting of a nucleobase sequence complementary to an isomeric moiety at positions 126-141, 126-143, 127-142, 127-143, 127-144, 127-146, 128-143, 128-144, 128-147, 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, 1474-1488, 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 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of No. 1. In certain embodiments, the modified oligonucleotide is a gapmer and consists of a nucleobase sequence complementary to an isomeric moiety at positions 128-143, 232-247, 233-248, 1309-1323, 1480-1495 from the 5'-end of the nucleobases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing.

[0242] In certain embodiments, the modified oligonucleotide is a gapmer and consists of a nucleobase sequence set forth in any one of SEQ ID NOs: 2-4, 7-64, 69-97, 102-112 in the Sequence Listing. In certain embodiments, the modified oligonucleotide is a gapmer and consists of a nucleobase sequence set forth in any one of SEQ ID NOs: 2-4, 75, 78 in the Sequence Listing.

[0243] In certain embodiments, the modified oligonucleotide is a gapmer and is the modified oligonucleotide described in any one of Compound Numbers 1 to 112, 114 to 132, and 137 to 246. Also, in certain embodiments, the modified oligonucleotide is a gapmer and is the modified oligonucleotide described in any one of Compound Numbers 1 to 3, 123, 157, 204, 221, and 231.

[0244] In this specification, in symbols such as "Gls" representing nucleotides, the abbreviation shown in the left position means the nucleobase part, the abbreviation shown in the central position means the sugar part, and the abbreviation shown in the right position means the mode of internucleoside linkage.

[0245] In certain embodiments, the modified oligonucleotide is a gapmer and has the following formula: GlsMlsMlsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMlsGlsGl; which is a modified oligonucleotide represented by wherein each nucleobase is represented by the following symbols: A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine, as shown according to; each sugar moiety is represented by the following symbols: l = LNA, d = 2'-deoxyribose, as shown according to; each internucleoside linkage is represented by the following symbol: s = phosphorothioate as shown according to, which is a modified oligonucleotide.

[0246] In certain embodiments, the modified oligonucleotide is a gapmer and has the following formula: GmsMmsMmsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMmsGmsGm; which is a modified oligonucleotide represented by wherein, each nucleobase is represented by the following symbols: A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine, is shown according to; each sugar moiety is represented by the following symbols: m = ALNA[Ms], d = 2'-deoxyribose, is shown according to; each internucleoside linkage is represented by the following symbol: s = phosphorothioate, and is a modified oligonucleotide.

[0247] In certain embodiments, the modified oligonucleotide is a gapmer and has the formula: GmsMmsAmsGdsTdsTdsCdsTdsCdsCdsGdsCdsGmsGmsTm; which is a modified oligonucleotide represented by wherein each nucleobase is represented by the following symbols: A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine, is shown according to; each sugar moiety is represented by the following symbols: m = ALNA[Ms], d = 2'-deoxyribose, is shown according to; each internucleoside linkage is represented by the following symbol: s = phosphorothioate and is a modified oligonucleotide shown according to.

[0248] In certain embodiments, the modified oligonucleotide is a gapmer and has the formula: MlsGlsAlsGdsAdsTdsTdsCdsCdsCdsGdsCdsCdsGlsGlsTl; which is a modified oligonucleotide represented by wherein each nucleobase is represented by the following symbols: A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine, is shown according to; each sugar moiety is represented by the following symbols: l = LNA, d = 2'-deoxyribose, is shown according to; each internucleoside linkage is the following symbol: s = phosphorothioate, is a modified oligonucleotide shown according to.

[0249] Certain embodiments provide a method of reducing the level of DUX4 mRNA and / or DUX4 protein (e.g., intramuscular level) in an animal with a DUX4-related disease, the method comprising administering to the animal an effective amount of a modified oligonucleotide against DUX4.

[0250] Certain embodiments provide a method of reducing the level of DUX4 mRNA and / or DUX4 protein (e.g., intramuscular level) in an animal with FSHD, the method comprising administering to the animal an effective amount of a modified oligonucleotide against DUX4. Also certain embodiments provide a method of reducing muscle impairment and / or improving motor function in an animal with FSHD, the method comprising administering to the animal an effective amount of a modified oligonucleotide against DUX4.

[0251] Certain embodiments provide a method of treating FSHD, i.e., a method of treating, preventing, improving, or alleviating, the method comprising administering to the animal an effective amount of a modified oligonucleotide against DUX4.

[0252] Certain embodiments provide a method of preventing, improving, or alleviating various symptoms of FSHD (e.g., facial muscle weakness, ptosis, inability to whistle, decreased facial expression, depressed or angry facial expressions, difficulty pronouncing words, scapular muscle weakness (deformations such as winged scapula, shoulder patting, etc.), lower limb weakness, hearing loss, and heart disease), the method comprising administering to the animal an effective amount of a modified oligonucleotide against DUX4.

[0253] Certain embodiments provide a method of reducing the level of mRNA and / or protein (e.g., intracellular level in blood B cells) resulting from the fusion of the DUX4 gene with the IGH gene in an animal such as B-cell acute lymphoblastic leukemia, the method comprising administering to the animal an effective amount of a modified oligonucleotide against DUX4. Also, certain embodiments provide a method of treating, i.e., treating, preventing, ameliorating, or reducing, B-cell acute lymphoblastic leukemia and the like, the method comprising administering to the animal an effective amount of a modified oligonucleotide against DUX4.

[0254] Certain embodiments provide a method of reducing the level of mRNA and / or protein (e.g., intratumoral level) resulting from the fusion of the DUX4 gene with the CIC gene in an animal such as dedifferentiated round cell sarcoma, the method comprising administering to the animal an effective amount of a modified oligonucleotide against DUX4. Also, certain embodiments provide a method of treating, i.e., treating, preventing, ameliorating, or reducing, dedifferentiated round cell sarcoma and the like, the method comprising administering to the animal an effective amount of a modified oligonucleotide against DUX4.

[0255] Certain embodiments provide a method of reducing the level of mRNA and / or protein (e.g., intratumoral level) resulting from the fusion of the DUX4 gene with the EWSR1 gene in an animal such as embryonal rhabdomyosarcoma, the method comprising administering to the animal a modified oligonucleotide against DUX4. Also, certain embodiments provide a method of treating, i.e., treating, preventing, ameliorating, or reducing, embryonal rhabdomyosarcoma and the like, the method comprising administering to the animal an effective amount of a modified oligonucleotide against DUX4.

[0256] Certain embodiments provide a method for treating, preventing, ameliorating, or reducing DUX4-related diseases with fewer side effects, the method comprising administering a modified oligonucleotide to an animal. In certain embodiments, side effects include injection site reactions, abnormal liver function tests, abnormal kidney function, hepatotoxicity (histopathological abnormal findings: hepatocyte degeneration and necrosis, hepatocyte hypertrophy, etc.), nephrotoxicity (histopathological abnormal findings, 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 bile acids) in the blood may indicate hepatotoxicity or abnormal liver function. For example, an increase in bilirubin may indicate hepatotoxicity or abnormal liver function. Also, an increase in urinary protein, creatinine, or UN in the blood may indicate nephrotoxicity or abnormal kidney function.

[0257] Certain embodiments provide the use of any compound described herein or a pharmaceutical composition containing the compound in the manufacture of a medicament for use in any of the treatment methods described herein. For example, certain embodiments treat FSHD, i.e., cure, Provided is the use of a compound described herein or a pharmaceutical composition containing the compound in the manufacture of a medicament for preventing, delaying or improving. Certain embodiments provide the use of a compound described herein or a pharmaceutical composition containing the compound in the manufacture of a medicament for inhibiting the expression of DUX4 and treating, i.e., curing, preventing, delaying or improving DUX4-related diseases and / or their symptoms. Certain embodiments provide the use of a compound described herein or a pharmaceutical composition containing the compound in the manufacture of a medicament for reducing the expression of DUX4 in an animal. Certain embodiments provide the use of a compound described herein or a pharmaceutical composition containing the compound in the manufacture of a medicament for reducing the level of DUX4-FL mRNA (e.g., intramuscular level) preferentially in an animal and alleviating myotonia. Certain embodiments provide the use of a compound described herein or a pharmaceutical composition containing the compound in the manufacture of a medicament for treating an animal having FSHD. Certain embodiments provide the use of a compound described herein or a pharmaceutical composition containing the compound in the manufacture of a medicament for treating one or more symptoms and outcomes associated with the development of FSHD, including myotonia, myotonia, facial muscle weakness, ptosis, inability to whistle, decreased facial expression, depressive or angry facial expressions, difficulty pronouncing words, scapular muscle weakness (deformations such as winged scapula, shoulder stroking, etc.), lower limb weakness, hearing loss, and heart disease. Certain embodiments provide the use of a compound described herein or a pharmaceutical composition containing the compound in the manufacture of a medicament for preventing the expression of DUX4 protein by leading to the transcription of the DUX4 gene, the translation of DUX4 mRNA, and the cleavage of DUX4 mRNA.

[0258] Certain embodiments provide a kit for treating FSHD as described herein, i.e., curing, preventing or improving, comprising a) a compound described herein, and optionally b) a further agent or therapy described herein. The kit may further comprise instructions or a label for using the kit for the purpose of treating FSHD, i.e., curing, preventing or improving.

[0259] Certain embodiments provide for the use of any of the compounds described herein or a pharmaceutical composition containing the compound in the manufacture of a medicament for use in any of the treatment methods described herein. For example, certain embodiments provide for the use of the compounds described herein or a pharmaceutical composition containing the compound in the manufacture of a medicament for treating, i.e., treating, ameliorating, or preventing, FSHD. Certain embodiments provide for the use of the compounds described herein or a pharmaceutical composition containing the compound in the manufacture of a medicament for inhibiting the expression of DUX4 and for treating, i.e., treating, preventing, delaying or ameliorating, DUX4-related diseases and / or their symptoms. Certain embodiments provide for the use of the compounds described herein or a pharmaceutical composition containing the compound in the manufacture of a medicament for reducing the expression of DUX4 in an animal. Certain embodiments provide for the use of the compounds described herein or a pharmaceutical composition containing the compound in the manufacture of a medicament for preferentially reducing the level of DUX4-FL mRNA (e.g., intramuscular level) in an animal and for reducing myotonia. Certain embodiments provide for the use of the compounds described herein or a pharmaceutical composition containing the compound in the manufacture of a medicament for treating an animal having FSHD. Certain embodiments provide for the compounds described herein or a pharmaceutical composition containing the compound for treating one or more symptoms and outcomes associated with the development of FSHD, including myotonia, myotonia, facial muscle weakness, ptosis, inability to whistle, decreased facial expression, depressive or angry facial expressions, difficulty pronouncing words, scapular muscle weakness (deformations such as winged scapula, shrugging shoulders, etc.), lower limb weakness, hearing loss, and heart disease. Certain embodiments provide a modified oligonucleotide having a nucleobase sequence consisting of the nucleobase sequences of SEQ ID NOs: 2-4, 7-64, 69-97 or 102-112 in the Sequence Listing, or a compound of Compound Nos. 1-112, 114-132 or 137-246.

[0260] In certain embodiments, when described in the 5' to 3' direction, the modified oligonucleotide has a nucleobase sequence that includes the reverse complementary strand of the target segment of the target nucleic acid that it targets. In certain such embodiments, when described in the 5' to 3' direction, the modified oligonucleotide has a nucleobase sequence that includes the reverse complementary strand of the target segment of the target nucleic acid that it targets.

[0261] In certain embodiments, the modified oligonucleotides described herein that target DUX4 are 12 to 30 nucleotides in length. In other words, in some embodiments, the modified oligonucleotides are linked nucleobases of 12 to 30 residues. In other embodiments, the modified oligonucleotides are 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 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, 14 to 15 residues of linked nucleobases. In certain such embodiments, the modified oligonucleotides comprise linked nucleobases of a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 residues, or a range defined by any two of the above values. In certain embodiments, modified oligonucleotides of any of these lengths comprise 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 nucleobases of the nucleobase sequence of any one of the exemplary modified oligonucleotides described herein (e.g., SEQ ID NOs: 2-4, 7-64, 69-97, or 102-112 in the Sequence Listing), including at least 8 consecutive nucleobases of the nucleobase sequence set forth in any one of them.

[0262] The length of an antisense compound such as an antisense oligonucleotide can be increased or decreased and / or mismatched bases can be introduced without abolishing its activity. For example, according to Woolf et al. (Proc. Natl. Acad. Sci. USA 89 :7305-7309, 1992), a series of antisense oligonucleotides with lengths of 13 to 25 nucleobases were tested in an oocyte injection model for their ability to induce cleavage of the target RNA, and it was reported that a 25-nucleobase antisense oligonucleotide having 8 or 11 mismatched bases near the ends of the antisense oligonucleotide could direct specific cleavage of the target mRNA, although to a lesser extent than a mismatch-free antisense oligonucleotide. Similarly, it has been reported that target-specific cleavage was achieved using 13-nucleobase antisense oligonucleotides, including those having 1 or 3 mismatches.

[0263] According to Gautschi et al. (J. Natl. Cancer Inst. 93:46 3-471, March 2001), an oligonucleotide having 100% complementarity to bcl-2 mRNA and 3 mismatches to bcl-xL mRNA was reported to show the ability to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide was reported to show potent antitumor activity in vivo.

[0264] Target nucleic acid, target region, and nucleotide sequence Examples of the nucleotide sequence encoding DUX4 include, but are not limited to, the following sequences. · The sequence described in GenBank accession number NM_001293798.2 (incorporated herein as SEQ ID NO: 1 in the Sequence Listing). The splicing variant of SEQ ID NO: 1 in the Sequence Listing is also referred to as DUX4-FL1 or the mature mRNA of DUX4. ·The sequence described in GenBank accession number NM_001306068.2 (incorporated herein as SEQ ID NO: 5 in the Sequence Listing). The splicing variant of SEQ ID NO: 5 in the Sequence Listing is also referred to as DUX4-FL2. ·The sequence described in GenBank accession number NM_001363820.1 (incorporated herein as SEQ ID NO: 6 in the Sequence Listing). The splicing variant of SEQ ID NO: 6 in the Sequence Listing is also referred to as DUX4-s. ·SNPs of each of the above splicing variants. The sequences described in each SEQ ID NO in the Sequence Listing of the examples included in this specification are independent of any modification to the sugar moiety, internucleoside linkage, or nucleobase. Accordingly, the modified oligonucleotides defined by the SEQ ID NOs in the Sequence Listing can independently include one or more modifications to the sugar moiety, internucleoside linkage, or nucleobase. The modified oligonucleotides described by compound numbers indicate a combination of nucleobase sequences and motifs.

