Compounds, methods and pharmaceutical compositions for modulating expression of DUX4

Abstract

The purpose of the present invention is to provide a compound, a method, and a pharmaceutical composition for normalizing dual homologous frame 4 (DUX4) in an individual in which the DUX4 gene is abnormally expressed, and to provide a modified oligonucleotide comprising 12-30 residues, comprising at least 8 contiguous nucleic acid base sequences, the modified oligonucleotide has a nucleic acid base sequence which is complementary to the isometric portion of the nucleic acid base of the mature mRNA of DUX4 of SEQ ID NO: 1 from the 5'terminal thereof at positions 126th to 147th, positions 232th to 248, positions 1306th to 1325th, or positions 1472nd to 1495th, and the nucleic acid base sequence of the modified oligonucleotide has at least 90% complementarity with the isometric portion of the nucleic acid base sequence of the mature mRNA of DUX4 of SEQ ID NO: 1.

Description

[0001] This application is a divisional application of the Chinese patent application with the application number 202080025889.2 (the original invention name was "Compounds, methods and pharmaceutical compositions for regulating the expression of DUX4", and the application date of the original application was March 27, 2020). Technical Field

[0002] The present invention relates to compounds for reducing the expression of DUX4 mRNA and protein in animals, methods using such compounds, and pharmaceutical compositions containing such compounds. The methods of the present invention are useful for treating, preventing or alleviating DUX4-related diseases, such as facioscapulohumeral muscular dystrophy (FSHD). Background Art

[0003] 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 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. In about 20% of cases, patients are wheelchair-bound before the age of 40 (even in adulthood, there may be only mild facial muscle weakness). Pain, sensorineural deafness, and retinopathy are also common complications. Approximately 90% of patients develop the disease before the age of 20. In severe patients (about 4%), muscle weakness is present from infancy.

[0004] Based on the causative gene, FSHD is classified into two types, FSHD1 and FSHD2. FSHD1 accounts for about 95% of all FSHD patients. In patients with FSHD1, the D4Z4 repeat region on 4q35 is genetically shortened (1 - 10 D4Z4 repeats), which causes abnormal expression of DUX4 encoded by the D4Z4 repeat region (in healthy individuals, DUX4 is not expressed). FSHD2 accounts for about 5% of all FSHD patients and is caused by mutations in SMCHD1 (a DNA methyltransferase), resulting in abnormal expression of DUX4. DUX4 has a function similar to a transcription factor and causes the expression of genes downstream that induce apoptosis or muscle atrophy in muscle cells. The abnormal expression of DUX4 is due to the presence of an allele called 4qA among the two alleles 4qA and 4qB. The polyadenylation site present in 4qA is essential for the stabilization of DUX4 mRNA (Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3).

[0005] Antisense technology has gradually become known as an effective means for regulating the expression of certain gene products, and thus there is a possibility of being found very useful in several therapeutic, diagnostic, and research applications for regulating DUX4.

[0006] A method of suppressing the expression of the DUX4 gene using an adeno-associated virus encoding DUX4 miRNA has been reported (Patent Document 1), but the preparation of this adeno-associated virus is complicated and it is difficult to deliver it to the whole body muscles that need it.

[0007] A method of suppressing the expression of the DUX4 gene using a lentivirus encoding DUX4 shRNA has been reported (Non-Patent Document 2), but the preparation of this lentivirus is complicated and it is difficult to deliver it to the whole body muscles that need it. Furthermore, there is a residual activity of 21% and 44% in the gene suppression effect in vitro in quadriceps or trapezius muscle cells, and the effect cannot be said to be sufficient.

[0008] A compound linked with multiple antisense oligonucleotides against DUX4 has been reported (Patent Document 2), but it is not a modified oligonucleotide and its inhibitory effect cannot be said to be sufficient.

[0009] An antisense oligonucleotide compound that binds to the splicing site of DUX4 mRNA has been reported (Patent Document 3), but since these compounds are selective for pre-mRNA containing introns, the inhibitory effect on mature mRNA is weak. In addition, it is difficult to administer them to organisms due to knockdown based on the liposome transfection method.

[0010] As the current treatment methods for FSHD, there are rehabilitation (stretching, exercise), administration of NSAIDs, respiratory care, etc. as symptomatic therapies, but the effects are not sufficient and the burden on patients is large. Therefore, the object of the present specification is to provide compounds, compositions, and methods for treating FSHD.

[0011] Prior art documents

[0012] Patent documents

[0013] Patent Document 1: International Publication No. 2013 / 016352

[0014] Patent Document 2: International Publication No. 2017 / 007886

[0015] Patent Document 3: US Publication No. 2012 / 0225034

[0016] Non-patent documents

[0017] Non-Patent Document 1: Snider et al., PLoS 2010, Vol.6(10)p1

[0018] Non-Patent Document 2: Ferreboeuf et al., Human Molecular Genetics 2013, Vol.23(1), p171

[0019] Non-Patent Document 3: Sacconi et al., Biochim. Biophys. Acta 2015, p607 Summary of the Invention

[0020] The object of the present invention is to provide compounds, methods and pharmaceutical compositions for suppressing the expression of DUX4 and for treating, i.e., treating, preventing, delaying or improving DUX4-related diseases and / or their symptoms. The compounds and pharmaceutical compositions disclosed in this specification also suppress variant DUX4 such as SNPs or splicing variants of DUX4.

[0021] Some embodiments provide a method for reducing the expression of DUX4 in an animal (including a human), which includes administering to the animal a compound containing a modified oligonucleotide targeting DUX4 or a pharmaceutical composition containing the compound as described in this specification.

[0022] Some embodiments provide a method of administering a compound or pharmaceutical composition containing a modified oligonucleotide targeting DUX4 to an animal for nuclease (such as RNase H)-mediated knockdown in the nucleus. Additionally, a method is provided of administering a compound containing the modified oligonucleotide to inhibit the transcription of DUX4 mRNA or the translation of DUX4 protein. The modified oligonucleotide is preferably distributed in muscle, particularly preferably in skeletal muscle.

[0023] Some embodiments provide a method of treating an animal with FSHD. In some embodiments, the method of the present invention further includes administering to the animal a compound or pharmaceutical composition containing a therapeutically effective amount of a modified oligonucleotide targeting DUX4 as described in this specification. In some embodiments, the method of the present invention includes identifying an animal with FSHD1 and / or FSHD2.

[0024] Some embodiments provide a method of treating, i.e., treating, preventing, delaying or improving muscle atrophy and muscle strength decline. Alleviate the lack of facial expression / eye-opening while sleeping / difficulty in raising the upper limb / winging of the scapula caused by muscle strength decline in the cheeks, shoulders and upper arms, or delay the deterioration. Additionally, it is preferable to prevent muscle strength decline in the waistband and lower limbs, and more preferably to prevent the complication of nerve deafness and retinopathy.

[0025] In certain embodiments, the DUX4 mRNA has the sequence set forth in GenBank accession number NM_001293798.2 (incorporated herein by reference as SEQ ID NO: 1 in the Sequence Listing). The splice 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 set forth in GenBank accession number NM_001306068.2 (incorporated herein by reference as SEQ ID NO: 5 in the Sequence Listing). The splice 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 set forth in GenBank accession number NM_001363820.1 (incorporated herein by reference as SEQ ID NO: 6 in the Sequence Listing). The splice 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 is referred to as the SNP of the above splice variant.

[0026] The present invention relates to the non-limiting numbered embodiments described in the following items [1] to

[27] , but is not limited thereto.

[0027] Item [1] A modified oligonucleotide consisting of 12 to 30 residues,

[0028] comprising at least 8 consecutive nucleic acid base sequences that are complementary to the nucleic acid base sequences of positions 126 to 147, 232 to 248, 1306 to 1325, or 1480 to 1495 from the 5'-end of the nucleic acid base sequence of the mature mRNA of DUX4 of SEQ ID NO: 1,

[0029] The nucleic acid base sequence of the above modified oligonucleotide has at least 90% complementarity with the nucleic acid base sequence of the mature mRNA of DUX4 of SEQ ID NO: 1.

[0030] In the case where the above at least 8 consecutive nucleic acid base sequences contain a nucleic acid base sequence complementary to the nucleic acid base sequence of positions 1480 to 1495 from the 5'-end of the nucleic acid base sequence of SEQ ID NO: 1, the above modified oligonucleotide consists of a nucleic acid base sequence having a complementary base to the base at position 1480 from the 5'-end of the nucleic acid base sequence of SEQ ID NO: 1 at the 3'-end.

[0031] Item [2] The modified oligonucleotide according to item [1], wherein one or more modified nucleotides in the above modified oligonucleotide contain a modified sugar.

[0032] Item [3] The modified oligonucleotide according to item [2], wherein the above modified sugar is selected from bicyclic sugars, sugars modified by 2'-O-methoxyethyl, and sugars modified by 2'-O-methyl.

[0033] Item [4]. The modified oligonucleotide according to item [3], wherein the bicyclic sugar is selected from LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], and ALNA[Tr z].

[0034] Item [5]. A modified oligonucleotide composed of 12 to 30 residues,

[0035] comprising at least 8 consecutive nucleic acid base sequences that are complementary to the corresponding length portion starting from the 1472nd to 1495th positions of the nucleic acid base sequence of the mature mRNA of DUX4 of SEQ ID NO: 1,

[0036] the nucleic acid base sequence of the above-mentioned modified oligonucleotide has at least 90% complementarity with the corresponding length portion of the nucleic acid base sequence of the mature mRNA of DUX4 of SEQ ID NO: 1,

[0037] the above-mentioned modified oligonucleotide contains at least 1 nucleoside comprising a modified sugar selected from GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], and ALNA[Trz].

[0038] Item [6]. The modified oligonucleotide according to item [5], wherein the above-mentioned modified oligonucleotide further comprises a sugar modified by 2'-O-methoxyethyl and / or a sugar modified by 2'-O-methyl.

[0039] Item [7]. The modified oligonucleotide according to any one of items [1] to [6], wherein at least 1 modified nucleotide in the above-mentioned modified oligonucleotide contains a modified nucleic acid base.

[0040] Item [8]. The modified oligonucleotide according to item [7], wherein the above-mentioned modified nucleic acid base is 5-methylcytosine.

[0041] Item [9]. The modified oligonucleotide according to any one of items [1] to [8], wherein at least 1 internucleoside bond is a modified internucleoside bond.

[0042] Item

[10] . The modified oligonucleotide according to item [9], wherein the above-mentioned modified internucleoside bond is a phosphorothioate internucleoside bond.

[0043] Item

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

[10] , wherein the above-mentioned modified oligonucleotide comprises:

[0044] 1) a gap segment,

[0045] 2) a 5' flanking segment, and

[0046] 3) a 3' flanking segment;

[0047] The above-mentioned spacer segment is located between the above-mentioned 5'-flanking segment and the above-mentioned 3'-flanking segment.

[0048] The nucleosides of the above-mentioned 5'-flanking segment and 3'-flanking segment each contain at least one modified sugar.

[0049] The nucleosides of the above-mentioned spacer segment are only nucleosides without modified sugars, or are nucleosides containing 1 or 2 nucleosides containing modified sugars and without modified sugars other than these.

[0050] Item

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

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

[0051] Item

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

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

[0052] Item

[14] The modified oligonucleotide according to Item

[13] is represented by the following formula:

[0053] GlsMlsMlsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMlsGlsGl.