[0265] In certain embodiments, the target region is a structurally defined region of the target nucleic acid. For example, the target region can include one or more of 3’UTR, 5’UTR, exon, intron, exon / intron junction, coding region, translation initiation region, translation termination region, or other defined nucleic acid regions. Structurally defined regions for DUX4 can be obtained by accession number from sequence databases such as NCBI, and such information is incorporated herein by reference. In certain embodiments, the target region can include the sequence from the 5’ target site of one target segment within the target region to the 3’ target site of another target segment within the target region.

[0266] Targeting involves determining at least one target segment to which the modified oligonucleotide hybridizes and as a result a desired effect occurs. In certain embodiments, the desired effect is a reduction in the mRNA target nucleic acid level. In certain embodiments, the desired effect is a reduction in the level of the protein encoded by the target nucleic acid, or a change in the phenotype associated with the target nucleic acid.

[0267] The target region can include one or more target segments. Multiple target segments within the target region may overlap. Alternatively, they may not overlap. In certain embodiments, the target segments within the target region are separated by about 300 nucleotides or less. In certain embodiments, the target segments within the target region are 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides of the target nucleic acid, or approximately these numbers of nucleotides, or less than these numbers of nucleotides, or within a range defined by any two of the foregoing values, and are separated by a number of nucleotides. In certain embodiments, the target segments within the target region are separated by 5 or less or about 5 or less nucleotides of the target nucleic acid. In certain embodiments, the target segments are contiguous. A target region defined by a range having a starting nucleic acid that is either a 5' target site or a 3' target site listed herein is contemplated.

[0268] Suitable target segments can be found within the 5'UTR, coding region, 3'UTR, intron, exon, or exon / intron junction. Target segments that include a start codon or a stop codon are also suitable target segments. Suitable target segments can specifically exclude certain structurally defined regions such as start codons or stop codons.

[0269] Determination of suitable target segments can include comparison of the sequence of the target nucleic acid with other sequences across the genome. For example, the BLAST algorithm can be used to identify similar regions between different nucleic acids. This comparison can prevent the selection of modified oligonucleotide sequences that can hybridize in a non-specific manner to sequences other than the selected target nucleic acid (i.e., non-target sequences or off-target sequences).

[0270] ​There can be variations in the activity of the modified oligonucleotide within the active target region (e.g., as determined by the percent reduction in target nucleic acid level). In certain embodiments, a reduction in the level of DUX4 mRNA serves as an indicator of inhibition of DUX4 protein expression. A reduction in the level of DUX4 protein also serves as an indicator of inhibition of target mRNA expression. Furthermore, changes in phenotype, such as reducing myotonia and reducing muscle impairment, can serve as indicators of inhibition of DUX4 mRNA and / or protein expression.

[0271] Hybridization In some embodiments, hybridization occurs between the modified oligonucleotide disclosed herein and the DUX4 nucleic acid. The most common mechanism of hybridization involves hydrogen bonding between complementary nucleobases of nucleic acid molecules (e.g., Watson-Crick, Hoogsteen or reverse Hoogsteen type hydrogen bonds). The strength of hybridization can be represented by the melting temperature Tm. Tm is the temperature at which 50% of the hybridized double-stranded nucleic acid dissociates into single-stranded nucleic acid, and it varies depending on the salt conditions of the solution, the length and base sequence of the nucleic acid, etc. The higher the Tm value, the stronger the hybridization.

[0272] Hybridization occurs under various conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules that will hybridize.

[0273] Methods for determining whether a sequence can specifically hybridize to a target nucleic acid are well known in the art (Sambrooke and Russell, Molecular Cl oning:A Laboratory Manual, 3rd Ed., 2001). A In certain embodiments, the modified oligonucleotide provided herein is capable of specifically hybridizing to the DUX4 nucleic acid.

[0274] Complementarity If a sufficient number of nucleobases of the modified oligonucleotide can hydrogen bond with the corresponding nucleobases of the target nucleic acid such that the desired effect occurs (e.g., antisense inhibition of a target nucleic acid such as a DUX4 nucleic acid), the modified oligonucleotide and the target nucleic acid are complementary to each other.

[0275] The modified oligonucleotide can hybridize across one or more segments of the DUX4 nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., loop structures, mismatches or hairpin structures).

[0276] In certain embodiments, the modified oligonucleotides provided herein, or particular portions thereof, are 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to a DUX4 nucleic acid, target region, target segment or particular 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, target region, target segment or particular portions thereof, and includes 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 nucleobases of the nucleobase sequence of any one of the exemplary modified oligonucleotides described herein (e.g., at least 8 consecutive nucleobases of the nucleobase sequences set forth in any one of SEQ ID NOs: 2, 3, 4, 7-64, 69-97 or 102-112 of the Sequence Listing). The percent complementarity of the modified oligonucleotide to the target nucleic acid can be determined using conventional methods and is measured over the entire length of the modified oligonucleotide.

[0277] For example, a modified oligonucleotide in which 18 out of 20 nucleobases are complementary to the target region and thus are likely to hybridize specifically corresponds to 90 percent complementarity. In this example, the remaining non-complementary nucleobases may be clustered, may be interspersed with complementary nucleobases, and need not be contiguous with each other or with the complementary nucleobases. The percent complementarity of the modified oligonucleotide to 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, 403 410; Zhang and Madden, Genome Res., 1997, 7, 6 49656). Percent homology, sequence identity or complementarity can be determined, for example, using the gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genet ics Computer Group, University Research Park, Madison Wis.) with the default settings using the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489). The positions of non-complementary nucleobases may be at the 5' end or the 3' end of the modified oligonucleotide. Alternatively, the non-complementary nucleobase(s) may be at an internal position within the modified oligonucleotide. When two or more non-complementary nucleobases are present, these may be contiguous (i.e., linked) or non-contiguous. In one embodiment, the non-complementary nucleobases are located in the wing segments of the gapmer-modified oligonucleotide.

[0278]

[0279] ​In certain embodiments, modified oligonucleotides that are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length, or modified oligonucleotides up to these nucleobase lengths, contain three or fewer, two or fewer, or one or fewer non-complementary nucleobase(s) to a target nucleic acid, such as a DUX4 nucleic acid or a particular portion thereof.

[0280] The modified oligonucleotides provided herein also include those that are complementary to a portion of a target nucleic acid. As used herein, "portion" refers to a defined number of contiguous (i.e., linked) nucleobases within a region or segment of a target nucleic acid. "Portion" can also refer to a defined number of contiguous nucleobases of a modified oligonucleotide. In certain embodiments, the modified oligonucleotide is complementary to at least an 8-nucleobase portion of a target segment. In certain embodiments, the modified oligonucleotide is complementary to at least a 10-nucleobase portion of a target segment. In certain embodiments, the modified oligonucleotide is complementary to at least a 15-nucleobase portion of a target segment. Modified oligonucleotides that are 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 nucleobase portions of a target segment, or to ranges defined by any two of these values, are also contemplated. are also contemplated.

[0281] Identity The modified oligonucleotides provided herein can also have a defined percentage identity to a specific nucleotide sequence, a compound represented by an accession number in the Sequence Listing or a specific compound number, or a portion thereof. As used herein, a modified oligonucleotide is identical to a sequence disclosed herein if it has the same nucleic acid base pairing ability. For example, an RNA containing uracil instead of thymidine in a disclosed DNA sequence is considered identical to that DNA sequence because both uracil and thymidine pair with adenine. Also contemplated are shortened and extended versions of the modified oligonucleotides described herein and compounds having bases that are not identical to those of the modified oligonucleotides provided herein. The non-identical bases may be adjacent to each other or may be dispersed throughout the modified oligonucleotide. The percentage identity of a modified oligonucleotide is calculated according to the number of bases having identical base pairing to the sequence being compared.

[0282] In certain embodiments, a 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 or accession numbers in the Sequence Listing or portions thereof disclosed herein.

[0283] modification A nucleoside is a combination of a base and a sugar. The nucleobase (also known as the base) portion of a nucleoside is usually a heterocyclic base portion. A nucleotide is a nucleoside that further contains a phosphate group covalently bonded to the sugar portion of the nucleoside. For nucleosides containing pentofuranosyl sugar, the phosphate group can be linked to the 2', 3' or 5'-hydroxyl portion of the sugar. An oligonucleotide is formed through the covalent bonding of adjacent nucleosides to each other to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate group is generally said to form the internucleoside bond of the oligonucleotide.

[0284] Modifications in modified oligonucleotides include substitutions or changes to internucleoside bonds, sugar moieties or nucleobases. Modified oligonucleotides are often preferred over their natural counterparts for reasons such as enhanced cellular uptake, enhanced affinity for nucleic acid targets, increased stability in the presence of nucleases or increased inhibitory activity.

[0285] Modified internucleoside bonds The naturally occurring internucleoside bonds in RNA and DNA are 3'-5' phosphodiester bonds. Modified oligonucleotides having one or more modified, i.e., non-naturally occurring, internucleoside bonds are often selected over modified oligonucleotides having naturally occurring internucleoside bonds for reasons such as enhanced cellular uptake, enhanced affinity for target nucleic acids and increased stability in the presence of nucleases.

[0286] Oligonucleotides having modified internucleoside bonds include internucleoside bonds that retain a phosphorus atom and internucleoside bonds that do not have a phosphorus atom. Representative phosphorus-containing internucleoside bonds include, but are not limited to, one or more of phosphodiester, phosphotriester, methylphosphonate, phosphoramidate and phosphorothioate. Methods for preparing phosphorus-containing and non-phosphorus-containing bonds are well known.

[0287] In certain embodiments, the modified oligonucleotides targeting DUX4 nucleic acid include one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkage is a phosphorothioate linkage. In certain embodiments, each internucleoside linkage of the modified oligonucleotide is a phosphorothioate internucleoside linkage.

[0288] Modified sugar moiety The modified oligonucleotides of the present invention are preferably those in which at least one nucleoside constituting the oligonucleotide contains a modified sugar. The modified sugar in the present invention refers to a sugar moiety that has been modified. Modified oligonucleotides containing one or more of the modified sugars have advantageous features such as enhanced nuclease stability and increased binding affinity. At least one of the modified sugars preferably has a bicyclic sugar or a substituted sugar moiety.

[0289] Examples of nucleosides having a modified sugar 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. Substituents at the 2'-position include allyl, amino, azido, thio, O-allyl, O-C1-C 10 alkyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-O-N(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 is independently H or substituted or unsubstituted C1-C 10 alkyl).

[0290] Examples of nucleosides having bicyclic sugars include, but are not limited to, nucleosides that include a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the oligonucleotides provided herein include nucleosides having one or more bicyclic sugars where the bridge includes one of the following formulas: 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' and 4'-CH(CH2OCH3)-O-2' (and their analogs; see U.S. Patent No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and their analogs; see WO2009 / 006478); 4'-CH2-N(OCH3)-2' (and their analogs; see WO2008 / 150729); 4'-CH2-O-N(CH3)-2' (and their analogs; see US2004-0171570); 4'-CH2-N(R)-O-2' (wherein R is H, C1-C12 alkyl or a protecting group) (see U.S. Patent No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (and their analogs; see Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and their analogs; see WO2008 / 154401).

[0291] Additional nucleosides having bicyclic sugars have been reported in the published literature (e.g., Srivastava et al., J. Am. Chem. Soc., 2007, 129(2 6) 8362-8379; Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372; Elayadi et al., C urr. 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. U.S.A., 2000, 97, 5633-5638; Singh et al., Che m. Commun., 1998, 4, 455-456; Koshkin et al., T etrahedron, 1998, 54, 3607-3630; Kumar et al. , Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al.,; U.S. Patent US7,399,845; 6,770,748; 6,525,191; 6,268,490; U.S. US2008-0039618; US2007-0287831; US2004-0171570; US2009-0012281; WO2010 / 036698; WO 2009 / 067647; WO2009 / 067647; WO2007 / 134181; WO2005 / 021570; WO2004 / 106356; WO94 / 14226; WO 2009 / 006478 No.; WO2008 / 154401; and WO2008 / 150729). Each of the nucleosides having the aforementioned bicyclic sugar can be prepared having one or more stereochemical sugar configurations including, for example, α-L-ribofuranose and β-D-ribofuranose.

[0292] The GuNA of the nucleoside having a bicyclic sugar has been reported as an artificial nucleoside having a guanidine bridge (see WO2014 / 046212, WO2017 / 047816). The ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Trz] and ALNA[Oxz] of the bicyclic nucleoside have been reported as cross-linked artificial nucleic acid amino LNA (ALNA) (see Japanese Patent Application No. 2018-212424).

[0293] In certain embodiments, the nucleoside having a bicyclic sugar contains a bridge between the 4' and 2' carbon atoms of the pentofuranosyl sugar moiety, including, but not limited to, -[C(R a )(R b )] n -, -C(R a )=C(R b )-, -C(R a )=N-, -C(=NR a )-, -C(=O)-, -C(=S)-, -N(R a )-, -O-, -Si(R a )2- and -S(=O) x -; where X is 0, 1 or 2; n is 1, 2, 3 or 4; each R a and R b is independently H, a protecting group, hydroxyl, C1-C 12 alkyl, substituted C1-C 12 alkyl, C2-C 12 alkenyl, substituted C2-C 12 alkenyl, C2-C 12 alkynyl, substituted C2-C 12 alkynyl, an aromatic ring group, a substituted aromatic ring group, a heterocyclic group, a substituted heterocyclic group, a C5-C7 cycloaliphatic group, a substituted C5-C7 cycloaliphatic group, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1) or sulfoxyl (S(=O)-J1), each 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 12 alkynyl, an aromatic ring group, a substituted aromatic ring group, acyl (C(=O)-H), substituted acyl, a heterocyclic group, a substituted heterocyclic group, C1-C 12 aminoalkyl, substituted C1-C 12 aminoalkyl or a protecting group.