[0054] In the formula,

[0055] Each nucleic acid base is represented by the following notations:

[0056] A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine,

[0057] Each sugar moiety is represented by the following notations:

[0058] l = LNA, d = 2'-deoxyribose,

[0059] Each internucleoside bond is represented by the following notations:

[0060] s = phosphorothioate.

[0061] Item

[15] The modified oligonucleotide according to item

[13] is represented by the following formula:

[0062] GmsMmsMmsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMmsGmsGm.

[0063] In the formula,

[0064] Each nucleic acid base is represented by the following notations:

[0065] A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine,

[0066] Each sugar moiety is represented by the following notations:

[0067] m = ALNA[Ms], d = 2'-deoxyribose,

[0068] Each internucleoside bond is represented by the following notations:

[0069] s = phosphorothioate.

[0070] Item

[16] The modified oligonucleotide according to item

[13] is represented by the following formula:

[0071] GmsMmsAmsGdsTdsTdsCdsTdsCdsCdsGdsCdsGmsGmsTm.

[0072] In the formula,

[0073] Each nucleic acid base is represented by the following notations:

[0074] A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine,

[0075] Each sugar moiety is represented by the following notations:

[0076] m = ALNA[Ms], d = 2'-deoxyribose,

[0077] Each internucleoside bond is represented by the following notations:

[0078] s = phosphorothioate.

[0079] Item

[17] The modified oligonucleotide according to item

[13] is represented by the following formula:

[0080] MlsGlsAlsGdsAdsTdsTdsCdsCdsCdsGdsCdsCdsGlsGlsTl。

[0081] Wherein,

[0082] Each nucleic acid base is represented by the following notations:

[0083] A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine;

[0084] Each sugar moiety is represented by the following notations:

[0085] l = LNA, d = 2'-deoxyribose,

[0086] Each internucleoside linkage is represented by the following notations:

[0087] s = phosphorothioate.

[0088] Item

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

[14] is represented by the following formula:

[0089]

[0090] Item

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

[15] is represented by the following formula:

[0091]

[0092] Item

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

[16] is represented by the following formula:

[0093]

[0094] Item

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

[17] is represented by the following formula:

[0095]

[0096] Item

[22] A pharmaceutical composition comprising the modified oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of items [1] to

[21] , and a pharmaceutically acceptable carrier.

[0097] Item

[23] The pharmaceutical composition according to item

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

[0098] Item

[24] The pharmaceutical composition according to item

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

[0099] Item

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

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

[0100] Item

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

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

[0101] Item

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

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

[0102] 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

[0103] Figure 1 It is a drawing showing the inhibitory effect on DUX4 gene expression brought about by administering various DUX4-modified oligonucleotides.

[0104] Figure 2 It is a drawing showing the inhibitory effect on DUX4 gene expression brought about by administering various DUX4-modified oligonucleotides.

[0105] Figure 3 It is a drawing showing the dose-dependent inhibitory effect on DUX4 gene expression brought about by administering DUX4-modified oligonucleotides.

[0106] Figure 4 It is a drawing showing the inhibitory effect on DUX4 gene expression brought about by administering various DUX4-modified oligonucleotides.

[0107] Figure 5 It is a drawing showing the inhibitory effect on creatine kinase and DUX4 mRNA in DUX4 Tg mice brought about by administering DUX4-modified oligonucleotide (compound number 3).

[0108] Figure 6 It is a drawing showing the inhibitory effect on creatine kinase and DUX4 mRNA in DUX4 Tg mice brought about by administering DUX4-modified oligonucleotides (compound number 123, compound number 247, and compound number 113). DETAILED DESCRIPTION OF THE INVENTION

[0109] Both the foregoing summary and the following detailed description are exemplary and explanatory only and are to be construed as non-limiting of the claimed invention. In this specification, unless otherwise specified, the use of the singular form includes the plural form. In this specification, unless otherwise specified, 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 specified, terms such as "element" include elements containing one unit and elements containing more than one subunit.

[0110] The section headings 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 as they relate to the portions and the whole of the documents discussed in this specification.

[0111] (Definitions)

[0112] Unless otherwise defined, the nomenclatures and their sequences and practices utilized in connection with analytical chemistry, organic synthetic chemistry, and medical and pharmaceutical chemistry as described in this specification are well known and commonly used in the art. Standard practices may be used in chemical synthesis and chemical analysis as used in this specification. Where permitted, all patents, applications, published applications, and other publications referred to throughout the disclosure of this specification, GenBank accession numbers and related sequence information, and other data available through databases such as the National Center for Biotechnology Information (NCBI), are hereby expressly incorporated by reference in their entirety and in part as they relate to the portions and the whole of the documents discussed in this specification.

[0113] In addition, this specification is filed together with a sequence listing in electronic format, and the information of the sequence listing recorded in this electronic format is hereby incorporated by reference in its entirety.

[0114] Unless otherwise indicated, the following terms have the following meanings.

[0115] "Nucleic acid" refers to a molecule composed of monomeric nucleotides. Examples of nucleic acids include ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acid (siRNA), and microRNA (miRNA), but are not limited thereto. A nucleic acid may also contain a combination of these elements in a single molecule.

[0116] "Nucleic acid base" refers to a heterocyclic moiety capable of pairing with the base of another nucleic acid. Nucleic acid bases include "modified nucleic acid bases" and "unmodified nucleic acid bases".

[0117] "Nucleic acid base sequence" refers to the sequence of consecutive nucleic acid bases that is arbitrary and has no relation to glycosidic bonds or nucleic acid base modifications.

[0118] "Nucleoside" refers to a nucleic acid base linked to a sugar. In certain embodiments, the nucleoside is linked to a phosphate group.

[0119] "Nucleotide" refers to a nucleoside having a phosphate group and the like covalently bonded to the sugar moiety of the nucleoside. The sugar moiety of naturally occurring nucleotides is ribose or deoxyribose, and is covalently bonded via a phosphate group by a phosphodiester bond.

[0120] "Oligomeric compound" or "oligomer" refers to a polymer of linked monomer subunits capable of hybridizing to at least one region of a nucleic acid molecule.

[0121] "Oligonucleotide" refers to a polymer of linked nucleosides where each nucleoside and each internucleoside bond can be independently modified or unmodified.

[0122] "Unmodified nucleotide" refers to a nucleotide composed of naturally occurring nucleic acid bases, a sugar moiety, and internucleoside bonds. In certain embodiments, the unmodified nucleotide is an RNA nucleotide (i.e., β-D-ribonucleoside) or a DNA nucleotide (i.e., β-D-deoxyribonucleoside), but is not limited thereto.

[0123] "Modified nucleotide" refers to a nucleotide independently having a modified sugar moiety, a modified internucleoside bond, or a modified nucleic acid base. "Modified nucleoside" refers to a nucleoside independently having a modified sugar moiety or a modified nucleic acid base.

[0124] "Internucleoside bond" refers to the chemical bond between nucleosides.

[0125] "Linked nucleosides" refer to adjacent nucleosides bonded or linked by an internucleoside bond.

[0126] "Naturally occurring internucleoside bond" refers to the 3'-5' phosphodiester bond.

[0127] "Modified internucleoside bond" refers to a substitution or arbitrary variation from the naturally occurring internucleoside bond (i.e., the phosphodiester internucleoside bond). For example, there is a phosphorothioate internucleoside bond, but is not limited thereto.

[0128] "Phosphorothioate internucleoside bond" refers to the internucleoside bond that modifies the phosphodiester bond by substituting one non-bridging oxygen atom with a sulfur atom. The phosphorothioate bond is an example of this modified internucleoside bond.

[0129] "Modified nucleic acid base" refers to any nucleic acid base other than adenine, cytosine, guanine, thymine, or uracil. For example, 5-methylcytosine, but not limited thereto. "Unmodified nucleic acid base" refers to adenine (A) and guanine (G) which are purine bases, and thymine (T), cytosine (C), and uracil (U) which are pyrimidine bases.

[0130] "Modified oligonucleotide" refers to an oligonucleotide containing at least 1 of such modified nucleosides and / or the internucleoside bonds.

[0131] "Salt" is a general term for compounds obtained by substituting one or more dissociable hydrogen ions contained in an acid with cations such as metal ions or ammonium ions. As salts of modified oligonucleotides, salts formed with inorganic ions (e.g., sodium ion, magnesium ion) on the sulfur (S) group of the phosphorothioate bond or the functional group (e.g., amino group) in the modified nucleic acid base (e.g., sodium salt, magnesium salt) can be cited, but not limited thereto.

[0132] "Sugar" or "sugar moiety" refers to a natural sugar moiety or a modified sugar moiety.

[0133] "Natural sugar moiety" refers to the sugar visible in DNA (2'-H) or RNA (2'-OH).

[0134] "Modified sugar" refers to a substitution or change from the natural sugar moiety. As modified sugars, for example, substituted sugar moieties and surrogate sugar moieties can be cited.

[0135] "Substituted sugar moiety" refers to a furanosyl group other than the natural sugar of RNA or DNA.

[0136] "2'-O-methoxyethyl" (the same applies to 2'-MOE and 2'-O(CH2)2-OCH3) refers to the O-methoxy-ethyl modification at the 2'-position of the furanosyl ring. The sugar modified with 2'-O-methoxyethyl is a modified sugar.

[0137] "2'-O-methoxyethyl nucleotide" refers to a nucleotide containing a sugar moiety modified with 2'-O-methoxyethyl.

[0138] "2'-O-methyl" (the same applies to 2'-OMe and 2'-OCH3) refers to the O-methyl modification at the 2'-position of the furanosyl ring. The sugar modified with 2'-O-methyl is a modified sugar.

[0139] "2'-O-methyl nucleotide" refers to a nucleotide containing a sugar moiety modified with 2'-O-methyl.

[0140] A "sugar substitute" for a "sugar substitute moiety" refers to a substitution of only the sugar unit (furanose ring). The sugar substitute can replace the naturally occurring sugar moiety of a nucleoside, and as a result, the resulting nucleoside subunits can be linked to each other and / or to other nucleosides to form an oligomeric compound capable of hybridizing with a complementary oligomeric compound. Such a structure includes: a ring containing a different number of atoms from the furanosyl group (e.g., a 4-, 6- or 7-membered ring); substitution of the oxygen of the furanosyl group with a non-oxygen atom (e.g., carbon, sulfur or nitrogen); or both a change in the number of atoms and substitution of oxygen. Such a structure may also include substitutions corresponding to those described for the substituted sugar moiety (e.g., a 6-membered carbocyclic bicyclic sugar substitute optionally containing further substituents). Sugar substitutes also include substitutions of more complex sugars (e.g., acyclic peptide nucleic acids). Examples of sugar substitutes include, but are not limited to, morpholino, cyclohexenyl and cyclohexitol.

[0141] A "bicyclic sugar" refers to a furanosyl ring modified by crosslinking of two different carbon atoms present in the same ring. Preferably, a "bicyclic sugar" refers to a modified sugar in which the 2'-position and 4'-position of the furanosyl ring are modified by crosslinking. A "bicyclic nucleic acid" refers to a nucleoside or nucleotide in which the furanosyl moiety of the nucleoside or nucleotide contains a "bicyclic sugar".