[0294] In certain embodiments, the bridge of the bicyclic sugar moiety is -[C(R a )(R b )] n -, -[C(R a )(R b )] n -, -O-, -C(R a R b )-N(R)-O- or -C(R a R b )-O-N(R)-. In certain embodiments, the bridge is 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2' (the nucleoside having the bicyclic sugar in this case is also referred to as LNA), 4'-(CH2)2-O-2', 4'-CH2-O-N(R)-2' and 4'-CH2-N(R)-O-2', wherein each R is independently H, a protecting group or C1-C 12 alkyl.

[0295] In certain embodiments, the bridge 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-tri azol-3-yl (ALNA[Trz]), -CO-NH-CH(CH3)2 (AL NA[ipU]), 5-methyl-1,2,4-oxadiazol-3-yl (ALNA Oxz]) (Japanese Patent Application No. 2018-212424).

[0296] In certain embodiments, the nucleoside having a bicyclic sugar is further defined by the isomeric configuration. For example, the nucleoside containing a 4'-(CH2)-O-2' bridge may exist in the α-L configuration or in the β-D configuration.

[0297] In certain embodiments, nucleosides having a bicyclic sugar include those having a 4'-2' bridge, such bridges including, but not limited to, α-L-4'-(CH2)-O-2', β-D-4'-CH2-O-2', 4'-(CH2)2-O-2', 4'-CH2-O-N(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, C1-C 12 alkyl, or a urea or guanidine optionally substituted with C1-C 12 alkyl).

[0298] In certain embodiments, nucleosides having a bicyclic sugar have the following formula:

[0299] [Chemical formula]

[0300] wherein Bx is a heterocyclic base moiety; T a and T b are each independently a hydrogen atom, a protecting group for a hydroxyl group, an optionally substituted phosphate group, a phosphorus moiety or a covalent bond to a support, etc.; Z a 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.

[0301] In certain embodiments, each of the substituents is independently halogen, oxo, hydroxyl, OJ c , NJ c J d , SJ c , N3, OC(=X)J c and NJ e C(=X)NJ c J d (wherein each J c, J d and J e are each independently H, C1-C6 alkyl or substituted C1-C6 alkyl, and X is O or N-J c and is monosubstituted or polysubstituted with a substituent independently selected from ().

[0302] In certain embodiments, the nucleoside having a bicyclic sugar has the following formula:

[0303] [Chemical formula]

[0304] wherein Bx is a heterocyclic base moiety; T a and T b are each independently a hydrogen atom, a protecting group for a hydroxyl group, an optionally substituted phosphate group, a phosphorus moiety or a covalent bond to a support, etc.; Z b is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl or substituted acyl (C(=O)-).

[0305] In certain embodiments, the nucleoside having a bicyclic sugar has the following formula:

[0306] [Chemical formula]

[0307] wherein Bx is a heterocyclic base moiety; T a and T b are each independently a hydrogen atom, a protecting group for a hydroxyl group, an optionally substituted phosphate group, a phosphorus moiety or a covalent bond to a support, etc.; R dis C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; each q a 、q b 、q c and q d is independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, C1-C6 alkoxyl, substituted C1-C6 alkoxyl, acyl, substituted acyl, C1-C6 aminoalkyl or substituted C1-C6 aminoalkyl.

[0308] In certain embodiments, the nucleoside having a bicyclic sugar has the following formula:

[0309]

Chemical formula

[0310] wherein Bx is a heterocyclic base moiety; T a and T b are each independently a hydrogen atom, a protecting group for a hydroxyl group, an optionally substituted phosphate group, a phosphorus moiety or a covalent bond to a support, etc.; q a 、q b 、q e and q f are each independently hydrogen, halogen, C1-C 12 alkyl, substituted C1-C 12 alkyl, C2-C 12 alkenyl, substituted C2-C 12 alkenyl, C2-C 12 alkynyl, substituted C2-C 12 alkynyl, C1-C 12 alkoxyl, substituted C1-C 12 alkoxyl, OJ j 、SJ j 、SOJ j 、SO2J j 、NJ jJ k , N3, CN, C(=O)OJj, C(=O)NJ j J k , C(=O)J j , O-C(=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 ; or, q e and q f both are =C(q g )(q h ) q g and q h are each independently H, halogen, C1-C 12 alkyl or substituted C1-C 12 alkyl

[0311] The synthesis and preparation of adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil bicyclic nucleosides (also referred to as LNA) having a 4’-CH2-O-2’ bridge are described together with their oligomerization and nucleic acid recognition properties (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). The synthesis of nucleosides having a bicyclic sugar is also described in WO98 / 39352 and WO99 / 14226.

[0312] Various bicyclic nucleoside analogs having 4’-2’ bridging groups such as 4’-CH2-O-2’ (in this case the bicyclic nucleoside is also referred to as LNA) and 4’-CH2-S-2’ have also been prepared (Kumar et al., Bioorg. Med. Chem. Le tt., 1998, 8, 2219 - 2222). The preparation of oligodeoxyribonucleotide duplexes containing bicyclic nucleosides for use as substrates of nucleic acid polymerases is also described (Wengelet al., WO99 / 14226). Furthermore, 2'-ami no - BNA (in this case, the bicyclic nucleoside is also referred to as ALNA), that is, the synthesis of stereochemically restricted high - affinity oligonucleotide analogs has been described in the art (Singhet al., J. Org. Chem., 1998, 63, 10035 - 1 0039). Furthermore, 2'-amino - and 2'-methylamino - BNA have been prepared, and the thermal stability of duplexes with complementary RNA and DNA strands has been previously reported.

[0313] In certain embodiments, the nucleoside having a bicyclic sugar has the following formula:

[0314]

Chemical formula

[0315] Wherein, Bx is a heterocyclic base moiety; T a and T b are each independently a hydrogen atom, a protecting group for a hydroxyl group, an optionally substituted phosphate group, a phosphorus moiety or a covalent bond to a support, etc.; each q i 、q j 、q k and q l are independently H, halogen, C1 - C 12 alkyl, substituted C1 - C 12 alkyl, C2 - C 12 alkenyl, substituted C2 - C 12 alkenyl, C2 - C 12 alkynyl, substituted C2 - C 12 alkynyl, C1 - C 12 alkoxyl, substituted C1 - C 12 alkoxyl, OJ j 、SJ j 、SOJj , SO2J j , NJ j J k , N3, CN, C(=O)OJ j , C(=O)NJ j J k , C(=O)J j , O-C(=O)NJ j J k , N(H)C(=NH)NJ j J k , N(H)C(=O)NJ j J k or N(H)C(=S)NJ j J k ; and q i and q j or q l and q k are both =C(q g )(q h ), where q g and q h are each independently H, halogen, C1-C 12 alkyl or substituted C1-C 12 alkyl.

[0316] One carbocyclic bicyclic nucleoside having 4'-(CH2)3-2' bridging and alkenyl analog bridging 4'-CH=CH-CH2-2' has been described (Frier et al., Nucleic Acids Research, 1997, 25(22), 442 9-4443 and Albaek et al., J. Org. Chem., 2006, 71 , 7731-7740). The synthesis and preparation of the carbocyclic bicyclic nucleoside, along with its oligomerization and biochemical studies, have also been described (Srivastava et al., J . Am. Chem. Soc. 2007, 129(26), 8362-8379). .

[0317] In certain embodiments, the nucleosides having a bicyclic sugar include, but are not limited to, those as shown below, (A) α-L-methyleneoxy (4’-CH2-O-2’) BNA, (B) β-D-methyleneoxy (4’-CH2-O-2’) BNA, (C) ethyleneoxy (4’-(CH2)2-O-2’) BNA, (D) aminooxy (4’-CH2-O-N(R)-2’) BNA, (E) oxyamino (4’-CH2-N(R)-O-2’) BNA, (F) methyl (methyleneoxy) (4’-CH(CH3)-O-2’) BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4’-CH2-S-2’) BNA, (H) methylene-amino (4’-CH2-N(R)-2’) BNA, (I) methyl carbocyclic (4’-CH2-CH(CH3)-2’) BNA, (J) propylene carbocyclic (4’-(CH2)3-2’) BNA, and (K) vinyl BNA.

[0318] [Chemical Structure]

[0319] In the formula, Bx is a base moiety, and R is independently a protecting group, C1-C6 alkyl, or C1-C6 alkoxy.

[0320] In certain embodiments (LNA), nucleosides having a bicyclic sugar have the following general formula:

[0321] [Chemical Structure]

[0322] [In the formula, B is a nucleobase; X and Y can each independently be a nucleoside represented by a hydrogen atom, a protecting group for a hydroxyl group, an optionally substituted phosphate group, a phosphorus moiety, or a covalent bond to a support (see WO98 / 39352). Typical specific examples are the following formula:

[0323]

Chemical formula

[0324] and nucleotides represented by it can be mentioned.

[0325] In certain embodiments (GuNA), nucleosides containing a bicyclic ring are represented by the following general formula:

[0326]

Chemical formula

[0327] [wherein B is a nucleobase, R3, R4, R5, R6 are each independently a hydrogen atom or a C 1-6 alkyl group optionally substituted with one or more substituents, R7, R8 are each independently a hydrogen atom, a protecting group for a hydroxyl group, an optionally substituted phosphate group, a phosphorus moiety, or a covalent bond to a support, etc., and R9, R 10 , R 11 are each independently a hydrogen atom, a C 1-6 alkyl group optionally substituted with one or more substituents, or a protecting group for an amino group.] and are nucleosides represented by it (see, for example, International Publication No. 2014 / 046212, International Publication No. 2017 / 047816).

[0328] In certain embodiments (ALNA[mU]), nucleosides containing a bicyclic sugar are represented by the following general formula (I):

[0329]

Chemical formula

[0330] [wherein, B is a nucleobase; R1, R2, R3 and R4 are each independently a hydrogen atom or a C 1-6 alkyl group which may be substituted with one or more substituents; R5 and R6 are each independently a hydrogen atom, a protecting group for a hydroxyl group, an optionally substituted phosphate group, a phosphorus moiety or a covalent bond to a support, etc.; m is 1 or 2; X is a group represented by the following formula (II-1):

[0331]

Chemical formula

[0332] ; The symbols described in formula (II-1):

[0333]

Chemical formula

[0334] indicate the bonding point with the 2'-amino group; One of R7 and R8 is a hydrogen atom and the other is a methyl group which may be substituted with one or more substituents.] It is a nucleoside represented by (see, for example, 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.

[0335] In certain embodiments (ALNA[ipU]), the nucleoside containing a bicyclic sugar is a nucleoside having the general formula (I) defined in the above ALNA[mU], wherein X is a group represented by the following formula (II-1):

[0336]

Chemical formula

[0337] is a group represented by; 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 (see, for example, 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.

[0338] In certain embodiments (ALNA[Trz]), the bicyclic-containing nucleoside is a nucleoside having the above general formula (I), wherein X is the following formula (II-2):

[0339] [Chemical formula]

[0340] is a group represented by; A is a triazolyl 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 nucleoside in which A is a triazolyl group which may have one or more methyl groups, and more specifically, 1,5-di methyl-1,2,4-triazol-3-yl group.

[0341] In certain embodiments (ALNA[Oxz]), it is a nucleoside having the general formula (I) defined in the above ALNA[mU], wherein X is the following formula (II-2):

[0342] [Chemical formula]

[0343] is a group represented by; A is an oxadiazolyl group which may be substituted with one or more substituents (see, for example, Japanese Patent Application No. 2018-212424). A typical specific example is a nucleoside or nucleotide in which A is an oxadiazolyl group which may have one or more methyl groups, and more specifically, a 5-methyl-1,2,4-oxadiazol-3-yl group.

[0344] In certain embodiments (ALNA[Ms]), the bicyclic-containing nucleoside is a nucleoside having the above general formula (I), wherein X is the following general formula (II-3):

[0345]

Chemical formula

[0346] a group represented by; M is a sulfonyl group substituted with a methyl 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[Ms] is a nucleoside in which M is a sulfonyl group substituted with an unsubstituted methyl group.

[0347] In certain embodiments, the nucleoside is modified by replacement of the ribosyl ring with a sugar surrogate. Such modifications include, but are not limited to, replacement of the ribosyl ring with a surrogate ring system (sometimes referred to as a DNA analog), such as a morpholino ring, a cyclohexenyl ring, a cyclohexyl ring or a tetrahydropyranyl ring, such as replacement with one having one of the following formulas:

[0348]

Chemical formula

[0349] In certain embodiments, a sugar surrogate having the following formula is selected:

[0350]

Chemical formula

[0351] wherein Bx is a complex cyclic base moiety; T3 and T4 are each independently a nucleoside internucleoside linking group that links a tetrahydropyran nucleoside analog to an oligomeric compound, or one of T3 and T4 is a nucleoside internucleoside linking group that links a tetrahydropyran nucleoside analog to an oligomeric compound or oligonucleotide, and the other of T3 and T4 is H, a hydroxyl protecting group, a linking conjugate group or a 5' or 3'-terminal group; q1, q2, q3, q4, q5, q6 and q7 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; One of R1 and R2 is hydrogen and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN (wherein X is O, S or NJ1, and each J1, J2 and J3 is independently H or C1-C6 alkyl).

[0352] In certain embodiments, q1, q2, q3, q4, q5, q6 and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is other than H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is methyl. In certain embodiments, one of R1 and R2 A THP nucleoside in which R1 is F is provided. In certain embodiments, R1 is fluoro and R2 is H; R1 is methoxy and R2 is H; and R1 is methoxyethoxy and R2 is H.

[0353] Such sugar substitutes include, but are not limited to, those referred to in the art as hexitol nucleic acid (HNA), altolitol nucleic acid (ANA), and mannitol nucleic acid (MNA) (see Leumann, C.J., Bioorg. & Med. Chem . , 2002, 10, 841 - 854).

[0354] In certain embodiments, the sugar substitute includes a ring having more than five atoms and more than one heteroatom. For example, its use in nucleoside and oligomer compounds containing a morpholino sugar moiety has been reported (e.g., see Braaschet al., Biochemistry ., 2002, 41, 4503 - 4510; and U.S. Patents 5,698,685; 5,166,315; 5,185,444; and 5,034,506).

[0355] As used herein, the term "morpholino" means a sugar substitute having the following structure:

[0356]

Chemical formula

[0357] In certain embodiments, for example, morpholino can be modified by adding or changing various substituents from the above - mentioned morpholino structure. Such sugar substitutes are referred to herein as "modified morpholino".

[0358] In certain embodiments, the oligonucleotide includes one or more modified cyclohexenyl nucleosides that are nucleosides having a 6 - membered cyclohexenyl in place of the pentofuranosyl residue of a naturally occurring nucleoside. Modified cyclohexenyl nucleosides include, but are not limited to, those described in the art (e.g., WO2010 / 036696 published on April 10, 2010 regarding covalent bonds, Robeynset al., J. Am. Chem. Soc., 2008, 130(6), 19 79 - 1984; Horvath et al., Tetrahedron Letters , 2007, 48, 3621 - 3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30), 9340 - 9348; Guet al., Nucleosides, Nucleotides & Nucleic Acids, 2005, 24(5 - 7), 993 - 998; Nauwelaerts et al. , Nucleic Acids Research, 2005, 33(8), 2452 - 2 463; Robeyns et al., Acta Crystallographica , Section F: Structural Biology and Crystall ization Communications, 2005, F61(6), 585 - 5 86; Guet et al., Tetrahedron, 2004, 60(9), 2111 - 2123; Guet et al., Oligonucleotides, 2003, 13(6 ), 479 - 489; Wang et al., J. Org. Chem., 2003, 68 , 4499 - 4505; Verbeure et al., Nucleic Acids Research, 2001, 29(24), 4941 - 4947; Wang et al., J. Org. Chem., 2001, 66, 8478 - 82; Wang et al., Nucleosides, Nucleotides & Nucleic Acids, 200 1, 20(4 - 7), 785 - 788; Wang et al., J. Am. Chem., 2000, 122, 8595 - 8602; See WO06 / 047842 and WO01 / 049687; the entire text of each is incorporated herein by reference).

[0359] Certain modified cyclohexenyl nucleosides have the following formula:

[0360] [Chemical formula]

[0361] In the formula: Bx is a heterocyclic base moiety; T3 and T4 are each independently a internucleoside linking group that links a cyclohexenyl nucleoside analog to an oligonucleotide compound, or one of T3 and T4 is a internucleoside linking group that links a tetrahydropyran nucleoside analog to an oligonucleotide compound, and the other of T3 and T4 is H, a hydroxyl protecting group, a linking conjugate group or a 5'- or 3'-terminal group; q1, q2, q3, q4, q5, q6, q7, q8 and q9 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or other sugar substituents.

[0362] Many other bicyclic and tricyclic sugar surrogate ring systems are known in the art that can be used to modify nucleosides for incorporation into oligonucleotides (see, for example, review: Leumann, Christian J., Bioorg. & Med. Chem., 2002, 10, 841-854). Such ring systems can undergo various further substitutions to enhance activity.

[0363] Methods for preparing modified sugars are well known to those skilled in the art. Some representative U.S. patents that teach the preparation of such modified sugars include, but are not limited to, U.S.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5 ,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032 and WO2005 / 121371 are mentioned, each of which is hereby incorporated by reference in its entirety.

[0364] In nucleotides having a modified sugar moiety, the nucleobase moiety (natural, modified or combinations thereof) is maintained during hybridization with a suitable nucleic acid target.

[0365] Modified nucleobase Modification or substitution of a nucleobase (or base) is structurally distinguishable from naturally occurring or synthetic unmodified nucleobases and is further functionally compatible with such unmodified nucleobases. Both natural and modified nucleobases can participate in hydrogen bonding. Such nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological property to the modified oligonucleotide. Modified nucleobases include synthetic and natural nucleobases such as 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitution, are particularly useful for increasing the binding affinity of the modified oligonucleotide to the target nucleic acid. For example, 5-methylcytosine substitution has been shown to increase the duplex stability of nucleic acids by 0.6 - 1.2 °C (Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276 - 278).

[0366] Additional modified nucleobases include 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azauracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine.

[0367] The heterocyclic base moiety can also include those in which the purine or pyrimidine base is replaced by another heterocycle, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of modified oligonucleotides include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines (including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine).

[0368] In certain embodiments, the modified oligonucleotide targeting the DUX4 nucleic acid comprises one or more modified nucleobases. In certain embodiments, the modified oligonucleotide targeting the DUX4 nucleic acid comprises one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is 5-methylcytosine.

[0369] Certain modified oligonucleotide motifs In certain embodiments, modified oligonucleotides targeting DUX4 nucleic acids have chemically modified subunits arranged in a pattern or motif to confer properties such as enhanced inhibitory activity, increased binding affinity for the target nucleic acid, or resistance to degradation by in vivo nucleases to the modified oligonucleotide.

[0370] Chimeric modified oligonucleotides typically include at least one modified region to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and / or increased inhibitory activity. The second region of the chimeric modified oligonucleotide can optionally function as a substrate for the intracellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

[0371] A modified oligonucleotide having a gapmer motif is a chimeric modified oligonucleotide. In a gapmer, an internal region having a plurality of nucleotides that assist RNaseH cleavage is located between an external region having a plurality of nucleotides that are chemically different from the nucleosides of the internal region. In the case of a modified oligonucleotide having a gapmer motif, the gap segment generally serves as a substrate for cleavage by an endonuclease, while the wing segment contains modified nucleosides. In certain embodiments, the gapmer regions are distinguished by the type of sugar moiety that includes different regions. In some embodiments, the types of sugar moieties used to distinguish the gapmer regions can include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2'-modified nucleosides (such 2'-modified nucleosides can include, among others, 2'-MOE and 2'-O-CH3), and bicyclic sugar-modified nucleosides (such bicyclic sugar-modified nucleosides can include those having LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Trz] and / or ALNA[Oxz]). The wing-gap-wing motif is often described as "X-Y-Z", where "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. As used herein, a gapmer described as "X-Y-Z" has a conformation such that the gap segment is located immediately adjacent to each of the 5' wing segment and the 3' wing segment. Thus, there are no intervening 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 described herein may have a gapmer motif. In some embodiments, X and Z are the same, and in other embodiments they are different. In a preferred embodiment, Y is from 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.Thus, examples of gapmers include, but are not limited to, 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, 5-8-4, or 7-6-3.

[0372] In certain embodiments, the modified oligonucleotide targeting DUX4 nucleic acid has a 3-10-3 gapmer motif. In certain embodiments, the modified oligonucleotide targeting DUX4 nucleic acid has a 3-9-3 gapmer motif. In certain embodiments, the modified oligonucleotide targeting DUX4 nucleic acid has a 3-9-4 gapmer motif. In certain embodiments, the modified oligonucleotide targeting DUX4 nucleic acid has a 3-8-5 gapmer motif.

[0373] In certain embodiments, 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 nucleobases of the nucleobase sequence of any of the exemplary modified oligonucleotides described herein (e.g., at least 8 consecutive nucleobases of the nucleobase sequence set forth in any one of SEQ ID NOs: 2, 3, 4, 7-64, 69-97, or 102-112 of the Sequence Listing).

[0374] In certain embodiments, the invention provides oligomeric compounds comprising oligonucleotides. In certain embodiments, such oligonucleotides comprise one or more chemical modifications. In certain embodiments, the chemically modified oligonucleotides comprise one or more modified sugars. In certain embodiments, the chemically modified oligonucleotides comprise one or more modified nucleobases. In certain embodiments, the chemically modified oligonucleotides comprise one or more modified internucleoside linkages. In certain embodiments, the chemical modifications (sugar modifications, nucleobase modifications and / or linkage modifications) define a pattern or motif. In certain embodiments, the patterns of chemical modifications of the sugar moiety, internucleoside linkages and nucleobases are each independent of one another. Thus, an oligonucleotide can be characterized by its sugar modification motif, internucleoside linkage motif and / or nucleobase modification motif (as used herein, a nucleobase modification motif describes a chemical modification to a nucleobase, independent of the sequence of nucleobases).

[0375] Certain sugar motifs In certain embodiments, an oligonucleotide comprises one or more types of modified sugar moieties and / or naturally occurring sugar moieties disposed along the oligonucleotide or a region thereof in a defined pattern or sugar modification motif. Such motifs can include any of the sugar modifications discussed herein and / or other known sugar modifications.

[0376] In certain embodiments, the oligonucleotide comprises or consists of a region having a gapmer sugar modification motif, which includes two external regions, namely the "wing segments", and one internal region, namely the "gap segment". The three regions of the gapmer motif (5' wing segment, gap segment and 3' wing segment) form a continuous sequence of nucleosides, where at least some of the sugar moieties of the nucleosides in each wing segment are different from at least some of the sugar moieties of the nucleosides in the gap segment. In particular, at least the sugar moieties of the nucleosides in each wing segment closest to the gap segment (the most 3' nucleoside of the 5' wing segment and the most 5' nucleoside of the 3' wing segment) are different from the sugar moieties of the nucleosides in the adjacent gap segment, thus defining the boundary sites between the wing segment and the gap segment. In certain embodiments, the sugar moieties within the gap segment are the same as each other. In certain embodiments, the gap segment includes one or more nucleosides having a sugar moiety different from the sugar moieties of one or more other nucleosides of the gap segment. In certain embodiments, the sugar modification motifs of the two wing segments are the same as each other (symmetric gapmer). In certain embodiments, the sugar modification motif of the 5' wing segment is different from the sugar modification motif of the 3' wing segment (asymmetric gapmer).

[0377] Certain 5' wing segments In certain embodiments, the 5' wing segment of the gapmer consists of from 1 to 5 linked nucleosides. In certain embodiments, the 5' wing of the gapmer The ment 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.

[0378] In certain embodiments, the 5' wing segment of the gapmer comprises at least one bicyclic nucleoside. In certain embodiments, the 5' wing segment of the gapmer comprises at least two bicyclic nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises at least three bicyclic nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises at least four bicyclic nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 5' wing segment of the gapmer comprises at least one 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 certain embodiments, each nucleoside of the 5' wing segment of the gapmer is an 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.

[0379] In certain embodiments, the 5’-wing segment of the gapmer comprises at least one acyclic 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, e.g., 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 an acyclic 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.

[0380] In certain embodiments, the 5’-wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one acyclic 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.

[0381] In certain embodiments, the 5' wing segment of the gapmer comprises at least one constrained ethyl nucleoside and at least one acyclic 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.

[0382] 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 five 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.

[0383] In certain embodiments, the 5' wing segment of the gapmer comprises two LNA-containing nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises three LNA-containing nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises four LNA-containing nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises three ALNA[Ms]-containing nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises two GuNA-containing nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises three GuNA-containing nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises three ALNA[mU]-containing nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises three ALNA[ipU]-containing nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises two LNA-containing nucleosides and one GuNA-containing nucleoside. In certain embodiments, the 5' wing segment of the gapmer comprises three ALNA[Trz]-containing nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises three ALNA[Ms]-containing nucleosides and one 2'-OMe nucleoside. In certain embodiments, the 5' wing segment of the gapmer comprises three ALNA[Ms]-containing nucleosides and two 2'-OMe nucleosides.

[0384] In certain embodiments, the 5' wing segment of the gapmer comprises three constrained ethyl nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises two bicyclic nucleosides and two non-bicyclic modified nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises two constrained ethyl nucleosides and two 2'-OMe nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises two bicyclic nucleosides and two non-bicyclic modified nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises two constrained ethyl nucleosides and two 2'-OMe nucleosides. In certain embodiments, the 5' wing segment of the gapmer comprises two constrained ethyl nucleosides and three 2'-OMe nucleosides.

[0385] In certain embodiments, the 5' wing segment of the gapmer consists of one ALNA[Ms]-containing nucleoside. In certain embodiments, the 5' wing segment of the gapmer consists of two linked ALNA[Ms]-containing nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of three linked ALNA[Ms]-containing nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of four linked ALNA[Ms]-containing nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of five linked ALNA[Ms]-containing nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of linked one, two or three ALNA[Ms]-containing nucleosides and one 2'-OMe nucleo It consists of reosid. In certain embodiments, the 5'-wing segment of the gapmer consists of 1, 2, or 3 linked ALNA[Ms]-containing nucleosides and 2 2'-OMe nucleosides. In certain embodiments, the 5'-wing segment of the gapmer consists of 1, 2, or 3 linked ALNA[Ms]-containing nucleosides and 3 2'-OMe nucleosides. In certain embodiments, the 5'-wing segment of the gapmer consists of 3 linked ALNA[Ms]-containing nucleosides and 1 2'-MOE nucleoside. In certain embodiments, the 5'-wing segment of the gapmer consists of 2 linked ALNA[Ms]-containing nucleosides and 2 2'-MOE nucleosides. In certain embodiments, the 5'-wing segment of the gapmer consists of 3 linked LNA-containing nucleosides and 2 2'-MOE nucleosides. In certain embodiments, the 5'-wing segment of the gapmer consists of 2 linked LNA-containing nucleosides and 2 2'-MOE nucleosides. In certain embodiments, the 5'-wing segment of the gapmer consists of 1, 2, or 3 linked ALNA[Ms]-containing nucleosides and contains 1 5-methylcytosine. In certain embodiments, the 5'-wing segment of the gapmer consists of 1, 2, or 3 linked ALNA[Ms]-containing nucleosides and contains 2 5-methylcytosines. In certain embodiments, the 5'-wing segment of the gapmer consists of 1, 2, or 3 linked ALNA[Ms]-containing nucleosides and contains 3 5-methylcytosines.

[0386] Certain 3'-wing segments In certain embodiments, the 3' wing segment of the gapmer consists of from 1 to 8 linked nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of from 2 to 5 linked nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of from 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 from 1 to 4 linked nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of from 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 from 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.