[0142] "LNA" refers to a nucleoside or nucleotide commonly known as 2',4'-locked nucleic acid, and examples thereof include the general formula:

[0143]

[0144] [wherein,

[0145] B is a nucleic acid base,

[0146] X and Y are each independently a hydrogen atom, a protecting group for a hydroxyl group, a phosphoryl group which may be substituted, a covalent bond to a phosphorus moiety or a support, etc.]

[0147] a nucleoside or nucleotide represented by (see WO98 / 39352). Typical specific examples include the following formula:

[0148]

[0149] a nucleoside or nucleotide represented by.

[0150] "GuNA" is the following formula:

[0151]

[0152] [wherein, B is a nucleic acid base, and R3, R4, R5, and R6 are each independently a hydrogen atom or a C which may be substituted with one or more substituents 1-6An alkyl group, R7 and R8 are each independently a hydrogen atom, a protecting group for a hydroxyl group, a phosphoryl group which may be substituted, a covalent bond to a phosphorus moiety or a support, etc., and R9, R 10 , R 11 are each independently a hydrogen atom, a C 1-6 alkyl group which may be substituted by one or more substituents, or a protecting group for an amino group]

[0153] represents a nucleoside or nucleotide. (For example, refer to International Publication No. 2014 / 046212 and International Publication No. 2017 / 047816).

[0154] “ALNA[mU]” is the following general formula (I):

[0155]

[0156] [In the formula,

[0157] B is a nucleic acid base;

[0158] R1, R2, R3 and R4 are each independently a hydrogen atom, or a C 1-6 alkyl group which may be substituted by one or more substituents,

[0159] R5 and R6 are each independently a hydrogen atom, a protecting group for a hydroxyl group, or a phosphoryl group which may be substituted, a covalent bond to a phosphorus moiety or a support, etc.,

[0160] m is 1 or 2;

[0161] X is the following formula (II-1):

[0162]

[0163] represents a group,

[0164] notations described in formula (II-1):

[0165]

[0166] represents the bonding point to the 2'-amino group,

[0167] one of R7 and R8 is a hydrogen atom, and the other is a methyl group which may be substituted by one or more substituents]

[0168] represents a nucleoside or nucleotide (for example, refer to 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.

[0169] “ALNA[ipU]” is a nucleoside or nucleotide represented by the general formula (I) defined in the above-mentioned “ALNA[mU]”. In this formula,

[0170] X is the following formula (II-1):

[0171]

[0172] The group represented by,

[0173] One of R7 and R8 is a hydrogen atom, and the other is an isopropyl group that may be substituted with one or more substituents (for example, refer to Japanese Patent Application No. 2018-212424). A typical specific example is a nucleoside or nucleotide in which one of R7 and R8 is a hydrogen atom and the other is an unsubstituted isopropyl group.

[0174] “ALNA[Trz]” is a nucleoside or nucleotide represented by the general formula (I) defined in the above-mentioned “ALNA[mU]”. In this formula,

[0175] X is the following formula (II-2):

[0176]

[0177] The group represented by,

[0178] A is a triazolyl group that may be substituted with one or more substituents (for example, refer to Japanese Patent Application No. 2018-212424). A typical specific example is a nucleoside or nucleotide in which A is a triazolyl group that may have one or more methyl groups, and more specifically, 1,5-dimethyl-1,2,4-triazol-3-yl.

[0179] “ALNA[Oxz]” is a nucleoside or nucleotide represented by the general formula (I) defined in the above-mentioned “ALNA[mU]”. In this formula,

[0180] X is the following formula (II-2):

[0181]

[0182] The group represented by,

[0183] A is a diazolyl group that may be substituted with one or more substituents (for example, refer to Japanese Patent Application No. 2018-212424). A typical specific example is a diazolyl group that may have one or more methyl groups, and more specifically, 5-methyl-1,2,4- diazol-3-yl nucleoside or nucleotide.

[0184] “ALNA[Ms]” is a nucleoside or nucleotide represented by the general formula (I) defined in the above-mentioned “ALNA[mU]”. In this formula,

[0185] X is the following formula (II-3):

[0186]

[0187] a group represented by,

[0188] M is a sulfonyl group that can be substituted by a methyl group, and the methyl group can be substituted by one or more substituents (for example, refer to Japanese Patent Application No. 2018-212424). A typical specific example is a nucleoside or nucleotide in which M is a sulfonyl group substituted by an unsubstituted methyl group.

[0189] “5-Methylcytosine” refers to cytosine modified by a methyl group bonded to the 5-position. 5-Methylcytosine is a modified nucleic acid base.

[0190] “Single-stranded oligonucleotide” refers to an oligonucleotide that is not hybridized to a complementary strand.

[0191] “DUX4” refers to a nucleic acid or protein of a transcription factor also known as Double homeobox4. Examples of DUX4 include various splice variants transcribed from the DUX4 gene, or single-base substitutions (SNPs) thereof, and may also be such variants and / or SNPs.

[0192] For DUX4 mRNA, a large number of splice variants 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 according 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 has DUX4-FL1 (SEQ ID NO: 1 in the sequence listing) as a mature mRNA without intron 1 and DUX4-FL2 (SEQ ID NO: 5 in the sequence listing) containing intron 1, both of which encode a full-length DUX4 protein and are considered to be the cause of FS HD when expressed in muscle (refer to the above-mentioned Non-Patent Document 2).

[0193] DUX4 mRNA is also expressed in normal testes. In addition to DUX4-FL, splice variants of exon 1, exon 2, exon 6, exon 7 and / or splice variants of exon 1, exon 2, exon 4, exon 5, exon 6, exon 7 are also expressed (refer to the above-mentioned Non-Patent Document 1).

[0194] The DUX4 protein functions as a transcription factor. Genes regulated by DUX4, such as MBD3L2, ZSCAN4, TRIM43, DEFB103, ZNF217, etc., can be cited (see Non-Patent Document 2 above).

[0195] The target DUX4 mRNA of the modified oligonucleotide of the present invention is preferably human DUX4, more preferably DUX4-FL, and further preferably DUX4-FL1 described in SEQ ID NO: 1 of the Sequence Listing. In addition, as the target site of the modified oligonucleotide of the present invention for DUX4, exon 1, intron 1, exon 2, intron 2, and exon 3 are preferred.

[0196] It is known that the DUX4 gene fuses with other genes due to chromosomal abnormalities such as translocation and is expressed. As other genes, for example, 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 they are respectively considered to be the causative genes of B-cell acute lymphoblastic leukemia, differentiated round cell sarcoma, embryonal rhabdomyosarcoma, etc. The modified oligonucleotide of the present invention also includes compounds targeting their fusion genes.

[0197] "Expression of DUX4" refers to the level of mRNA transcribed from the gene encoding DUX4 or the level of protein translated from the mRNA. The expression of DUX4 can be determined by methods known in the art such as Northern blotting, Western blotting, PCR, etc.

[0198] "DUX4 nucleic acid" refers to any nucleic acid encoding DUX4. For example, in a certain embodiment, the DUX4 nucleic acid includes a DNA sequence encoding DUX4, an RNA sequence transcribed from the DNA encoding DUX4 (including genomic DNA containing introns and exons), and a precursor mRNA or spliced mature mRNA encoding DUX4. In addition, in a certain embodiment, it includes a DNA sequence and an RNA sequence of a gene generated by the fusion of the DUX4 gene with other genes.

[0199] "DUX4 mRNA" refers to the mRNA encoding the DUX4 protein.

[0200] "Continuous nucleic acid bases", "consecutive nucleic acid bases" refer to nucleic acid bases that are directly adjacent to each other.

[0201] "Complementary" refers to the ability of nucleic acid bases of a first nucleic acid to pair with those of a second nucleic acid.

[0202] "Fully complementary (also referred to as complementarity)" or "100% complementary (also referred to as complementarity)" means that each nucleic acid base of the nucleic acid base sequence of the first nucleic acid has a complementary nucleic acid base in the second nucleic acid base 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.

[0203] "Hybridization" refers to the annealing of complementary nucleic acid molecules. In certain embodiments, examples of complementary nucleic acid molecules include modified oligonucleotides and target nucleic acids.

[0204] "Specifically hybridizable" refers to a modified oligonucleotide having a degree of complementarity sufficient to induce a desired effect between the modified oligonucleotide and the target nucleic acid, while specific binding exhibits minimal or no effect on non-target nucleic acids under the desired conditions, i.e., physiological conditions in in vivo assays and therapeutic treatments.

[0205] "Mismatch" or "non-complementary nucleic acid base" refers to the situation where the nucleic acid base of the first nucleic acid cannot pair with the corresponding nucleic acid base of the second nucleic acid or the target nucleic acid.

[0206] "Targeting" or "target" refers to the process of designing and selecting a modified oligonucleotide that specifically hybridizes to a target nucleic acid and induces a desired effect.

[0207] "Target nucleic acid", "target RNA", and "target RNA transcript" all refer to a nucleic acid to which a modified oligonucleotide can be targeted. In certain embodiments, the target nucleic acid includes a region of DUX4 nucleic acid.

[0208] "Target fragment" refers to the nucleotide sequence of the target nucleic acid to which a modified oligonucleotide is targeted. The "5' target site" refers to the most 5'-side nucleotide of the target fragment. The "3' target site" refers to the most 3'-side nucleotide of the target fragment.

[0209] "Active target region" or "target region" refers to a region to which one or more active modified oligonucleotides are targeted. An "active modified oligonucleotide" refers to a modified oligonucleotide that reduces the level of target nucleic acid or protein.

[0210] "Antisense inhibition" refers to a reduction in the level of target nucleic acid or target protein in the presence of a modified oligonucleotide complementary to the target nucleic acid compared to the level of target nucleic acid or target protein in the absence of the modified oligonucleotide.

[0211] "siRNA" refers to a double-stranded RNA oligonucleotide having a nucleic acid base sequence capable of hybridizing to a corresponding region or fragment of a target nucleic acid.

[0212] "shRNA" refers to a hairpin-shaped single-stranded RNA oligonucleotide having a nucleic acid base sequence capable of hybridizing to a corresponding region or fragment of a target nucleic acid.

[0213] "snoRNA" refers to a non-coding RNA present in the nucleolus, having a nucleic acid base sequence capable of hybridizing to a corresponding region or fragment of a target RNA nucleic acid, and being a single-stranded oligonucleotide that guides chemical modifications such as methylation or pseudouridylation of the target RNA nucleic acid.

[0214] "miRNA" refers to a non-coding RNA that regulates the expression of other genes, and is a single-stranded or double-stranded RNA oligonucleotide having a nucleic acid base sequence capable of hybridizing to a corresponding region or fragment of a target nucleic acid.

[0215] "Cap structure" or "terminal cap portion" refers to a chemical modification incorporated into either end of a modified oligonucleotide.

[0216] "Chemically heterogeneous region" refers to a region within a modified oligonucleotide that is chemically different at some point from other regions within the same modified oligonucleotide. For example, a region having 2'-O-methoxyethyl nucleotides is chemically different from a region having nucleotides that are not modified with 2'-O-methoxyethyl.

[0217] "Chimeric modified oligonucleotide" refers to a modified oligonucleotide having at least two chemically heterogeneous regions.

[0218] "Motif" refers to the pattern of chemically heterogeneous regions within a modified oligonucleotide.

[0219] "Gapmer" refers to a modified oligonucleotide in which an internal region having multiple nucleosides that support cleavage by RNase H is located between outer regions having one or more nucleosides, and the nucleosides constituting the internal region are chemically different from the nucleosides (which may be multiple) constituting the outer regions. The internal region may be referred to as the "gap segment," and the outer regions may be referred to as the "wing segments." The wing segment located at a position more 5' than the gap segment may be referred to as the "5' wing segment," and the wing segment located at a position more 3' than the gap segment may be referred to as the "3' wing segment."

[0220] "Directly adjacent" means that there is no intervening element between directly adjacent elements.

[0221] "Nuclear ribonuclease" refers to ribonuclease visible in the nucleus. As nuclear ribonucleases, RNase H including RNase H1 and RNase H2, double-stranded RNase Drosha, and other double-stranded RNases can be mentioned, but are not limited to these.

[0222] In certain embodiments, a gapmer is a modified oligonucleotide having a 5' flanking segment, a 3' flanking segment, and a gap segment; the number of nucleotides in the 5' flanking segment and the 3' flanking segment is each from 1 to 8; the gap segment is located between the two flanking segments and is directly adjacent to them, the number of nucleotides is 6 or more, and the gap segment consists of only nucleotides without modified sugars, or contains 1 or 2 nucleotides with modified sugars and otherwise consists of nucleotides without modified sugars.

[0223] "Agent" refers to an active substance that can confer a therapeutic benefit when administered to an animal. "First agent" refers to the therapeutic compound of the present invention. For example, the first agent may also be a modified oligonucleotide targeting DUX4. "Second agent" refers to the second therapeutic compound of the present invention (e.g., a second modified oligonucleotide targeting DUX4) and / or a therapeutic compound not targeting DUX4.

[0224] "Pharmaceutically acceptable salt" refers to 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 an undesired toxic effect thereto.

[0225] "Diluent" refers to a component in a composition that, although lacking pharmacological activity, is necessary or desirable in medicine. For example, the diluent in an injected composition can be a liquid, such as physiological saline.

[0226] "DUX4-related disease" refers to a disease 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. Facioscapulohumeral muscular dystrophy, B-cell acute lymphoblastic leukemia, differentiated round cell sarcoma, embryonal rhabdomyosarcoma, etc. can be mentioned, but are not limited to these.

[0227] "Facioscapulohumeral muscular dystrophy" or "FSHD" refers to muscular dystrophy that develops due to decreased muscle strength in the facial, scapular, and humeral muscles. In humans, it is thought to be mainly associated with the shortening of genomic repeat sequences (D4Z4) near the telomere (the end of the chromosome) of chromosome 4. As a result, the genomic structure changes, and the DUX4 gene, which is not normally expressed in muscle (precursor) cells, is ectopically expressed, leading to cell death (FSHD1). Additionally, in some FSHD patients, gene mutations are found in SMCHD1, one of the genomic structure control factors that suppress gene expression (FSHD2).

[0228] 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 humeral muscles. However, it can also develop in the pelvis, waist, and lower limbs. The symptoms of FSHD usually appear between the ages of 10 and 26, but it is not rare for them to develop much later. In some cases, there may be no symptoms at all. The typical symptoms are mild, and the rate of deterioration is very slow. Common symptoms include facial muscle weakness, which may cause drooping eyelids, inability to whistle, reduced facial expression changes, sad or angry facial expressions, difficulty in speech pronunciation, scapular muscle weakness (which causes deformities such as winged scapula and sloping shoulders), lower limb weakness, concerns about hearing loss and heart disease, etc.

[0229] "Active pharmaceutical agent" refers to the substance(s) in a pharmaceutical composition that provide a therapeutic benefit when administered to an animal. For example, in certain embodiments, a modified oligonucleotide targeting DUX4 is an active pharmaceutical agent.

[0230] "Co - administration" refers to the co - administration of two agents in any pattern in which the pharmacological effects caused by the two agents are manifested simultaneously in a patient. Co - administration does not require the two agents to be in a single pharmaceutical composition, in the same dosage form, or administered via the same route of administration. The pharmacological effects caused by the two agents do not need to be manifested simultaneously. The pharmacological effects only need to be repeated within a certain period and do not need to have the same breadth.

[0231] "Administration" refers to the administration of an agent to an animal, and examples include administration by a medical professional and self - administration, but are not limited to these.

[0232] "Improve" means to reduce at least one indicator, symptom, or sign 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.

[0233] "Animal" refers to humans, or non-human animals including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates including monkeys and chimpanzees.

[0234] "Co-administration" refers to administering 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. The two or more agents may be administered separately via the same or different administration routes. Co-administration includes concurrent or sequential administration.

[0235] "Dosage" refers to a specific quantity of a pharmaceutical agent administered in a single dose or over a specific period. In certain embodiments, the dosage may be administered by bolus, tablet, or injection one, two, or more times. For example, in certain embodiments where subcutaneous administration is desired, the desired dosage may be a volume that is not easily accommodated in a single injection, and thus, multiple injections may be used to achieve the desired dosage. In certain embodiments, the pharmaceutical agent is administered by infusion over a long or continuous period. The dosage may be expressed as the amount of the pharmaceutical agent per hour, day, week, or month.

[0236] "Effective amount" or "therapeutically effective amount" refers to the amount of an active pharmaceutical agent sufficient to achieve a desired physiological result in an individual in need of the agent. The effective amount may vary between individuals depending on the health and physical condition of the individual being treated, the classification group of the individual being treated, the formulation of the composition, the assessment of the medical condition of the individual, and other relevant factors.

[0237] "Animal determined to have facioscapulohumeral muscular dystrophy (FSHD)" refers to an animal diagnosed with the disorder or condition of FSHD or an animal determined to be predisposed to the disorder or condition of FSHD. For example, an individual with a family history may be predisposed to the disorder or condition of FSHD.

[0238] Such determination can be achieved by any method including reviewing the medical records of the individual and standard clinical examinations or evaluations. It should be noted that although FSHD is known to have FSHD1 and FSHD2 based on the pathogenesis, FSHD includes both.

[0239] "Individual" refers to a human or non-human animal selected for treatment or therapy.

[0240] "Myotonia" refers to an abnormally slow relaxation of muscle after voluntary contraction or electrical stimulation.

[0241] "Non-oral administration" refers to administration by injection or infusion. Examples of non-oral administration include subcutaneous administration, intravenous administration, intramuscular administration, intra-arterial administration, intraperitoneal administration, or intracranial administration, such as intramedullary or intraventricular administration. The administration may be continuous or long-term, or short-term or intermittent.

[0242] "Pharmaceutical composition" refers to a mixture of substances suitable for administration to an individual. For example, a pharmaceutical composition may comprise one or more active agents and a sterile aqueous solution.

[0243] "Prevention" means delaying or preventing the onset or occurrence of a disease, disorder or undesirable health condition, or one or more symptoms associated with the disease, disorder or undesirable health condition, for a period ranging from a few minutes to an indefinite period. Prevention also refers to reducing the risk of developing a disease, disorder or undesirable health condition.

[0244] "Treatment" 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 relieving or eradicating one or more causes of the disease, disorder or undesirable health condition itself.

[0245] "Disposal" means including the above prevention or treatment. For example, it also includes administering the pharmaceutical composition of the present invention in order to change or improve the disease, disorder or undesirable health condition.

[0246] "Prodrug" refers to a therapeutic agent prepared in an inactive form, which is converted into an active form in vivo or within its cells by the action of endogenous enzymes or other chemical substances or conditions.

[0247] "Side effect" refers to a physiological reaction that may be caused by disposal other than the desired effect. In certain embodiments, examples of 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 transferase (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. In addition, an increase in urinary protein, an increase in creatinine and blood urea nitrogen (UN) may indicate nephrotoxicity or renal function abnormalities.

[0248] "Subcutaneous administration" means administration just under the skin.

[0249] "Therapeutically effective amount" means a dose of a drug that confers a therapeutic benefit to an individual.

[0250] (Specific embodiments)

[0251] Embodiment

[0252] Certain specific embodiments shown below will provide compounds for inhibiting DUX4 expression, methods using the compounds and pharmaceutical compositions containing the compounds, but are not limited to these.

[0253] Certain embodiments will provide a method for reducing DUX4 expression in an animal, comprising administering to the animal a compound comprising a modified oligonucleotide targeting DUX4.

[0254] Certain embodiments will provide a method for impeding the accumulation of the pathogenic DUX4 transcription factor by inhibiting the transcription of the DUX4 gene, or inhibiting the translation of DUX4 mRNA, or directing the cleavage of DUX4 mRNA, by administering a modified oligonucleotide.

[0255] Certain embodiments provide a method of treating an animal having facioscapulohumeral muscular dystrophy, comprising the steps of: a) identifying said animal having facioscapulohumeral muscular dystrophy, and b) administering to said animal a therapeutically effective amount of a compound comprising a modified oligonucleotide targeting DUX4.

[0256] Certain embodiments provide a method of alleviating myotonia in a subject in need thereof. The method comprises administering to the subject a modified oligonucleotide complementary to DUX4 mRNA, which, when bound to DUX4 mRNA, activates a ribonuclease or an endoribonuclease, thereby alleviating myotonia. In certain embodiments, the subject has or is suspected of having facioscapulohumeral muscular dystrophy, or has high expression of DUX4 mRNA or a reduced number of repeats of D4Z4 on human chromosome 4, or has or is suspected of having a SMCHD1 (DNA methylase) variant.

[0257] In certain embodiments, the modified oligonucleotide used in the method of the present invention is a chimera. In certain embodiments, the modified oligonucleotide of the method used in the present invention is a gapmer.

[0258] In certain embodiments of the method of the present invention described in this specification, the administration is subcutaneous administration. In certain embodiments, the administration is intravenous or intramuscular administration.

[0259] In certain embodiments, the modified oligonucleotide used in the method of the present invention targets the DUX4 protein-coding region, intron, 5'UTR or 3'UTR of DUX4 mRNA. In certain embodiments, the modified oligonucleotide used in the method of the present invention targets exon 1, exon 2, exon 3, intron 1, intron 2 of DUX4 mRNA.

[0260] In certain embodiments of the method of the present invention described in this specification, DUX4 mRNA is cleaved by the endoribonuclease RNase H1.

[0261] In certain embodiments of the method of the present invention, DUX4 mRNA is reduced in muscle tissue. In certain embodiments, DUX4-FL1 (SEQ ID NO: 1 in the Sequence Listing), DUX4-FL2 (SEQ ID NO: 5 in the Sequence Listing), which are splicing variants, are preferentially reduced.

[0262] In certain embodiments, DUX4 mRNA has the sequence described in GenBank accession number NM_001293798.2 (incorporated into the present specification 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 into the present specification 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 into the present specification 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.

[0263] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide composed of 12 to 30 residues and has a nucleic acid base sequence containing the modified oligonucleotide, and the modified oligonucleotide is a nucleic acid base sequence containing at least 8 consecutive nucleic acid base sequences complementary to the isologous portions at positions 126 - 147, 232 - 248, 1306 - 1325, or 1472 - 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. In certain embodiments, the modified oligonucleotide is a modified oligonucleotide composed of 12 to 30 residues and has a nucleic acid base sequence containing the modified oligonucleotide, and the modified oligonucleotide is a nucleic acid base sequence containing at least 9, 10, 11, or 12 consecutive nucleic acid base sequences complementary to the isologous portions at positions 126 - 147, 232 - 248, 1306 - 1325, or 1472 - 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.