[0387] In certain embodiments, the 3' wing segment of the gapmer comprises at least one bicyclic nucleoside. In certain embodiments, the 3' wing segment of the gapmer comprises at least two bicyclic nucleosides. In certain embodiments, the 3' wing segment of the gapmer comprises at least three bicyclic nucleosides. In certain embodiments, the 3' wing segment of the gapmer comprises at least four bicyclic nucleosides. In certain embodiments, the 3' wing segment of the gapmer comprises at least one constrained ethyl nucleoside. In certain embodiments, the 3' wing segment of the gapmer comprises at least one 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 certain embodiments, each nucleoside of the 3' wing segment of the gapmer is an 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. de, ALNA[Trz]-containing nucleoside, and / or ALNA[Oxz]-containing nucleoside.

[0388] In certain embodiments, the 3' wing segment of the gapmer comprises at least one acyclic 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 an acyclic 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.

[0389] In certain embodiments, the 3' wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one acyclic 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.

[0390] 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.

[0391] 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 2'-MOE nucleosides, 2'-OMe nucleosides, LNA-containing nucleosides, GuNA-containing nucleosides, ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, A The LNA[ipU]-containing nucleoside, the ALNA[Trz]-containing nucleoside, and / or the ALNA[Oxz]-containing nucleoside contain at least three modified nucleosides selected therefrom. In certain embodiments, the 3' wing segment of the gapmer contains 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 contains at least five 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 contains at least six 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 contains at least seven 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.

[0392] In certain embodiments, the 3’ wing segment of the gapmer comprises two LNA-containing nucleosides. In certain embodiments, the 3’ wing segment of the gapmer comprises three LNA-containing nucleosides. In certain embodiments, the 3’ wing segment of the gapmer comprises four LNA-containing nucleosides. In certain embodiments, the 3’ wing segment of the gapmer comprises three ALNA[Ms]-containing nucleosides. In certain embodiments, the 3’ wing segment of the gapmer comprises two GuNA-containing nucleosides. In certain embodiments, the 3’ wing segment of the gapmer comprises three GuNA-containing nucleosides. In certain embodiments, the 3’ wing segment of the gapmer comprises three ALNA[mU]-containing nucleosides. In certain embodiments, the 3’ wing segment of the gapmer comprises three ALNA[ipU]-containing nucleosides. In certain embodiments, the 3’ wing of the gapmer comprises two LNA-containing nucleosides and one GuNA-containing nucleoside. In certain embodiments, the 3’ wing segment of the gapmer comprises three ALNA[Trz]-containing nucleosides. In certain embodiments, the 3’ wing segment of the gapmer comprises three ALNA[Ms]-containing nucleosides and one 2’-OMe nucleoside. In certain embodiments, the 3’ wing segment of the gapmer comprises three ALNA[Ms]-containing nucleosides and two 2’-OMe-containing nucleosides. In certain embodiments, the 3’ wing segment of the gapmer comprises three 2’-MOE nucleosides. In certain embodiments, the 3’ wing segment of the gapmer comprises four 2’-MOE nucleosides. In certain embodiments, the 3’ wing segment of the gapmer comprises five 2’-MOEs. In certain embodiments, the 3’ wing segment of the gapmer The T contains three ALNA[Ms]-containing nucleosides and one 2’-MOE-containing nucleoside. In certain embodiments, the 3’ wing segment of the gapmer contains three ALNA[Ms]-containing nucleosides and two 2’-MOE nucleosides. In certain embodiments, the 3’ wing segment of the gapmer contains two ALNA[Ms]-containing nucleosides and two 2’-MOE nucleosides. In certain embodiments, the 3’ wing segment of the gapmer contains two ALNA[Ms]-containing nucleosides and three 2’-MOE nucleosides. In certain embodiments, the 3’ wing segment of the gapmer contains three ALNA[Ms]-containing nucleosides and five 2’-MOE nucleosides. In certain embodiments, the 3’ wing segment of the gapmer contains one ALNA[Ms]-containing nucleoside and three 2’-MOE nucleosides. In certain embodiments, the 3’ wing segment of the gapmer contains two LNA-containing nucleosides and two 2’-MOE nucleosides.

[0393] In certain embodiments, the 3’ wing segment of the gapmer contains three constrained ethyl nucleosides. In certain embodiments, the 3’ wing segment of the gapmer contains two bicyclic nucleosides and two non-bicyclic modified nucleosides. In certain embodiments, the 3’ wing segment of the gapmer contains two constrained ethyl nucleosides and two 2’-OMe nucleosides. In certain embodiments, the 3’ wing segment of the gapmer contains two bicyclic nucleosides and two non-bicyclic modified nucleosides. In certain embodiments, the 3’ wing segment of the gapmer contains two constrained ethyl nucleosides and two 2’-OMe nucleosides. In certain embodiments, the 3’ wing segment of the gapmer contains two constrained ethyl nucleosides and three 2’-OMe nucleosides.

[0394] In certain embodiments, the 3' wing segment of the gapmer consists of one ALNA[Ms] - containing nucleoside. In certain embodiments, the 3' wing segment of the gapmer consists of two linked ALNA[Ms] - containing nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of three linked ALNA[Ms] - containing nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of four linked ALNA[Ms] - containing nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of five linked ALNA[Ms] - containing nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of three, four or five linked 2'-MOE nucleosides. In certain embodiments, the 3' wing segment of the gapmer consists of one, two or three linked ALNA[Ms] It consists of a nucleoside and one 2'-OMe nucleoside. In certain embodiments, the 3'-wing segment of the gapmer consists of linked 1, 2, or 3 ALNA[Ms]-containing nucleosides and two 2'-OMe nucleosides. In certain embodiments, the 3'-wing segment of the gapmer consists of linked 1, 2, or 3 ALNA[Ms]-containing nucleosides and three 2'-OMe nucleosides. In certain embodiments, the 3'-wing segment of the gapmer consists of linked 1, 2, or 3 ALNA[Ms]-containing nucleosides and one 2'-MOE nucleoside. In certain embodiments, the 3'-wing segment of the gapmer consists of linked 1, 2, or 3 ALNA[Ms]-containing nucleosides and two 2'-MOE nucleosides. In certain embodiments, the 3'-wing segment of the gapmer consists of linked 1, 2, or 3 ALNA[Ms]-containing nucleosides and three 2'-MOE nucleosides. In certain embodiments, the 3'-wing segment of the gapmer consists of linked 1, 2, or 3 ALNA[Ms]-containing nucleosides and five 2'-MOE nucleosides. In certain embodiments, the 3'-wing segment of the gapmer consists of linked two LNA-containing nucleosides and two 2'-MOE nucleosides. In certain embodiments, the 3'-wing segment of the gapmer is linked 1, 2, or 3 ALNA[Ms]-containing nucleosides and contains one 5-methylcytosine. In certain embodiments, the 3'-wing segment of the gapmer consists of linked 1, 2, or 3 ALNA[Ms]-containing nucleosides and contains two 5-methylcytosines. In certain embodiments, the 3'-wing segment of the gapmer consists of linked 1, 2, or 3 ALNA[Ms]-containing nucleosides and contains three 5-methylcytosines. In certain embodiments, the 3'-wing segment of the gapmer consists of linked 1, 2, 3, 4, or 5 2'-MOE nucleosides and contains one 5-methylcytosine.

[0395] In certain embodiments, the 5' wing segment of the gapmer consists of one ALNA[Ms]-containing nucleoside, and the 3' wing segment consists of one ALNA[Ms]-containing nucleoside. In certain embodiments, the 5' wing segment of the gapmer consists of two linked ALNA[Ms]-containing nucleosides, and the 3' wing segment consists of two linked ALNA[Ms]-containing nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of three linked ALNA[Ms]-containing nucleosides, and the 3' wing segment consists of three linked ALNA[Ms]-containing nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of four linked ALNA[Ms]-containing nucleosides, and the 3' wing segment consists of four linked ALNA[Ms]-containing nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of five linked ALNA[Ms]-containing nucleosides, and the 3' wing segment consists of five linked ALNA[Ms]-containing nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of one, two or three linked ALNA[Ms]-containing nucleosides and one 2'-OMe nucleoside, and the 3' wing segment consists of one, two or three linked ALNA[Ms]-containing nucleosides and one 2'-OMe nucleoside. In certain embodiments, the 5' wing segment of the gapmer consists of one, two or three linked ALNA[Ms]-containing nucleosides and two 2'-OMe nucleosides, and the 3' wing segment consists of one, two or three linked ALNA[Ms]-containing nucleosides and two linked 2'-OMe nucleosides. In certain embodiments, the 5' wing segment of the gapmer consists of one, two or three linked ALNA[Ms]-containing nucleosides and three 2'-OMe nucleosides, and the 3' wing segment consists of one, two or three linked ALNA[Ms]-containing nucleosides and three 2'-OMe nucleosides. In certain embodiments, the 5’ wing segment of the gapmer consists of 1, 2, or 3 linked ALNA[Ms]-containing nucleosides and 1 or 2 2’-MOE nucleosides, and the 3’ wing segment consists of 1, 2, or 3 linked ALNA[Ms]-containing nucleosides and 1 or 2 2’MOE nucleosides. In certain embodiments, the 5’ wing segment of the gapmer consists of 1, 2, or 3 linked ALNA[Ms]-containing nucleosides, and the 3’ wing segment consists of 1, 2, or 3 linked ALNA[Ms]-containing nucleosides and 1, 2, or 3 2’-MOE nucleosides. In certain embodiments, the 5’ wing segment of the gapmer consists of 1, 2, or 3 linked ALNA[Ms]-containing nucleosides, and the 3’ wing segment consists of 1, 2, 3, 4, or 5 linked 2’-MOE nucleosides. 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].

[0396] In certain embodiments, the 5’ wing segment of the gapmer consists of 1, 2 or or consists of three ALNA[Ms] - containing nucleosides and contains one 5 - methylcytosine. Further, the 3' wing segment of the gapmer consists of one, two or three linked ALNA[Ms] - containing nucleosides and contains one 5 - methylcytosine. In certain embodiments, the 5' wing segment of the gapmer consists of one, two or three linked ALNA[Ms] - containing nucleosides and contains two 5 - methylcytosines, and the 3' wing of the gapmer consists of one, two or three linked ALNA[Ms] - containing nucleosides and contains one 5 - methylcytosine. In certain embodiments, the 5' wing segment of the gapmer consists of one, two or three linked ALNA[Ms] - containing nucleosides and contains two 5 - methylcytosines, and the 3' wing segment of the gapmer consists of one, two or three linked ALNA[Ms] - containing nucleosides and contains two 5 - methylcytosines. In certain embodiments, the 5' wing segment of the gapmer consists of one, two or three linked ALNA[Ms] - containing nucleosides and contains three 5 - methylcytosines, and the 3' wing segment of the gapmer consists of one, two or three linked ALNA[Ms] - containing nucleosides and contains three 5 - methylcytosines. In certain embodiments, the 5' wing segment of the gapmer consists of one, two or three linked ALNA[Ms] - containing nucleosides and contains one 5 - methylcytosine, and the 3' wing segment of the gapmer consists of one, two, three, four or five linked 2'-MOE nucleosides and contains one 5 - methylcytosine. In certain embodiments, the 5' wing segment of the gapmer consists of one, two or three linked ALNA[Ms] - containing nucleosides, and the 3' wing segment of the gapmer consists of one, two or three linked ALNA[Ms] - containing nucleosides and one, two or three linked 2'-MOE nucleosides and contains one 5 - methylcytosine.

[0397] In certain embodiments, the gap segments of the gapmer comprise 10 contiguous nucleosides, include a 2'-OMe nucleoside at either the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th or 10th nucleoside, and the remaining nucleosides are deoxynucleosides.

[0398] Compositions and methods for formulating pharmaceutical compositions For the preparation of a pharmaceutical composition or formulation, a modified oligonucleotide can be mixed with one or more pharmaceutically acceptable active or inactive substances. The compositions and methods for formulating a pharmaceutical composition are determined by several criteria including, but not limited to, the route of administration, the degree of the disease or the dose to be administered.

[0399] By combining a modified oligonucleotide with a suitable pharmaceutically acceptable diluent or carrier, a modified oligonucleotide targeting DUX4 nucleic acid can be utilized in a pharmaceutical composition. Examples of pharmaceutically acceptable diluents include phosphate buffered saline (PBS). PBS is a diluent suitable for use in compositions delivered parenterally. Thus, in one embodiment, a pharmaceutical composition comprising a modified oligonucleotide targeting DUX4 nucleic acid and a pharmaceutically acceptable diluent is used in the methods described herein. In certain embodiments, the pharmaceutically acceptable diluent is PBS.

[0400] A pharmaceutical composition comprising a modified oligonucleotide can include 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 when administered to an animal including a human. Thus, for example, the present disclosure also relates to pharmaceutically acceptable salts of modified oligonucleotides, prodrugs, pharmaceutically acceptable salts of such prodrugs and other biological equivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium salts and potassium salts.

[0401] A prodrug can include incorporating additional nucleosides at one or both ends of a modified oligonucleotide that is cleaved by an endogenous nuclease in the body to form an active modified oligonucleotide.

[0402] Conjugated modified oligonucleotide 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. Exemplary conjugation groups include cholesterol moieties and lipid moieties. Additional conjugation groups include carbohydrates, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0403] The modified oligonucleotide can also be modified to have one or more stabilizing groups generally attached to one or both ends of the modified oligonucleotide to enhance properties such as nuclease stability. Cap structures are included in the stabilizing groups. These terminal modifications protect the modified oligonucleotide having terminal nucleic acids from degradation by exonucleases and can aid in intracellular delivery and / or localization. The cap can be present at the 5' end (5' cap), the 3' end (3' cap), or both ends. Cap structures are well known in the art and include, for example, inverted deoxyabasic caps. Additional 3' and 5' stabilizing groups that can be used to cap one or both ends of the modified oligonucleotide to confer nuclease stability include those disclosed in WO03 / 004602.