[0264] The modified oligonucleotide may consist of at least 8 consecutive nucleic acid base sequences complementary to the isologous portions at positions 126 - 147, 232 - 248, 1306 - 1325, or 1472 - 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. In addition to this nucleic acid base sequence, additional sequences may also be present on the 5'-end side and / or 3'-end side.

[0265] 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, or 22 consecutive nucleic acid base sequences complementary to an equi-length portion 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.

[0266] In certain embodiments, the modified oligonucleotide is a nucleic acid base sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 consecutive nucleic acid base sequences complementary to an equi-length portion 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.

[0267] 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, or 20 consecutive nucleic acid base sequences complementary to an equi-length portion 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.

[0268] 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 equi-length portion 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.

[0269] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues, comprising a nucleic acid base sequence complementary to the 126th to 147th positions of the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementarity to SEQ ID NO: 1 in the Sequence Listing in the equal-length portion. Additionally, in certain embodiments, it is a 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 as described above.

[0270] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues, and contains a nucleic acid base sequence complementary to the 232nd to 248th positions of the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and in the equal-length portion, it 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. Additionally, in certain embodiments, it is a 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 as described above.

[0271] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues, and contains a nucleic acid base sequence complementary to the 1306th to 1325th residues 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 in the equivalent-length portion, 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. Additionally, in certain embodiments, it is a 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 as described above.

[0272] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide composed of 12 to 30 residues, and has a nucleic acid base sequence that includes a nucleic acid base sequence complementary to an equi-length portion of 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 at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to SEQ ID NO: 1 in the equi-length portion. In addition, in certain embodiments, it is a modified oligonucleotide composed 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 of the above-mentioned modified oligonucleotide.

[0273] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide composed of 12 to 30 residues, and has a nucleic acid base sequence that includes a nucleic acid base sequence of at least 8 consecutive nucleic acid base sequences complementary to an equi-length portion of positions 128 to 143, 233 to 248, 1309 to 1323 or 1480 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. In certain embodiments, the modified oligonucleotide is a modified oligonucleotide composed of 12 to 30 residues, and has a nucleic acid base sequence that includes a nucleic acid base sequence of at least 9, 10, 11, 12 consecutive nucleic acid base sequences complementary to an equi-length portion of positions 128 to 143, 233 to 248, 1309 to 1323 or 1480 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.

[0274] In certain embodiments, the modified oligonucleotide is a nucleic acid base sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive nucleic acid base sequences complementary to an equal-length portion of the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, starting from positions 128 to 143 from the 5'-end, and is a modified oligonucleotide within 30 residues.

[0275] In certain embodiments, the modified oligonucleotide is a nucleic acid base sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive nucleic acid base sequences complementary to an equal-length portion of the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, starting from positions 233 to 248 from the 5'-end, and is a modified oligonucleotide within 30 residues.

[0276] In certain embodiments, the modified oligonucleotide is a nucleic acid base sequence comprising at least 8, 9, 10, 11, 12, 13, 14, or 15 consecutive nucleic acid base sequences complementary to an equal-length portion of the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, starting from positions 1309 to 1323 from the 5'-end, and is a modified oligonucleotide within 30 residues.

[0277] In certain embodiments, the modified oligonucleotide is at least 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive nucleic acid base sequences complementary to an equal-length portion of the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, starting from positions 1480 to 1495 from the 5'-end, and is a modified oligonucleotide within 30 residues having a nucleic acid base sequence with the complementary base of the base at position 1480 from the 5'-end of the nucleic acid bases of SEQ ID NO: 1 at the 3'-end.

[0278] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues, and contains a nucleic acid base sequence complementary to the 128th to 143rd positions of the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and in the equal-length portion, 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. Additionally, in certain embodiments, it is a 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 as described above.

[0279] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues, and contains a nucleic acid base sequence complementary to the 233rd to 248th positions of the nucleic acid bases 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 Sequence Listing in the equivalent length portion. In addition, in certain embodiments, it is a 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 as described above.

[0280] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues, which contains a nucleic acid base sequence complementary to the 1309th to 1323rd positions of the nucleic acid bases 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 Sequence Listing in the equivalent-length portion. Additionally, in certain embodiments, it is a 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 as described above.

[0281] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide composed of 12 to 30 residues, and is a nucleic acid base sequence complementary to the 1480th to 1495th positions of the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing, and is composed of a nucleic acid base sequence having a complementary base to the 1480th base from the 5'-end of the nucleic acid bases of SEQ ID NO: 1 at the 3'-end, and is a nucleic acid base sequence 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. In addition, in certain embodiments, it is the above-mentioned modified oligonucleotide composed 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.

[0282] In certain embodiments, the modified oligonucleotides provided in this specification target any one of the following regions in SEQ ID NO: 1 of 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 in this specification target any one of the following regions in SEQ ID NO: 1 of the sequence listing: positions 128 - 143, 232 - 247, 233 - 248, 1309 - 1323, and 1480 - 1495, counting from the 5'-end.

[0283] In certain embodiments, the modified oligonucleotides provided in this specification have a nucleic acid base sequence that includes a complementary region containing at least 8 consecutive nucleic acid bases complementary to a target region. In certain embodiments, the modified oligonucleotides provided in this specification have a nucleic acid base sequence that includes a complementary region containing at least 8 consecutive nucleic acid bases complementary to a target region, and the target region is targeted 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 of SEQ ID NO: 1 in the sequence listing. In certain embodiments, the modified oligonucleotides provided in this specification have a nucleic acid base sequence that includes a complementary region containing at least 8 consecutive nucleic acid bases complementary to a target region, and the target region is targeted at positions 128-143, 232-247, 233-248, 1309-1323 or 1480-1495 of SEQ ID NO: 1 in the sequence listing.

[0284] In certain embodiments, the modified oligonucleotides provided in this specification have a nucleic acid base sequence that includes a complementary region containing at least 10 consecutive nucleic acid bases complementary to a target region. In certain embodiments, the modified oligonucleotides provided in this specification have a nucleic acid base sequence that includes a complementary region containing at least 10 consecutive nucleic acid bases complementary to a target region, and the target region is targeted 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 counted from the 5'-end of SEQ ID NO: 1 in the Sequence Listing. In certain embodiments, the modified oligonucleotides provided in this specification have a nucleic acid base sequence that includes a complementary region containing at least 10 consecutive nucleic acid bases complementary to a target region, and the target region is targeted at 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.

[0285] In certain embodiments, the modified oligonucleotides provided in this specification have a nucleic acid base sequence that includes a complementary region containing at least 12 consecutive nucleic acid bases complementary to a target region. In certain embodiments, the modified oligonucleotides provided in this specification have a nucleic acid base sequence that includes a complementary region containing at least 12 consecutive nucleic acid bases complementary to a target region, and the target region is targeted 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 of SEQ ID NO: 1 in the Sequence Listing. In certain embodiments, the modified oligonucleotides provided in this specification have a nucleic acid base sequence that includes a complementary region containing at least 12 consecutive nucleic acid bases complementary to a target region, and the target region is targeted at positions 128-143, 232-247, 233-248, 1309-1323, or 1480-1495 of SEQ ID NO: 1 in the Sequence Listing.

[0286] In certain embodiments, the modified oligonucleotides provided in this specification have a nucleic acid base sequence that includes a complementary region containing at least 14 consecutive nucleic acid bases complementary to a target region. In certain embodiments, the modified oligonucleotides provided in this specification have a nucleic acid base sequence that includes a complementary region containing at least 14 consecutive nucleic acid bases complementary to a target region, and the target region is the target 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 counted from the 5'-end of SEQ ID NO: 1 in the sequence listing. In certain embodiments, the modified oligonucleotides provided in this specification have a nucleic acid base sequence that includes a complementary region containing at least 14 consecutive nucleic acid bases complementary to a target region, and the target region is the target at 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.

[0287] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide comprising a nucleic acid base sequence that is isocomplementary to the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing 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. Additionally, in certain embodiments, the modified oligonucleotide is a modified oligonucleotide comprising a nucleic acid base sequence that is isocomplementary to the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing at positions 128-143, 232-247, 233-248, 1309-1323, 1480-1495 from the 5'-end.

[0288] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide comprising the nucleic acid base sequence recited in any one of SEQ ID NOs: 2-4, 7-64, 69-97, 102-112 in the Sequence Listing. Additionally, in certain embodiments, the modified oligonucleotide is a modified oligonucleotide comprising the nucleic acid base sequence recited in any one of SEQ ID NOs: 2-4, 75, 78 in the Sequence Listing.

[0289] In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of the nucleic acid base sequence set forth in any one of SEQ ID NOs: 2 to 4, 7 to 64, 69 to 97, 102 to 112 in the Sequence Listing. In addition, in certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of the nucleic acid base sequence set forth in any one of SEQ ID NOs: 2 to 4, 75, 78 in the Sequence Listing.

[0290] In certain embodiments, the modified oligonucleotide has a nucleic acid base sequence comprising at least 8 consecutive nucleic acid bases of the nucleic acid base sequence set forth in any one of SEQ ID NOs: 2, 3, 4, 75, or 78 in the Sequence Listing.

[0291] In certain embodiments, the modified oligonucleotide has a nucleic acid base sequence comprising at least 10 consecutive nucleic acid bases of the nucleic acid base sequence set forth in any one of SEQ ID NOs: 2, 3, 4, 75, or 78 in the Sequence Listing.

[0292] In certain embodiments, the modified oligonucleotide has a nucleic acid base sequence comprising at least 12 consecutive nucleic acid bases of the nucleic acid base sequence set forth in any one of SEQ ID NOs: 2, 3, 4, 75, or 78 in the Sequence Listing.

[0293] In certain embodiments, the modified oligonucleotide has a nucleic acid base sequence comprising at least 14 consecutive nucleic acid bases of the nucleic acid base sequence set forth in any one of SEQ ID NOs: 2, 3, 4, 75, or 78 in the Sequence Listing.

[0294] In certain embodiments, the modified oligonucleotide has a nucleic acid base sequence comprising nucleic acid bases consisting of the nucleic acid base sequence set forth in any one of SEQ ID NOs: 2, 3, 4, 75, or 78 in the Sequence Listing.

[0295] In certain embodiments, the modified oligonucleotide has a nucleic acid base sequence consisting of the nucleic acid base sequence set forth in any one of SEQ ID NOs: 2, 3, 4, 75, or 78 in the Sequence Listing.

[0296] In certain embodiments, the animal is a human.

[0297] In certain embodiments, administration includes non-oral administration.

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

[0299] In certain embodiments, when determining the entire modified oligonucleotide, the nucleic acid base sequence of the modified oligonucleotide is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to the co - length portion of any one of the regions of SEQ ID NO: 1 in the sequence listing. In certain embodiments, when determining the entire modified oligonucleotide, the nucleic acid base sequence of the modified oligonucleotide is 100% complementary to the co - length portion of any one of the regions of SEQ ID NO: 1 in the sequence listing.

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

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

[0302] In certain embodiments, the sugar moiety of the modified oligonucleotide contains at least one modified sugar that is a 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].