[0404] Cell culture and modified oligonucleotide treatment The effects of modified oligonucleotides on the levels, activities or expression of DUX4 nucleic acids can be tested in vitro in various cell types. Cell types for such analyses are available from commercial suppliers (e.g., American Type Culture Collection, Manassus, VA; Zen-Bio, Inc., Research Triangle Park, NC; Clonetics Corporation, Walkersville, MD), and the cells are cultured according to the suppliers' instructions using commercially available reagents (e.g., Invitrogen Life Technologies, Carlsbad, CA). Exemplary cell types include, but are not limited to, C2C12 cells, HepG2 cells, Hep3B cells, primary hepatocytes, A549 cells, GM04281 fibroblasts, and LLC-MK2 cells. These cells can be used after transfection with a vector expressing human DUX4 mRNA. The vector preferably expresses as a fusion protein with a reporter gene such as luciferase or GFP, and examples include the psiCHECK-2 vector (Promega). obtainable, and the cells are cultured according to the suppliers' instructions using commercially available reagents (e.g., Invitrogen Life Tech nologies, Carlsbad, CA). Exemplary cell types include, but are not limited to, C2C12 cells, HepG2 cells, Hep3B cells, primary hepatocytes, A549 cells, GM04281 fibroblasts, and LLC-MK2 cells. These cells can be used after transfection with a vector expressing human DUX4 mRNA. The vector preferably expresses as a fusion protein with a reporter gene such as luciferase or GFP, and examples include the psiCHECK-2 vector (Promega).

[0405] In Vitro Testing of Modified Oligonucleotides Methods of treating cells with modified oligonucleotides are described herein and can be appropriately modified depending on the type of modified oligonucleotide.

[0406] Generally, cells are treated with the modified oligonucleotide when they reach about 60-80% confluence during culture.

[0407] The modified oligonucleotide is introduced into the cells, for example, by the lipofection method. One reagent commonly used to introduce modified oligonucleotides into cultured cells is the cationic lipid transfection reagent LIPOFECTIN® (Invitrogen, Carlsbad, CA). The modified oligonucleotide is Mix in OPTI-MEM® I (Invitrogen, Carlsbad, CA) with LIPOFECTIN® to obtain the desired final concentration of the modified oligonucleotide and a LIPOFECTIN® concentration ranging typically from 2 to 12 μg / mL per 100 nM of the modified oligonucleotide.

[0408] Another reagent used to introduce modified oligonucleotides into cultured cells is LIPOFECTAMINE 2000® (Invitrogen, Carlsbad, CA). Mix the modified oligonucleotide with LIPOFECTAMINE 2000® in OPTI-MEM® I serum-reduced medium (Invitrogen, Carlsbad, CA) to obtain the desired concentration of the modified oligonucleotide and a LIPOFECTAMINE® concentration ranging typically from 2 to 12 μg / mL per 100 nM of the modified oligonucleotide.

[0409] Another reagent used to introduce modified oligonucleotides into cultured cells is Cytofectin® (Invitrogen, Carlsbad, CA). Mix the modified oligonucleotide with Cytofectin® in OPTI-MEM® I serum-reduced medium (Invitrogen, Carlsbad, CA) to obtain the desired concentration of the modified oligonucleotide and a Cytofectin® concentration ranging typically from 2 to 12 μg / mL per 100 nM of the modified oligonucleotide.

[0410] Another technique used to introduce modified oligonucleotides into cultured cells is electroporation.

[0411] Modified oligonucleotides can be introduced into cells without using reagents such as LIPOFECTIN®. The method for suppressing the expression of the target gene of the modified oligonucleotide in this case is called the Gymnosis method.

[0412] Cells are treated with a modified oligonucleotide by conventional methods. Typically, cells are collected 16 to 48 hours after treatment with the modified oligonucleotide, at which point the RNA or protein level of the target nucleic acid is measured by methods known in the art and described herein. Generally, when performing the treatment in multiple replicates, the data is presented as the average of the replicate treatments.

[0413] The concentration of the modified oligonucleotide used varies depending on the cell line. Methods for determining the optimal modified oligonucleotide concentration for a particular cell line are well known in the art. When transfecting using LIPOFECTAMINE2000 (registered trademark), LIPOFECTIN (registered trademark) or Cytofectin (registered trademark), the modified oligonucleotide is typically used at a concentration ranging from 1 nM to 300 nM. When transfecting using electroporation, a higher concentration of the modified oligonucleotide ranging from 625 to 20,000 nM is used. From the inhibition rate of gene expression at each concentration, the concentration IC 50 of the modified oligonucleotide that suppresses 50% of gene expression can be calculated.

[0414] RNA Isolation RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods for RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using TRIzol (registered trademark) reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's recommended protocol.

[0415] Analysis of Target Level or Inhibition of Expression Inhibition of the level or expression of DUX4 nucleic acid can be assayed by various methods known in the art. For example, the target nucleic acid level 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 conventional in the art. Quantitative real-time PCR can be conveniently achieved using a commercially available ABI PRISM® 7600, 7700 or 790 0 sequence detection system available from PE-Applied Bio systems, Foster City, CA, according to the manufacturer's instructions .

[0416] Quantitative real-time PCR analysis of target RNA level Quantification of the target RNA level can be achieved by quantitative real-time PCR using an ABI PRISM® 7600, 7700 or 7900 sequence detection system (PE-Applied Bio systems, Foster City, CA) according to the manufacturer's instructions. Methods for quantitative real-time PCR are well known in the art.

[0417] Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction, thereby generating complementary DNA (cDNA) that is then used as a substrate for real-time PCR amplification. The RT and real-time PCR reactions are sequentially performed in the same sample well. The reagents for RT and real-time PCR are obtained from Invitrogen (Carlsbad, CA). The RT and real-time PCR reactions are performed by methods well known to those skilled in the art.

[0418] ​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® (Invitrogen, Inc. Carlsbad, CA). The expression of cyclophilin A is quantified by real-time PCR, simultaneously with the target, either multiplexed or separately. Total RNA is quantified using the RIBOGREEN® RNA quantification reagent (Invitrogen, Inc. Eugene, OR). The method for quantifying RNA by RIBOGREEN® is taught in Jones, L.J., et al., (Analytical Biochemistry, 1998, 265, 368-374). is. The RIBOGREEN® fluorescence is measured using a CYTOFLUOR® 4000 instrument (PE Applied Bios ystems).

[0419] Probes and primers are designed to hybridize to the DUX4 nucleic acid. Methods for designing probes and primers for real-time PCR are well known in the art and can include the use of software such as PRIMER EXPRESS® software (Applied Biosystems, Foster City, CA).

[0420] Analysis at the protein level By measuring the level of DUX4 protein, antisense inhibition of DUX4 nucleic acid can be evaluated. The level of DUX4 protein can be examined or quantified by various methods well-known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assay, protein activity assay (e.g., caspase activity assay), immunohistochemistry, immunocytochemistry, or fluorescence-activated cell sorting (FACS). Antibodies against the target can be identified and obtained from various sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or prepared by conventional monoclonal or polyclonal antibody production methods known in the art. It can be prepared by monoclonal or polyclonal antibody production methods.

[0421] Analysis of gene expression The level of DUX4 gene expression can also be measured using a reporter gene such as luciferase. For example, when using the psiCHECK-2 vector (Promega), the expression of the DUX4 gene can be measured by the luminescence amount of Renilla luciferase, which is a fusion protein with DUX4, and by correcting with the luminescence amount of Firefly luciferase present on the same vector, effects such as non-specific cell death can be excluded.

[0422] In vivo testing of modified oligonucleotides Modified oligonucleotides are tested in animals to evaluate their ability to inhibit DUX4 expression and change the phenotype. The tests are performed in normal animals or in experimental disease models, such as transgenic mouse models of DUX4 (Jones, T. et al. PLoS One. 2018;13(2), Article number e0192 657) or DUX4 gene expression mice using recombinant AAV virus (Wallace, LM et al. Mol Ther. 2012;20(7):1417, Wa It can be carried out as described in Lace, LM et al. Ann Neurol. 2011; 69(3):540).

[0423] For administration to an animal, the modified oligonucleotide is formulated in a pharmaceutically acceptable diluent, such as phosphate buffered saline. Administration includes parenteral administration routes. Following 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.

[0424] Certain antisense mechanisms FSHD is caused by abnormal expression of the DUX4 gene (especially the splicing variant of DUX4-FL) in muscle. On the other hand, DUX4 is expressed in healthy individuals, for example, in the testis. Among certain splicing variants of DUX4 expressed in the testis, in addition to DUX4-FL, splicing variants of exon 1, exon 2, exon 6, exon 7, and splicing variants of exon 1, exon 2, exon 4, exon 5, exon 6, exon 7 are expressed (Non-Patent Document 1 above).

[0425] Certain biomarkers At least in part, gene expression, such as MBD3L2, ZSCAN4, TRIM43, DEFB103, ZNF217, etc., is regulated by the accumulation level of DUX4 protein (Non-Patent Document 2 above). Also, serum creatine kinase can be measured as a marker of muscle disorder.

[0426] Certain indications In certain embodiments, provided herein is a method of treating an individual, the method comprising administering one or more pharmaceutical compositions described herein. In certain embodiments, the individual has FSHD.

[0427] Accordingly, provided herein are methods for ameliorating symptoms associated with FSHD in a subject in need thereof. In certain embodiments, methods for reducing the incidence of one or more symptoms associated with FSHD are provided. In certain embodiments, methods for reducing the severity of symptoms associated with FSHD are provided. In certain embodiments, symptoms associated with FSHD include muscle rigidity, myotonia, facial muscle weakness, ptosis, inability to whistle, decreased facial expression, depressive or angry facial expressions, difficulty pronouncing words, scapular muscle weakness (winged scapula, winging, etc. variations), lower limb weakness, hearing loss, and heart disease.

[0428] In certain embodiments, the methods of the invention comprise administering to an individual in need thereof a therapeutically effective amount of a compound that targets DUX4 nucleic acid.

[0429] In certain embodiments, administration of a modified oligonucleotide that targets DUX4 nucleic acid results in a reduction in DUX4 expression 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 in a range defined by any two of these values.

[0430] In certain embodiments, a pharmaceutical composition comprising a modified oligonucleotide that targets DUX4 is used to prepare a medicament for treating patients suffering from or susceptible to DUX4-related diseases such as FSHD.

[0431] In certain embodiments, the methods described herein comprise administering a compound comprising a modified oligonucleotide having a continuous nucleic acid base portion described herein of the sequences set forth in SEQ ID NOs: 2, 3, 4, 7-64, 69-97 or 102-112 of the Sequence Listing.

[0432] Administration In certain embodiments, the compounds and pharmaceutical compositions described herein are administered parenterally.

[0433] In certain embodiments, parenteral administration is by infusion. The infusion may be long-term or continuous, or short-term or intermittent. In certain embodiments, the infused pharmaceutical agent is delivered by a pump. In certain embodiments, parenteral administration is by injection (e.g., a bolus injection). The injectate may be delivered by a syringe.

[0434] Examples of parenteral administration include, for example, subcutaneous administration, intravenous administration, intramuscular administration, intra-arterial administration, intraperitoneal administration, or intracranial administration, for example, intrathecal or intraventricular administration. The administration may be continuous or long-term, or short-term or intermittent.

[0435] In certain embodiments, delivery of the compounds of the pharmaceutical compositions described herein results in at least 70% down-regulation of the level of the target mRNA and / or target protein. In certain embodiments, delivery of the compounds or compositions described herein results in 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% down-regulation of the level of the target mRNA and / or target protein over 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, 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.

[0436] In certain embodiments, the modified oligonucleotide is administered once daily, once every three days, once weekly , once every two weeks, once every three weeks, once monthly, once every two months, once every three months, once every six months, twice a year or once a year, by injection or infusion.

[0437] Certain combination therapies In certain embodiments, a first agent comprising a modified oligonucleotide of the invention is co-administered with one or more second agents. In certain embodiments, such second agents are designed to treat the same FSHD as the first agent described herein. In certain embodiments, such second agents are designed to treat a different disease, disorder or condition than the first agent described herein. In certain embodiments, such second agents are designed to treat an undesirable side effect of one or more of the pharmaceutical compositions described herein. In certain embodiments, the second agent is co-administered with the first agent to treat an undesirable effect of the first agent. In certain embodiments, the second agent is co-administered with the first agent to provide an additive effect. In certain embodiments, the second agent is co-administered with the first agent to provide a synergistic effect.

[0438] 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 formulated together in a single pharmaceutical formulation. In certain embodiments, the first agent and one or more second agents are formulated separately.

[0439] Certain compounds In certain embodiments, the compounds disclosed herein 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 methods 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 methods described in Japanese Patent Application No. 2018-212424.

[0440] In certain embodiments, the compounds disclosed herein enjoy the benefit of one or more improved in vitro and / or in vivo properties as compared to appropriate comparative compounds.

[0441] In certain embodiments, Compound No. 1 having the sequence (5' to 3') ngagattcccgccggt (where n is 5-methylcytosine and is incorporated herein as SEQ ID NO: 2), that is, a gapmer in which the 5' wing and the 3' wing each consist of 3 LNA-containing nucleosides, and each internucleoside bond is a phosphorothioate bond.

[0442] In certain embodiments, Compound No. 2 having the sequence (5' to 3') gnagttctccgcggt (where n is 5-methylcytosine and is incorporated herein as SEQ ID NO: 3 in the Sequence Listing), that is, a gapmer in which the 5' wing and the 3' wing each consist of 3 ALNA[Ms]-containing nucleosides, and each internucleoside bond is a phosphorothioate bond.

[0443] In certain embodiments, Compound No. 3 having the sequence (5' to 3') gnntagacagcgtngg (where n is 5-methylcytosine and is incorporated herein as SEQ ID NO: 4 in the Sequence Listing), that is, a compound in which the 5' wing and the 3' wing each consist of 3 LNA-containing nucleosides​ A gapmer compound in which each internucleoside linkage is a phosphorothioate linkage.

[0444] In certain embodiments, a compound of Compound No. 123 having the sequence (5' to 3') gnntagacagcgtngg (where n is 5-methylcytosine and is incorporated herein as SEQ ID NO: 4 in the Sequence Listing), i.e., a gapmer in which the 5' wing and the 3' wing each consist of three ALNA[Ms]-containing nucleosides, and in which each internucleoside linkage is a phosphorothioate linkage.

[0445] Non-limiting disclosure and incorporation by reference Certain compounds, compositions, and methods described herein are specifically described according to certain embodiments, but the following examples merely illustrate and are not intended to limit the compounds described herein. Each of the references, GENBANK accession numbers, etc. described in this application is incorporated herein by reference in its entirety.