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

[0304] In certain embodiments, the modified oligonucleotide is a gapmer and comprises the following fragments: a) a gap fragment composed of linked deoxynucleosides, b) a 5' flanking fragment composed of linked nucleosides, and c) a 3' flanking fragment composed of linked nucleosides. The gap fragment is located between the 5' flanking fragment and the 3' flanking fragment, and each nucleoside of each flanking fragment contains a modified sugar such as a 2'-O - methyl modified sugar, a 2'-O - methoxyethyl modified sugar or a bicyclic sugar.

[0305] In certain embodiments, the modified oligonucleotide is a gapmer and comprises the following segments: a) a gap segment comprising nucleosides that contain a modified sugar such as a 2'-O-methyl modified sugar or a 2'-O-methoxyethyl modified sugar, with 1 or 2 such modified sugars, and otherwise nucleosides that do not contain modified sugars; b) a 5' flanking segment comprising linked nucleosides; and c) a 3' flanking segment comprising linked nucleosides. The gap segment is located between the 5' flanking segment and the 3' flanking segment, and each nucleoside of each flanking segment contains a modified sugar such as a 2'-O-methyl modified sugar, a 2'-O-methoxyethyl sugar, or a bicyclic sugar.

[0306] In certain embodiments, the modified oligonucleotide is a gapmer and comprises the following segments: a) a gap segment having 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleosides, and the above nucleosides are only nucleosides that do not contain modified sugars, or a gap segment that contains 1 or 2 nucleosides that contain a modified sugar such as a 2'-O-methyl modified sugar or a 2'-O-methoxyethyl modified sugar and otherwise nucleosides that do not contain modified sugars; b) a 5' flanking segment having 2, 3, 4, 5, 6, or 7 nucleosides; and c) a 3' flanking segment having 2, 3, 4, 5, 6, 7, or 8 nucleosides.

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

[0308] 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.

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

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

[0311] In certain embodiments, the modified oligonucleotide is a gapmer, which is a modified oligonucleotide comprising 12 to 30 residues and having a nucleic acid base sequence comprising the modified oligonucleotide, and the modified oligonucleotide is a nucleic acid base sequence comprising at least 8 consecutive nucleic acid base sequences that are complementary to an equal-length portion of the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the sequence listing, positions 126 to 147, 232 to 248, 1306 to 1325, or 1472 to 1495 from the 5'-end. In certain embodiments, the modified oligonucleotide is a modified oligonucleotide consisting of 12 to 30 residues and having a nucleic acid base sequence comprising the modified oligonucleotide, and the modified oligonucleotide is a nucleic acid base sequence comprising at least 9, 10, 11, or 12 consecutive nucleic acid base sequences that are complementary to an equal-length portion of the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the sequence listing, positions 126 to 147, 232 to 248, 1306 to 1325, or 1472 to 1495 from the 5'-end.

[0312] In certain embodiments, the modified oligonucleotide is a gapmer, which is a nucleic acid base sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleic acid base sequences that are complementary to an equal-length portion of the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the sequence listing, positions 126 to 147 from the 5'-end, and is a modified oligonucleotide within 30 residues.

[0313] In certain embodiments, the modified oligonucleotide is a gapmer, which is a nucleic acid base sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 consecutive nucleic acid base sequences that are complementary to an equal-length portion of the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the sequence listing, positions 232 to 248 from the 5'-end, and is a modified oligonucleotide within 30 residues.

[0314] In certain embodiments, the modified oligonucleotide is a gapmer, which is a nucleic acid base sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleic acid base sequences that are complementary to an equal-length portion of the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the sequence listing, positions 1306 to 1325 from the 5'-end, and is a modified oligonucleotide within 30 residues.

[0315] In certain embodiments, the modified oligonucleotide is a gapmer and 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 bases complementary to the 1472-1495th positions 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.

[0316] In certain embodiments, the modified oligonucleotide is a gapmer of a modified oligonucleotide composed of 12-30 residues and is a nucleic acid base sequence comprising a nucleic acid base sequence complementary to the 126-147th positions 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 equivalent length portion. Additionally, in certain embodiments, it is a modified oligonucleotide 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.

[0317] In certain embodiments, the modified oligonucleotide is a gapmer of a modified oligonucleotide consisting of 12 to 30 residues, and contains a nucleic acid base sequence complementary to the 233rd to 248th positions of the nucleic acid bases 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 Sequence Listing in the equivalent-length portion. In addition, in certain embodiments, it is a 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 as described above.

[0318] In certain embodiments, the modified oligonucleotide is a gapmer of a modified oligonucleotide consisting of 12 to 30 residues, and contains a nucleic acid base sequence complementary to the 1309th to 1323rd positions of the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing. Among them, the 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 is a gapmer of a modified oligonucleotide consisting of 12 to 30 residues, and contains a nucleic acid base sequence complementary to the nucleic acid bases of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing. In addition, 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 as described above.

[0319] In certain embodiments, the modified oligonucleotide is a gapmer of a modified oligonucleotide composed of 12 to 30 residues, and contains a nucleic acid base sequence complementary to the 1472nd to 1495th positions of the nucleic acid bases 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 Sequence Listing in the equal-length portion. In addition, in certain embodiments, it is a 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. At this time, the oligonucleotide contained in the 5'-flanking fragment and / or the 3'-flanking fragment contains at least one nucleoside, and the nucleoside contains at least one modified sugar selected from GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz] and ALNA[Trz], and further may contain a sugar modified by 2'-O-methoxyethyl and / or a sugar modified by 2'-O-methyl.

[0320] In certain embodiments, the modified oligonucleotide is a gapmer of a modified oligonucleotide comprising 12 to 30 residues, has a nucleic acid base sequence complementary to the 1480th to 1495th positions 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 composed of a nucleic acid base sequence having a complementary base to the 1480th base from the 5'-end of the nucleic acid bases 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 equal-length portion. Further, in certain embodiments, it is the above-described modified oligonucleotide composed 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.

[0321] In certain embodiments, the modified oligonucleotides provided in this specification are gapmers, targeting 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 in this specification are gapmers, targeting 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.

[0322] In certain embodiments, the modified oligonucleotides provided in this specification are gapmers and have a nucleic acid base sequence that includes a complementary region containing at least 8 consecutive nucleic acid bases complementary to a target region. In certain embodiments, the modified oligonucleotides provided in this specification have a nucleic acid base sequence that includes a complementary region containing at least 8 consecutive nucleic acid bases complementary to a target region, and the target region is the target 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 of SEQ ID NO: 1 in the sequence listing. In certain embodiments, the modified oligonucleotides provided in this specification are gapmers and have a nucleic acid base sequence that includes a complementary region containing at least 8 consecutive nucleic acid bases complementary to a target region, and the target region is the target at positions 128-143, 232-247, 233-248, 1309-1323, or 1480-1495 of SEQ ID NO: 1 in the sequence listing.

[0323] In certain embodiments, the modified oligonucleotides provided in this specification are gapmers and have a nucleic acid base sequence that includes a complementary region containing at least 10 consecutive nucleic acid bases complementary to a target region. In certain embodiments, the modified oligonucleotides provided in this specification have a nucleic acid base sequence that includes a complementary region containing at least 10 consecutive nucleic acid bases complementary to a target region, and the target region is targeted 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 of SEQ ID NO: 1 in the sequence listing. In certain embodiments, the modified oligonucleotides provided in this specification are gapmers and have a nucleic acid base sequence that includes a complementary region containing at least 10 consecutive nucleic acid bases complementary to a target region, and the target region is targeted at positions 128-143, 232-247, 233-248, 1309-1323 or 1480-1495 of SEQ ID NO: 1 in the sequence listing.

[0324] In certain embodiments, the modified oligonucleotides provided in this specification are gapmers and have a nucleic acid base sequence that includes a complementary region containing at least 12 consecutive nucleic acid bases complementary to a target region. In certain embodiments, the modified oligonucleotides provided in this specification have a nucleic acid base sequence that includes a complementary region containing at least 12 consecutive nucleic acid bases complementary to a target region, and the target region is targeted 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 of SEQ ID NO: 1 in the sequence listing. In certain embodiments, the modified oligonucleotides provided in this specification are gapmers and have a nucleic acid base sequence that includes a complementary region containing at least 12 consecutive nucleic acid bases complementary to a target region, and the target region is targeted at positions 128-143, 232-247, 233-248, 1309-1323, or 1480-1495 of SEQ ID NO: 1 in the sequence listing.

[0325] In certain embodiments, the modified oligonucleotides provided in this specification are gapmers and have a nucleic acid base sequence that includes a complementary region containing at least 14 consecutive nucleic acid bases complementary to a target region. In certain embodiments, the modified oligonucleotides provided in this specification have a nucleic acid base sequence that includes a complementary region containing at least 14 consecutive nucleic acid bases complementary to a target region, and the target region is targeted 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 of SEQ ID NO: 1 in the sequence listing. In certain embodiments, the modified oligonucleotides provided in this specification are gapmers and have a nucleic acid base sequence that includes a complementary region containing at least 14 consecutive nucleic acid bases complementary to a target region, and the target region is targeted at positions 128-143, 232-247, 233-248, 1309-1323, or 1480-1495 of SEQ ID NO: 1 in the sequence listing.

[0326] In certain embodiments, the modified oligonucleotide is a gapmer, which is composed of nucleic acid base sequences that are isocomplementary to the nucleic acid bases 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 mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing. Additionally, in certain embodiments, the modified oligonucleotide is a gapmer, which is composed of nucleic acid base sequences that are isocomplementary to the nucleic acid bases at positions 128-143, 232-247, 233-248, 1309-1323, 1480-1495 from the 5'-end of the mature mRNA of DUX4 of SEQ ID NO: 1 in the Sequence Listing.

[0327] In certain embodiments, the modified oligonucleotide is a gapmer, which is composed of the nucleic acid base sequence recited 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, which is composed of the nucleic acid base sequence recited in any one of SEQ ID NOs: 2-4, 75, 78 in the Sequence Listing.

[0328] In certain embodiments, the modified oligonucleotide is a gapmer, which is the modified oligonucleotide described in any one of Compound Nos. 1 to 112, 114 to 132, and 137 to 246. Additionally, in certain embodiments, the modified oligonucleotide is a gapmer, which is the modified oligonucleotide described in any one of Compound Nos. 1 to 3, 123, 157, 204, 221, and 231.

[0329] In this specification, in notations such as "Gls" representing nucleotides, the abbreviation shown in the left position refers to the nucleobase part, the abbreviation shown in the central position refers to the sugar part, and the abbreviation shown in the right position refers to the type of internucleoside bond.

[0330] In certain embodiments, the modified oligonucleotide is a gapmer and has the following formula:

[0331] GlsMlsMlsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMlsGlsGl

[0332] represents the modified oligonucleotide,

[0333] wherein,

[0334] each nucleobase is represented by the following notations:

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

[0336] each sugar part is represented by the following notations:

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

[0338] each internucleoside bond is represented by the following notations:

[0339] s = phosphorothioate.

[0340] In certain embodiments, the modified oligonucleotide is a gapmer,

[0341] and has the following formula:

[0342] GmsMmsMmsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMmsGmsGm

[0343] represents the modified oligonucleotide,

[0344] wherein,

[0345] each nucleobase is represented by the following notations:

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

[0347] Each sugar moiety is represented by the following notations:

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

[0349] Each internucleoside linkage is represented by the following notations:

[0350] s = phosphorothioate.

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

[0352] GmsMmsAmsGdsTdsTdsCdsCdsCdsGdsCdsGmsGmsTm

[0353] The modified oligonucleotide represented by

[0354] wherein,

[0355] Each nucleobase is represented by the following notations:

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

[0357] Each sugar moiety is represented by the following notations:

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

[0359] Each internucleoside linkage is represented by the following notations:

[0360] s = phosphorothioate.

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

[0362] MlsGlsAlsGdsAdsTdsTdsCdsCdsCdsGdsCdsCdsGlsGlsTl

[0363] The modified oligonucleotide represented by

[0364] wherein,

[0365] Each nucleobase is represented by the following notations:

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

[0367] Each sugar moiety is represented by the following notations:

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

[0369] The bond between each nucleoside is represented by the following notations:

[0370] s = phosphorothioate.

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

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

[0373] Additionally, certain embodiments provide a method for treating, preventing, improving, or alleviating FSHD, comprising administering to the animal an effective amount of a modified oligonucleotide against DUX4.

[0374] Certain embodiments provide a method for preventing, improving, or alleviating various symptoms of FSHD (e.g., facial muscle weakness, ptosis, inability to whistle, reduced facial expression changes, sad or angry facial expressions, dysarthria, scapular muscle weakness (deformations such as winged scapula, sloping shoulders, etc.), lower limb weakness, hearing loss, and heart disease), comprising administering to the animal an effective amount of a modified oligonucleotide against DUX4.

[0375] Certain embodiments provide a method for reducing the level of mRNA and / or protein produced by the fusion of the DUX4 gene and the IGH gene (e.g., intracellular level in B cells in the blood) in an animal with B-cell acute lymphoblastic leukemia, etc., comprising administering to the animal an effective amount of a modified oligonucleotide against DUX4. Additionally, certain embodiments provide a method for treating, preventing, improving, or alleviating B-cell acute lymphoblastic leukemia, etc., comprising administering to the animal an effective amount of a modified oligonucleotide against DUX4.

[0376] Certain embodiments provide a method for reducing the level of mRNA and / or protein produced by the fusion of the DUX4 gene and the CIC gene (e.g., intratumoral level) in an animal with differentiated round cell sarcoma, etc., comprising administering to the animal an effective amount of a modified oligonucleotide against DUX4. Additionally, certain embodiments provide a method for treating, preventing, improving, or alleviating differentiated round cell sarcoma, etc., comprising administering to the animal an effective amount of a modified oligonucleotide against DUX4.

[0377] Certain embodiments provide a method for reducing the level of mRNA and / or protein produced by the fusion of the DUX4 gene and the EWSR1 gene (e.g., the level within a sarcoma) in an animal such as embryonal rhabdomyosarcoma, comprising administering a modified oligonucleotide targeting DUX4 to the animal. Additionally, certain embodiments provide a method for treating, preventing, ameliorating, or alleviating embryonal rhabdomyosarcoma, etc., comprising administering an effective amount of a modified oligonucleotide targeting DUX4 to the animal.

[0378] Certain embodiments provide a method for treating, preventing, ameliorating, or alleviating a DUX4-related disease with few side effects, the method comprising administering a modified oligonucleotide targeting DUX4 to an animal. In certain embodiments, examples of side effects include injection site reactions, abnormalities in liver function tests, renal function abnormalities, hepatotoxicity (abnormal histological findings: degeneration and necrosis of hepatocytes, hepatocyte hypertrophy, etc.), nephrotoxicity (abnormal histological findings, etc.), central nervous system abnormalities, myopathy, and malaise. 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 liver function abnormalities. For example, an increase in bilirubin may indicate hepatotoxicity or liver function abnormalities. Additionally, an increase in urinary protein, or an increase in creatinine or UN in the blood may indicate nephrotoxicity or renal function abnormalities.

[0379] Certain embodiments provide for the use of any compound described in this specification or a pharmaceutical composition comprising the compound in the manufacture of a medicament for use in any of the therapeutic methods described in this specification. For example, certain embodiments provide for the use of a compound described in this specification or a pharmaceutical composition comprising the compound in the manufacture of a medicament for treating, i.e., treating, preventing, delaying or improving FSHD. Certain embodiments provide for the use of a compound described in this specification or a pharmaceutical composition comprising the compound in the manufacture of a medicament for inhibiting DUX4 expression and for treating, i.e., treating, preventing, delaying or improving DUX4-related diseases and / or their symptoms. Certain embodiments provide for the use of a compound described in this specification or a pharmaceutical composition comprising the compound in the manufacture of a medicament for reducing the expression of DUX4 in an animal. Certain embodiments provide for the use of a compound described in this specification or a pharmaceutical composition comprising 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 alleviating myotonia. Certain embodiments provide for the use of a compound described in this specification or a pharmaceutical composition comprising the compound in the manufacture of a medicament for treating an animal having FSHD. Certain embodiments provide for the use of a compound described in this specification or a pharmaceutical composition comprising the compound in the manufacture of a medicament for treating one or more symptoms and outcomes associated with FSHD, including muscle stiffness, myotonia, facial muscle weakness, ptosis, inability to whistle, reduced facial expression changes, sad or angry facial expressions, dysarthria, scapular muscle weakness (deformations such as winged scapula, drooping shoulders, etc.), lower limb weakness, hearing loss and heart disease. Certain embodiments provide for the use of a compound described in this specification or a pharmaceutical composition comprising the compound in the manufacture of a medicament for interfering with DUX4 protein expression by guiding the transcription of the DUX4 gene, the translation of DUX4 mRNA, and the cleavage of DUX4 mRNA.

[0380] Certain embodiments provide a kit for treating, i.e., treating, preventing or improving FSHD as described in this specification, comprising a) a compound described in this specification and optionally b) a further agent or therapy described in this specification. The kit may further comprise instructions or a label for using the kit for the purpose of treating, i.e., treating, preventing or improving FSHD.

[0381] Certain embodiments provide for the use of any compound described in this specification or a pharmaceutical composition containing such compound for manufacturing a medicament for use in any of the treatment methods described in this specification. For example, certain embodiments provide for the use of a compound described in this specification or a pharmaceutical composition containing such compound for manufacturing a medicament for treating, i.e., treating, improving or preventing FSHD. Certain embodiments provide for the use of a compound described in this specification or a pharmaceutical composition containing such compound for manufacturing a medicament for inhibiting DUX4 expression and for treating, i.e., treating, preventing, delaying or improving DUX4-related diseases and / or their symptoms. Certain embodiments provide for the use of a compound described in this specification or a pharmaceutical composition containing such compound for manufacturing a medicament for reducing the expression of DUX4 in an animal. Certain embodiments provide for the use of a compound described in this specification or a pharmaceutical composition containing such compound for manufacturing a medicament for preferentially reducing the level of DUX4-FL mRNA (e.g., intramuscular level) in an animal and alleviating myotonia. Certain embodiments provide for the use of a compound described in this specification or a pharmaceutical composition containing such compound for manufacturing a medicament for treating an animal having FSHD. Certain embodiments provide a compound described in this specification or a pharmaceutical composition containing such compound for treating one or more symptoms and outcomes associated with the development of FSHD, including muscle stiffness, myotonia, facial muscle weakness, ptosis, inability to whistle, reduced facial expression changes, sad or angry facial expressions, dysarthria, scapular muscle weakness (deformations such as winged scapula, sloping shoulders, etc.), lower limb weakness, hearing loss, and heart disease. Certain embodiments provide a modified oligonucleotide having a nucleic acid base sequence consisting of the nucleic acid base sequences of SEQ ID NOs: 2 to 4, 7 to 64, 69 to 97, or 102 to 112 in the sequence listing, or a compound of Compound Nos. 1 to 112, 114 to 132, or 137 to 246.

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

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

[0384] It is possible to increase or decrease the length of an antisense compound such as an antisense oligonucleotide and / or introduce mismatched bases without loss of activity. For example, according to Woolf et al. (Proc. Natl. Acad. Sci. USA 89: 7305-7309, 1992), regarding a series of antisense oligonucleotides 13 to 25 nucleic acid bases in length for their ability to induce cleavage of target RNA in an oocyte injection model, it was reported that a 25-nucleic acid base-long antisense oligonucleotide having 8 or 11 mismatched bases near the end of the antisense oligonucleotide was able to direct specific cleavage of target mRNA, although to a lesser extent than an antisense oligonucleotide without mismatches. Similarly, it was reported that target-specific cleavage could be achieved using a 13-nucleic acid base antisense oligonucleotide including an antisense oligonucleotide having 1 or 3 mismatches.

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

[0386] Target nucleic acid, target region, and nucleotide sequence

[0387] As the nucleotide sequence encoding DUX4, there is no limitation, and the following sequences can be cited.

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

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

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

[0391] · SNPs of the above respective splice variants.

[0392] Each sequence recorded in the sequence listing of the embodiments included in this specification has nothing to do with any modification of the sugar moiety, internucleoside bond, or nucleic acid base. Therefore, the modified oligonucleotides specified by the sequence numbers in the sequence listing may independently contain one or more modifications of the sugar moiety, internucleoside bond, or nucleic acid base. The modified oligonucleotides recorded in the compound numbers represent the combination of the nucleic acid base sequence and the motif.

[0393] In certain embodiments, the target region is a structurally defined region of the target nucleic acid. For example, the target region may 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. The structurally defined regions for DUX4 can be obtained from sequence databases such as NCBI by accession numbers, and such information is incorporated herein by reference. In certain embodiments, the target region may include the sequence from the 5' target site of a certain target fragment within the target region to the 3' target site of another target fragment within the target region.

[0394] Targeting involves the determination of at least one target fragment to which a modified oligonucleotide hybridizes, resulting in a desired effect. 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 protein level encoded by the target nucleic acid or a change in the phenotype associated with the target nucleic acid.

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

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

[0397] Determination of an appropriate target fragment can involve comparison of the sequence of the target nucleic acid with other sequences throughout the genome as a whole. For example, the BLAST algorithm can be used to identify regions of similarity between different nucleic acids. This comparison can prevent selection of a modified oligonucleotide sequence that can hybridize in a non-specific manner to a sequence other than the selected target nucleic acid (i.e., a non-target sequence or off-target sequence).

[0398] There can be variation in the activity (e.g., as determined by the percentage reduction in the level of the target nucleic acid) of the modified oligonucleotide within the active target region. 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, a change in phenotype, such as alleviation of muscle rigidity or reduction of muscle dystrophy, can serve as an indicator of inhibition of DUX4 mRNA and / or protein expression.

[0399] Hybridization

[0400] In some embodiments, hybridization occurs between the modified oligonucleotide disclosed in this specification and the DUX4 nucleic acid. The most common mechanism of hybridization involves hydrogen bonding between complementary nucleic acid bases 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 refers to the temperature at which 50% of the hybridized double-stranded nucleic acid dissociates into single-stranded nucleic acids, and although it varies depending on the salt conditions of the solution, the length of the nucleic acid, or the base sequence, etc., the higher the Tm value, the stronger the hybridization.

[0401] Hybridization occurs under various conditions. Stringent conditions are sequence-dependent and are determined based on the nature and composition of the nucleic acid molecules being hybridized.