[0446] The Sequence Listing attached to this application identifies each sequence as either "RNA" or "DNA" as necessary, but in reality, those sequences can be modified with any combination of chemical modifications. Those skilled in the art will readily understand that designations such as "RNA" or "DNA" for describing modified oligonucleotides can be arbitrary in some cases. For example, an oligonucleotide containing a nucleoside with a 2'-OH sugar moiety and a thymine base can be described as DNA with a modified sugar (2'-OH relative to the natural 2'-H of DNA), or as RNA with a modified base (thymine (methylated uracil) relative to the natural uracil of RNA).

[0447] Accordingly, without limitation, the nucleic acid sequences provided herein, including those in the Sequence Listing, are intended to encompass nucleic acids that include any combination of natural or modified RNA and / or DNA, including, without limitation, such nucleic acids having modified nucleobases. Without limitation, as a further example, an oligomeric compound having a nucleobase sequence of "ATCGATCG" encompasses any oligomeric compound having such a nucleobase sequence, whether modified or unmodified, including, without limitation, such compounds that include RNA bases, such as those having the sequence "AUCGAUCG", as well as those having some DNA bases and some RNA bases, such as "AUCGATCG", and oligomeric compounds having other modified or naturally occurring bases, such as "ATmeCGAUCG" (where meC represents a cytosine base containing a methyl group at the 5-position).

Example

[0448] Non-limiting disclosure and incorporation by reference Certain compounds, compositions, and methods described herein are specifically described according to certain embodiments, but the following examples serve only to illustrate the compounds described herein and are not intended to limit them. Each of the references cited in this application is hereby incorporated by reference in its entirety into this specification.

[0449] The structures of each artificial nucleic acid used herein are shown in the following structural formulas together with their respective abbreviations. Structure and abbreviation of each artificial nucleic acid

[0450]

Chem.

[0451] Example 1 Synthesis and Purification of Modified Oligonucleotide Compounds for In Vitro Evaluation Various amidites (LNA amidites are from Chem Genes and Hongene Biotechnology Limited The amidites were purchased from Sigma-Aldrich, the 2'-OMe amidite was purchased from Sigma-Aldrich, GuNA was synthesized by the methods described in WO2014 / 046212 and WO2017 / 047816, and ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Trz] and ALNA[Oxz] were synthesized by the methods described in Japanese Patent Application No. 2018-212424. Using these, a modified oligonucleotide compound was synthesized on a CPG or polystyrene carrier at a scale of 0.2 or 1.0 μmol by a DNA / RNA oligonucleotide automatic synthesizer nS-8II (manufactured by Gene Design, Inc.). All amidites were adjusted to a 0.1 M acetonitrile solution, the coupling time for non-natural nucleosides was 10 minutes, and the other steps were carried out under the standard conditions of nS-8II. Activator42 (Sigma-Aldrich) was used as the activator, and Sulfurizing ReagentII (Gren Research Corporation) was used for thiolation. The synthesized oligonucleotide was reacted with 28% aqueous ammonia solution at 60 - 65 °C for 8 hours to cleave it from the carrier and deprotect the base moiety. After concentrating and distilling off ammonia, reverse phase HPLC purification was carried out.

[0452] Example 2 Synthesis and Purification of Modified Oligonucleotide Compounds for In Vivo Evaluation Using various amidites, a modified oligonucleotide compound was synthesized on a polystyrene carrier at a scale of 20 - 50 μmol by a DNA / RNA oligonucleotide automatic synthesizer AKTA oligopilot plus 10 (manufactured by GE Healthcare Japan Co., Ltd.). The DNA amidite was adjusted to a 0.1 M, and the non-natural amidite was adjusted to a 0.05 - 0.1 M acetonitrile solution. The coupling recycle time for non-natural nucleosides was 20 minutes. When introducing the first base to the universal carrier, the coupling, thiolation, and capping steps were each carried out twice continuously. The other steps were carried out according to AKTA ol igonucleotide automatic synthesizer AKTA oligopilot plus 10 (manufactured by GE Healthcare Japan Co., Ltd.). The DNA amidite was adjusted to a 0.1 M, and the non-natural amidite was adjusted to a 0.05 - 0.1 M acetonitrile solution. The coupling recycle time for non-natural nucleosides was 20 minutes. When introducing the first base to the universal carrier, the coupling, thiolation, and capping steps were each carried out twice continuously. The other steps were carried out according to AKTA ol It was carried out under the standard conditions of igopilot plus10. Activator42 (Sigma-Aldrich) was used as the activator, and Sulfurizing ReagentII (Gren Research Corporation) was used for thiolation. The synthesized oligonucleotide was subjected to descyanoethylation on the solid phase using 20% diethylamine / acetonitrile or 50% triethylamine / acetonitrile, and 28% aqueous ammonia solution was added and reacted at 60 - 65 °C for 8 - 24 hours to cleave from the support and deprotect the base moieties. After concentrating and distilling off ammonia, purification was performed using an anion exchange column. The excess salts contained after anion exchange were removed using a desalting column.

[0453] Example 3 Purity Confirmation of Modified Oligonucleotide Compounds Purification and purity confirmation of the synthesized modified oligonucleotide compounds were carried out under the following conditions by reverse-phase HPLC. The purity of all compounds was 85% or more. Reverse-phase HPLC (purification) Mobile phase: Solution A: 400 mM hexafluoroisopropanol, 15 mM triethylamine Solution B: methanol Gradient: A:B = 85:15 → 70:30 (10 min) Column used: Preparative Waters XBridge@ Oligonucleotide BEH C18 OBDTM Prep Column, 130 Å, 2.5 μm, 10 mm * 50 mm Flow rate: Preparative 5 mL / min Column temperature: 60 °C Detection: UV (260 nm) Reverse-phase HPLC (purity confirmation) Mobile phase: Solution A: 400 mM hexafluoroisopropanol, 15 mM aqueous triethylamine solution Solution B: methanol Gradient: A:B = 80:20 → 70:30 (6.5 min) Column used: Analysis Waters ACQUITY UPLC@ Oligonucleotide BEH C18 Column, 130 Åm 1.7 μm, 2.1 mm * 50 mm Flow rate: 0.2 mL / min Column temperature: 60 °C Detection: UV (260 nm) Anion exchange purification Mobile phase: Solution A: 1 mM NaOH 20% acetonitrile aqueous solution Solution B: 1 mM NaOH, 1.5 M NaCl / 20% acetonitrile aqueous solution Column used: TSKgel SuperQ-5PW(13) φ21.1 * 15 mm Flow rate: 7 mL / min Column temperature: Room temperature Detection: UV (260 nm) Desalting column Mobile phase: Solution A: 20% acetonitrile aqueous solution Solution B: 20% acetonitrile aqueous solution Column used: GE HiPrep 26 / 10 Desalting * 4 columns in series Flow rate: 12 mL / min Column temperature: Room temperature

[0454] Example 4 Measurement of molecular weight of modified oligonucleotide compound The molecular weight of the synthesized modified oligonucleotide compound was determined under the following conditions using Waters ZQ. Mobile phase: Solution A: 400 mM hexafluoroisopropanol, 15 mM triethylamine aqueous solution Solution B: Methanol Gradient: A:B = 80:20 → 70:30 (6.5 min) Column used: Waters ACQUITY UPLC@ Oligonucleotide BEH C18 Column, 130Åm 1.7μm, 2.1mm*50mm Flow rate: 0.2 mL / min Column temperature: 60 °C Detection: UV (260 nm)

[0455] Example 5 Molecular Weight of the Synthesized Modified Oligonucleotide Compounds The synthesized modified oligonucleotide compounds are shown in Table 1 below. The compounds are denoted by three letters for each nucleotide. However, the 3'-terminal nucleotide is denoted by two letters because there is no internucleoside bond. 1) The first letter is in uppercase and represents the following nucleobases: A = adenine, T = thymine, G = guanine, C = cytosine, U = uracil, M = 5-methylcytosine, 2) The second letter represents the following sugar moieties: 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, 3) The third letter represents the following internucleoside bonds: s = phosphorothioate, p = phosphodiester. As the target position, the 5'-target site of the DUX4 mature mRNA of the modified oligonucleotide (the position of SEQ ID NO: 1 in the sequence listing corresponding to the 3'-end of the modified oligonucleotide) is shown.

[0456]

Table 1-1

[0457]

Table 1-2

[0458]

Table 1-3

[0459]

Table 1-4

[0460]

Table 1-5

[0461]

Table 1-6

[0462] Example 6 In Vitro DUX4 Knockdown Activity Test (Lipofection Method) C2C12 cells were seeded onto a transfection reagent prepared by mixing DUX4-modified oligonucleotides and Lipofectamine RNAi Reagent at a density of 1.25×10 4 cells / cm 2 and cultured overnight in a CO2 incubator. The next day, a reporter plasmid in which the DUX4 sequence was cloned into the multiple cloning site of the psiCHECK-2 vector (Promega) was transfected into the cells using Lipofectamine 2000 Reagent, and the cells were 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 plate reader with the Dual-Glo Luciferase Assay System. To correct for the effects of transfection efficiency and cell number from the luminescence value due to Renilla luciferase activity, the ratio to the luminescence value due to Firefly luciferase activity was calculated. The inhibition rate was calculated as a percentage from the decrease rate of Renilla / Firefly when the modified oligonucleotide was added, and the IC 50 value was determined from the two concentrations sandwiching 50% and the inhibition rate at that time Calculated (Table 2). Compared with compounds (Compound Nos. 1 to 132) complementary to positions 232 to 248, 1306 to 1325, or 1472 to 1495 of mature DUX4 mRNA, Compound Nos. 133 (complementary to positions 214 to 227 of SEQ ID NO: 1 in the Sequence Listing), 134 (complementary to positions 1323 to 1336 of SEQ ID NO: 1 in the Sequence Listing), 135 (complementary to positions 1458 to 1471 of SEQ ID NO: 1 in the Sequence Listing), and 136 (complementary to positions 1495 to 1508 of SEQ ID NO: 1 in the Sequence Listing) were found to have significantly weaker inhibition rates.

[0463] Example 7 In Vitro DUX4 Knockdown Activity Test (Gymnosis Method) C2C12 cells were seeded into the DUX4-modified oligonucleotide solution at a density of 6×10 3 cells / cm 2 and cultured in a CO2 incubator for two nights. After two days, the medium containing the DUX4-modified oligonucleotide solution was removed from the cells, and the cells were washed with fresh medium. Then, using Lipofectamine 2000 Reagent, a reporter plasmid in which the DUX4 sequence was cloned into the multiple cloning site of the psiCHECK-2 vector (Promega) was transfected into the cells and cultured in a CO2 incubator for about 24 hours. Then, using the Dual-Glo Luciferase Assay System, the luminescence values of Firefly luciferase and Renilla luciferase in the cells were detected with a plate reader. To correct for the effects of transfection efficiency and cell number from the luminescence value due to Renilla luciferase activity, the ratio to the luminescence value due to Firefly luciferase activity was calculated. The inhibition rate was calculated as a percentage from the decrease rate of Renilla / Firefly when the modified oligonucleotide was added, and the IC 50Values were calculated. The results are shown in Table 2 below. Compared with compounds (Compound Nos. 1 to 132) containing nucleobase sequences complementary to the isometric portions within the regions of positions 232 to 248, 1306 to 1325, or 1472 to 1495 of the DUX4 mature mRNA, Compound No. 133 (complementary to positions 214 to 227 of SEQ ID NO: 1 in the Sequence Listing), 134 (complementary to positions 1323 to 1336 of SEQ ID NO: 1 in the Sequence Listing), 135 (complementary to positions 1458 to 1471 of SEQ ID NO: 1 in the Sequence Listing), and 136 (complementary to positions 1495 to 1508 of SEQ ID NO: 1 in the Sequence Listing) were found to have significantly weaker inhibition rates.

[0464]

Table 2-1

[0465]

Table 2-2

[0466]

Table 2-3

[0467]

Table 2-4

[0468] Example 8 Synthesis of Modified Oligonucleotide Compounds and In Vitro DUX4 Knockdown Activity Test (Gymnosis Method) Table 3 shows the results of the in vitro DUX4 knockdown activity test performed in the same manner as in Example 7 for the newly synthesized modified oligonucleotide compounds and the compounds. Shown.

[0469] In addition, the compounds in Table 3 are represented by three letters for each nucleotide. However, the 3'-terminal nucleotide is represented by two letters because there is no internucleoside bond. 1) The first letter is represented by a capital letter and indicates the following nucleobases: A = adenine, T = thymine, G = guanine, C = cytosine, U = uracil, M = 5-methylcytosine, 2) The second character indicates each of the following sugar moieties: l = LNA, m = ALNA[Ms], e = 2'-MOE, o = 2'-OMe, d = 2'-deoxyribose, 3) The third character indicates the following internucleoside linkage: s = phosphorothioate, p = phosphodiester. As the target position, the 5'-target site of the DUX4 mature mRNA of the modified oligonucleotide (the position of SEQ ID NO: 1 in the sequence listing corresponding to the 3'-end of the modified oligonucleotide) is shown.

[0470] Compared with compounds (Compound Nos. 137 to 247) containing a nucleobase sequence complementary to an isologous portion within the regions of positions 126 to 147, 232 to 248, 1306 to 1325, or 1472 to 1495 of the DUX4 mature mRNA, Compound Nos. 248 (complementary to positions 112 to 127 of SEQ ID NO: 1 in the sequence listing), 249 (complementary to positions 162 to 177 of SEQ ID NO: 1 in the sequence listing), 250 (complementary to positions 264 to 279 of SEQ ID NO: 1 in the sequence listing), and 251 (complementary to positions 1273 to 1288 of SEQ ID NO: 1 in the sequence listing) were found to have significantly weaker inhibition rates.

[0471] Table 3

[0472]

Table 3-1

[0473] Table 3. Continued

[0474]

Table 3-2

[0475] Example 9 In Vivo DUX4 Knockdown Activity Test Adeno-associated virus vector AAV-DUX4 incorporating the DUX4 mature mRNA of SEQ ID NO: 1 in the Sequence Listing (SignaGen Laboratories, Cat.#SL1 An AAV-DUX4 was prepared. The anterior tibial muscle of 8-week-old C57BL / 6J mice (male, Charles River Japan) was intramuscularly injected with 1E+10 VG / 50 μL of AAV-DUX4 under isoflurane (Fizer) anesthesia. Three days later, modified oligonucleotides targeting DUX4 were prepared with physiological saline to be 1, 3, 10, and 50 mg / 5 mL / kg, and were intravenously injected into the tail veins of 8-week-old C57BL / 6J mice (male, Charles River Japan). Seventy-two hours later, whole blood was collected from the abdominal vena cava under cervical dislocation or isoflurane (Fizer) anesthesia, and the mice were sacrificed. After sacrifice, the anterior tibial muscle was collected, immersed in RNAlater Soln. (invitrogen), and then frozen at -80°C. Homogenization buffer of the Maxwell RSC simplyRNA Tissue Kit (Promega) was added to the tissue, and the tissue was disrupted using a multi-bead shocker, and RNA was purified according to the protocol described in the kit. 400 ng of RNA was reverse-transcribed, and quantitative PCR was performed using the obtained cDNA. The knockdown activity of the modified oligonucleotide was shown as the relative value of the amount ratio of DUX4 to 18S rRNA with respect to the vehicle group. The results for the cases of 1, 3, 10, and 50 mg / 5 mL / kg are shown in Figures 1 to 4.Compound 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 1309 to 1323 of DUX4 mature mRNA), Compound No. 68 (sequence complementary to positions 1309 to 1323 of DUX4 mature mRNA), Compound No. 78 (sequence complementary to positions 1309 to 1324 of DUX4 mature mRNA), Compound No. 88 (sequence complementary to positions 1310 to 1323 of DUX4 mature mRNA), Compound No. 104 (sequence complementary to positions 1473 to 1488 of DUX4 mature mRNA), Compound No. 122 (sequence complementary to positions 1480 to 1495 of DUX4 mature mRNA) could suppress the expression of the DUX4 gene in muscle even when administered to a living body.

[0476] Example 10 Safety of Modified Oligonucleotides When Compound 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 findings such as hepatotoxicity (increase in ALT and AST in blood and pathological histological abnormalities), nephrotoxicity (increase in UN and creatinine in blood and pathological histological abnormalities), general symptom changes, and death were observed.

[0477] Example 11 In Vivo Tg Mouse DUX4 Knockdown Activity Test Male 9-week-old FLExDUX4-heteto / HSA-MCM-hetero:TG (DUX4-Tg) and FLExDUX4-wild / HSA-MCM-hetero:TG (MCM, control) were used (male, introduced from The Jackson Laboratory to Charles River Japan, Inc.). Modified oligonucleotides targeting DUX4 were prepared with physiological saline so that the dosage solution was 5 mL / kg, and administered intravenously into the tail vein once a week for 4 weeks. One week after the end of administration, under deep anesthesia with isoflurane, whole blood was collected from the abdominal vena cava and the mice were euthanized. EDTA plasma was separated and used for measurement of creatine kinase (CK). The muscles of the lower limbs were collected and the wet weight was measured and used for gene expression analysis. As shown in FIGS. 5 and 6, Compound No. 3 and Compound No. 123 suppressed the mRNA expression of DUX4. In addition, they decreased the blood levels of CK, a muscle disorder marker. On the other hand, no effects on the DUX4 mRNA expression and blood CK levels were observed for Compound No. 113 and Compound No. 247.

[0478] Example 12 Mouse Repeated Dose Toxicity Test In 6-week-old ICR mice (male, Charles River Japan), modified oligonucleotides targeting DUX4 were prepared with physiological saline so that it was 100 mg / 5 mL / kg, and administered ​It was administered via the caudal vein. Blood was collected from the posterior vena cava under isoflurane anesthesia 72 hours after the final administration, and clinical biochemical tests were performed. In addition, after sacrificing by bloodletting, autopsy was performed, and histopathological examinations of the liver and kidneys were carried out. For Compound No. 123, no changes were observed in mortality, general condition, food intake, and body weight after administration, and no hepatotoxicity (increase in ALT and AST in serum, and histopathological abnormalities) or nephrotoxicity (increase in UN and creatinine in serum, and histopathological abnormalities) was observed. On the other hand, for Compound No. 113 and Compound No. 247, although no changes were observed in mortality, general condition, food intake, and body weight after administration, both showed definite hepatotoxicity (increase in ALT, AST, GLDH, ALP, bilirubin, and bile acids in serum, histopathological abnormalities: degeneration and necrosis of hepatocytes, hepatocyte hypertrophy) and nephrotoxicity (increase in creatinine in serum). Also, the concentrations of Compound No. 123 in the liver and kidneys under the test conditions were 323 and 251 μg / g, respectively, and despite having tissue concentrations equal to or higher than those of Compound No. 247 (111 and 185 μg / g in the liver and kidneys) and Compound No. 113 (39.3 and 235 μg / g in the liver and kidneys), no liver and kidney toxicity was observed.

[0479]

Table 4

[0480]

Table 5

[0481] Reference Example Schemes for the synthesis methods 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. Note that the starting compounds 1a, 1d, 1g can be synthesized by the methods described in International Publication No. 2017 / 047816.

[0482] Synthesis of ALNA[Ms]-T

[0483]

Chem.

[0484] Synthesis of ALNA[Ms]-mC

[0485]

Chem.

[0486] Synthesis of ALNA[Ms]-G

[0487]

Chem.

[0488] Synthesis of ALNA[Ms]-A

[0489]

Chem.

[0490] Synthesis of ALNA[mU]-T

[0491]

Chem.

[0492] Synthesis of ALNA[mU]-mC

[0493]

Chem.

[0494] Synthesis of ALNA[mU]-G

[0495]

Chem.

[0496] Synthesis of ALNA[mU]-A

[0497]

Chem.

[0498] Synthesis of ALNA[ipU]-T

[0499]

Chem.

[0500] Synthesis of ALNA[ipU]-mC

[0501]

Chem.

[0502] Synthesis of ALNA[ipU]-G

[0503]

Chem.

[0504] Synthesis of ALNA[ipU]-A

[0505]

Chem.

[0506] Synthesis of ALNA[Trz]-T

[0507]

Chem.

[0508] Synthesis of ALNA[Trz]-mC

[0509]

Chem.

[0510] Synthesis of ALNA[Trz]-G

[0511] [Chemical formula]

[0512] Synthesis of ALNA[Trz]-A

[0513] [Chemical formula]

[0514] Synthesis of ALNA[Oxz]-T

[0515] [Chemical formula]

[0516] Synthesis of ALNA[Oxz]-mC

[0517] [Chemical formula] [Industrial Applicability]

[0518] The modified oligonucleotide of the present invention can be used as a compound useful for the treatment, prevention, or delay of the progression of DUX4-related diseases.

[0519] [Sequence Listing Free Text] SEQ ID NO: 1 in the sequence listing shows the nucleotide sequence of DUX4 mature mRNA. SEQ ID NOS: 2 to 4, and 7 to 112 in the sequence listing show the nucleotide sequences of the modified oligonucleotides. SEQ ID NOS: 5 to 6 in the sequence listing show the nucleotide sequences of DUX4-FL2 and DUX4-s, respectively, as splicing variants of SEQ ID NO: 1.

Claims

**Claim 1** A modified oligonucleotide consisting of 12 to 30 residues, comprising at least 8 consecutive nucleobase sequences that are complementary to the corresponding length portions at positions 126 to 147, 232 to 248, 1306 to 1325, or 1480 to 1495 from the 5'-end of the nucleobase sequence of the mature mRNA of DUX4 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide has at least 90% complementarity to the corresponding length portion in the nucleobase sequence of the mature mRNA of DUX4 of SEQ ID NO: 1, and when the at least 8 consecutive nucleobase sequences include a nucleobase sequence that is complementary to the corresponding length portion at positions 1480 to 1495 from the 5'-end of the nucleobase sequence of SEQ ID NO: 1, the modified oligonucleotide consists of a nucleobase sequence having the complementary base of the base at position 1480 from the 5'-end of the nucleobases of SEQ ID NO: 1 at the 3'-end, A modified oligonucleotide. **Claim 2** The modified oligonucleotide according to claim 1, wherein one or more of the modified nucleotides of the modified oligonucleotide contain a modified sugar. **Claim 3** The modified oligonucleotide according to claim 2, wherein the modified sugar is selected from the group consisting of bicyclic sugars, sugars modified with 2'-O-methoxyethyl, and sugars modified with 2'-O-methyl. **Claim 4** The modified oligonucleotide according to claim 3, wherein the bicyclic sugar is selected from the group consisting of LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], and ALNA[Trz]. **Claim 5** A modified oligonucleotide consisting of 12 to 30 residues, comprising at least 8 consecutive nucleobase sequences that are complementary to the corresponding length portion at positions 1472 to 1495 from the 5'-end of the nucleobase sequence of the mature mRNA of DUX4 of SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide has at least 90% complementarity to the corresponding length portion in the nucleobase sequence of the mature mRNA of DUX4 of SEQ ID NO: 1, and the modified oligonucleotide contains at least one nucleoside containing a modified sugar selected from GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], and ALNA[Trz], A modified oligonucleotide. **Claim 6** The modified oligonucleotide according to claim 5, further comprising a sugar modified with 2'-O-methoxyethyl and / or a sugar modified with 2'-O-methyl. **Claim 7** The modified oligonucleotide according to any one of claims 1 to 6, wherein at least one modified nucleotide of the modified oligonucleotide contains a modified nucleobase. **Claim 8** The modified oligonucleotide according to claim 7, wherein the modified nucleobase is 5-methylcytosine. **Claim 9** The modified oligonucleotide according to any one of claims 1 to 8, wherein at least one internucleoside bond is a modified internucleoside bond. **Claim 10** The modified oligonucleotide according to claim 9, wherein the modified internucleoside bond is a phosphorothioate internucleoside bond. **Claim 11** The modified oligonucleotide, 1) a gap segment, 2) a 5' wing segment, and 3) a 3' wing segment, and the gap segment is positioned between the 5' wing segment and the 3' wing segment, any of the nucleosides of the 5' wing segment and the 3' wing segment contains at least one modified sugar, the nucleosides of the gap segment are either only nucleosides that do not contain a modified sugar, or contain 1 or 2 nucleosides that contain a modified sugar and the rest are nucleosides that do not contain a modified sugar, The modified oligonucleotide according to any one of claims 1 to 10. **Claim 12** The modified oligonucleotide, from the 5' end of the nucleobase sequence of the mature mRNA of DUX4 of SEQ ID NO: 1 the nucleobase sequence at positions 128 to 143, the nucleobase sequence at positions 232 to 247, the nucleobase sequence at positions 233 to 248, the nucleobase sequence at positions 1309 to 1323 from the 5' end, or the nucleobase sequence at positions 1480 to 1495 from the 5' end, The modified oligonucleotide according to any one of claims 1 to 11, consisting of a nucleobase sequence complementary thereto. **Claim 13** The modified oligonucleotide, 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) The modified oligonucleotide according to any one of claims 1 to 12, comprising the nucleotide sequence of.

14. The following formula: GlsMlsMlsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMlsGlsGl; The modified oligonucleotide according to claim 13, represented by. In the formula,[[]] Each nucleobase is represented by the following symbols: A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine, Is represented according to; Each sugar moiety is represented by the following symbols: l = LNA, d = 2'-deoxyribose, Is represented according to; Each internucleoside linkage is represented by the following symbol: s = phosphorothioate is represented according to.

15. The following formula: GmsMmsMmsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMmsGmsGm; The modified oligonucleotide according to claim 13, represented by. In the formula,[[]] Each nucleobase is represented by the following symbols: A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine, Is represented according to; Each sugar moiety is represented by the following symbols: m = ALNA[Ms], d = 2'-deoxyribose, Is represented according to; Each internucleoside linkage is represented by the following symbol: s = phosphorothioate is represented according to.

16. The following formula: GmsMmsAmsGdsTdsTdsCdsTdsCdsCdsGdsCdsGmsGmsTm; The modified oligonucleotide according to claim 13, represented by. In the formula,[[]] Each nucleobase is represented by the following symbols: A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine, is represented according to; Each sugar moiety is represented by the following symbols: m = ALNA[Ms], d = 2'-deoxyribose, Is represented according to; Each internucleoside linkage is represented by the following symbol: s = phosphorothioate Is represented according to.

17. The following formula: MlsGlsAlsGdsAdsTdsTdsCdsCdsCdsGdsCdsCdsGlsGlsTl; The modified oligonucleotide according to claim 13, represented by. In the formula,[[]] Each nucleobase is represented by the following symbols: A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine, Is represented according to; Each sugar moiety is represented by the following symbols: l = LNA, d = 2'-deoxyribose, Is represented according to; Each internucleoside linkage is represented by the following symbol: s = phosphorothioate It is shown according to

18. The following formula: 【Chemical 1】 The modified oligonucleotide or a salt thereof according to claim 14, represented by

19. The following formula: [Chemical 2] The modified oligonucleotide or a salt thereof according to claim 15, represented by

20. The following formula: [Chemical 3] The modified oligonucleotide or a salt thereof according to claim 16, represented by

21. The following formula: 【Chemical Formula 4】 The modified oligonucleotide or a salt thereof according to claim 17, represented by

22. A pharmaceutical composition comprising the modified oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 21, and a pharmaceutically acceptable carrier.

23. The pharmaceutical composition according to claim 22, for the treatment, prevention, or delay of the progression of DUX4-related diseases.

24. The pharmaceutical composition according to claim 23, wherein the DUX4-related disease is facioscapulohumeral muscular dystrophy.

25. A method for the treatment, prevention, or delay of the progression of DUX4-related diseases in a subject, comprising administering an effective amount of the modified oligonucleotide according to any one of claims 1 to 21 to a subject in need thereof.

26. Use of the modified oligonucleotide according to any one of claims 1 to 21 in the manufacture of a medicament for the treatment, prevention, or delay of the progression of DUX4-related diseases.

27. Use of the modified oligonucleotide according to any one of claims 1 to 21 for the treatment, prevention, or delay of the progression of DUX4-related diseases.

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