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

[0403] Complementarity

[0404] A modified oligonucleotide and a target nucleic acid are complementary to each other when a sufficient number of nucleic acid bases of the modified oligonucleotide can form hydrogen bonds with the corresponding nucleic acid bases of the target nucleic acid to produce a desired effect (e.g., antisense inhibition of a target nucleic acid such as DUX4 nucleic acid).

[0405] The modified oligonucleotide can hybridize to one or more segments of the DUX4 nucleic acid in a manner such that intervening or flanking segments do not participate in the hybridization phenomenon (e.g., loop structures, mismatches, or hairpin structures).

[0406] In certain embodiments, the modified oligonucleotide or a particular portion thereof provided in this specification is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the DUX4 nucleic acid, target region, target segment, or a particular portion 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 the DUX4 nucleic acid, target region, target segment, or a defined portion thereof, comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleic acid bases of any nucleic acid base sequence exemplified in the modified oligonucleotides described in this specification (e.g., at least 8 consecutive nucleic acid bases of the nucleic acid base sequence recited in any one of SEQ ID NOs: 2, 3, 4, 7-64, 69-97, or 102-112 in the Sequence Listing). The percentage of complementarity of the modified oligonucleotide to the target nucleic acid can be determined using conventional methods and can be measured over the entire length of the modified oligonucleotide.

[0407] For example, 18 out of 20 nucleic acid bases of a modified oligonucleotide are complementary to the target region. Therefore, the modified oligonucleotide that undergoes specific hybridization is considered to have 90% complementarity. In this example, the remaining non-complementary nucleic acid bases can be clustered or can sandwich complementary nucleic acid bases, and do not need to be contiguous to each other or to the complementary nucleic acid bases. The percentage of 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, 649-656) known in the art. The percentage of homology, sequence identity or complementarity can be determined, for example, by the GAP program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using the default settings of the algorithm utilizing Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0408] The position of the non-complementary nucleic acid bases can be at the 5' end or the 3' end of the modified oligonucleotide. Alternatively, the non-complementary nucleic acid bases (which can be multiple) can also be at internal positions of the modified oligonucleotide. When there are two or more non-complementary nucleic acid bases, they can be contiguous (i.e., linked) or non-contiguous. In one embodiment, the non-complementary nucleic acid bases are located in the flanking segments of the spacer-modified oligonucleotide.

[0409] In certain embodiments, a modified oligonucleotide that is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleic acid bases in length or up to these nucleic acid bases in length contains three or fewer, two or fewer, or one or fewer non-complementary nucleic acid bases (which can be multiple) to a target nucleic acid, such as the DUX4 nucleic acid or a specific portion thereof.

[0410] The modified oligonucleotides provided in this specification also contain modified nucleic acid bases that are complementary to a portion of the target nucleic acid. As used in this specification, "portion" refers to a defined number of consecutive (i.e., contiguous) nucleic acid bases within a region or fragment of the target nucleic acid. "Portion" can also refer to a defined number of consecutive nucleic acid bases of the modified oligonucleotide. In certain embodiments, the modified oligonucleotide is at least 8 nucleic acid bases complementary to the target fragment. In certain embodiments, the modified oligonucleotide is at least 10 nucleic acid bases complementary to the target fragment. In certain embodiments, the modified oligonucleotide is at least 15 nucleic acid bases complementary to the target fragment. It also refers to a modified oligonucleotide that is complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or more nucleic acid bases of the target fragment, or a range determined by any two of these values.

[0411] Identity

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

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

[0414] Modification

[0415] A nucleoside is a base-sugar composition. The nucleic acid base (also known as a base) portion of a nucleoside is typically a heterocyclic base portion. A nucleotide is a nucleoside that further includes a phosphate group covalently bonded to the sugar portion of the nucleoside. For nucleosides containing a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′, or 5′ hydroxyl portion of the sugar. Oligonucleotides are formed by covalently bonding adjacent nucleosides to each other, forming a linear polymer oligonucleotide. Within the oligonucleotide structure, the phosphate groups are generally considered to form the internucleoside bonds of the oligonucleotide.

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

[0417] Modified internucleoside bonds

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

[0419] Oligonucleotides having modified internucleoside bonds include internucleoside bonds that retain a phosphorus atom and internucleoside bonds that do not have a phosphorus atom. As representative phosphorus-containing internucleoside bonds, one or more of phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate can be cited, but are not limited to these. Methods for preparing phosphorus-containing and non-phosphorus-containing bonds are well known.

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

[0421] Modified sugar portions

[0422] The modified oligonucleotides of the present invention preferably use modified oligonucleotides in which at least one nucleoside constituting the same includes a modified sugar. In the present invention, a modified sugar refers to a sugar in which the sugar portion is modified, and a modified oligonucleotide containing one or more of such modified sugars has advantageous characteristics 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 portion.

[0423] Examples of nucleosides with modified sugars include 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, but are not limited thereto. Substituents at the 2'-position may also be selected from allyl, amino, azido, thio, O-allyl, O-C1-C 10 alkyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-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).

[0424] Examples of nucleosides with bicyclic sugars include nucleosides containing a crosslink between the ribosyl ring atoms at the 4'- and 2'-positions, but are not limited thereto. In certain embodiments, the oligonucleotides provided herein contain a crosslinking nucleoside having one or more bicyclic sugars including 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 WO 2009 / 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-C 12 alkyl or a protecting group) (see U.S. Patent No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (and their analogs. See Chattopadhyayaetal., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and their analogs. See WO2008 / 154401).

[0425] Nucleosides having further bicyclic sugars have been reported in the published literature (see, for example, Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al.,; U.S. Patent Nos. 7,399,845; 6,770,748; 6,525,191; 6,268,490; U.S. 2008-0039618; U.S. 2007-0287831; U.S. 2004-0171570; U.S. 2009-0012281; WO2010 / 036698; WO 2009 / 067647; WO2009 / 067647; WO2007 / 134181; WO2005 / 021570; WO2004 / 106356; WO94 / 14226; WO 2009 / 006478; WO2008 / 154401; and WO2008 / 150729). The nucleosides having the above-mentioned bicyclic sugars can be prepared, for example, respectively, by including one or more stereochemical sugar configurations of α-L-ribofuranose and β-D-ribofuranose.

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

[0427] In certain embodiments, the nucleoside with a bicyclic sugar contains a crosslink between the 4' and 2' carbon atoms of the pentofuranosyl sugar moiety and contains a linking group selected independently from -[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 -, but is not limited to these. In the formula, 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, a hydroxyl group, a C1-C 12 alkyl group, a substituted C1-C 12 alkyl group, a C2-C 12 alkenyl group, a substituted C2-C 12 alkenyl group, a C2-C 12 alkynyl group, a substituted C2-C 12 alkynyl group, 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, a halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, an acyl group (C(=O)-H), a substituted acyl group, CN, a sulfonyl group (S(=O)2-J1), or a sulfinyl group (S(=O)-J1),

[0428] each J1 and J2 is independently H, a C1-C 12 alkyl group, a substituted C1-C 12 alkyl group, a C2-C 12 alkenyl group, a substituted C2-C 12 alkenyl group, a C2-C 12 alkynyl group, a substituted C2-C 12Alkynyl, aromatic ring group, substituted aromatic ring group, acyl group (C(=O)-H), substituted acyl group, heterocyclic group, substituted heterocyclic group, C1-C 12 Aminoalkyl, substituted C1-C 12 Aminoalkyl or a protecting group.

[0429] In certain embodiments, the crosslinking of the bicyclic sugar moiety is -[C(R a )(R b )] n -, -[C(R a )(R b )] n -O-, -C(R a R b )-N(R)-O- or -C(R a R b )-O-N(R). In certain embodiments, the crosslinking is 4’-CH2-2’, 4’-(CH2)2-2’, 4’-(CH2)3-2’, 4’-CH2-O-2’ (the nucleoside having a bicyclic sugar at this time is also called 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.

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

[0431] In certain embodiments, the nucleoside having a bicyclic sugar is further defined by the isomeric spatial configuration. For example, the nucleoside containing 4’-(CH2)-O-2’ crosslinking can exist in the α-L-spatial configuration or the β-D-spatial configuration.

[0432] In certain embodiments, a nucleoside having a bicyclic sugar includes a nucleoside having a 4′-2′ crosslink. Examples of such a crosslink include 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, a C1-C 12 alkyl group, or urea or guanidine that may be substituted with a C1-C 12 alkyl group), but are not limited thereto.

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

[0434]

[0435] wherein,

[0436] Bx is a heterocyclic base moiety,

[0437] T a and T b are each independently a hydrogen atom, a protecting group for a hydroxyl group, a phosphoryl group that may be substituted, a covalent bond to a phosphorus moiety or a support, etc.,

[0438] Z a is a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, a substituted C1-C6 alkyl group, a substituted C2-C6 alkenyl group, a substituted C2-C6 alkynyl group, an acyl group, a substituted acyl group, a substituted amide, a thiol or a substituted thiol.

[0439] In certain embodiments, the substituents are each independently monosubstituted or polysubstituted with a substituent selected independently from halogen, oxo group, hydroxyl group, 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 is independently H, a C1-C6 alkyl group or a substituted C1-C6 alkyl group, and X is O or NJ c ).

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

[0441]

[0442] In the formula,

[0443] Bx is a heterocyclic base moiety,

[0444] T a and T b are each independently a hydrogen atom, a protecting group for a hydroxyl group, a phosphoryl group which may be substituted, a covalent bond to a phosphorus moiety or a support, etc.,

[0445] 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)-).

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

[0447]

[0448] In the formula,

[0449] Bx is a heterocyclic base moiety,

[0450] T a and T b are each independently a hydrogen atom, a protecting group for a hydroxyl group, a phosphoryl group which may be substituted, a covalent bond to a phosphorus moiety or a support, etc.,

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

[0452] each q a 、q b 、q c and q d are 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 alkoxy, substituted C1-C6 alkoxy, acyl, substituted acyl, C1-C6 aminoalkyl or substituted C1-C6 aminoalkyl.

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

[0454]

[0455] In the formula,

[0456] Bx is a heterocyclic base moiety;

[0457] Ta and T b are each independently a hydrogen atom, a protecting group for a hydroxyl group, a phosphoryl group which may be substituted, a covalent bond to a phosphorus moiety or a support, etc.,

[0458] q a 、q b 、q e and q f are 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 alkoxy, substituted C1-C 12 alkoxy, OJ j 、SJ j 、SOJ j 、SO2J j 、NJ j J k 、N3, CN, C(=O)OJj, C(=O)NJ j J k 、C(=O)J j 、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 ,

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

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

[0461] The synthesis and preparation of adenine, cytosine, guanine, 5-methyl-cytosine, thymine, and uracil bicyclic nucleosides (also known as LNA) having a 4'-CH2-O-2' crosslink 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.

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

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

[0464]

[0465] wherein,

[0466] Bx is a heterocyclic base moiety,

[0467] T a and T b are each independently a hydrogen atom, a protecting group for a hydroxyl group, a phosphoryl group which may be substituted, a covalent bond to a phosphorus moiety or a support, etc.,

[0468] each q i 、q j 、q k and q l are independently H, halogen, C1-C 12 alkyl, substituted C1-C 12 alkyl, C2-C 12 alkenyl, substituted C2-C 12 alkenyl, C2-C 12 alkynyl, substituted C2-C12 Alkynyl, C1-C 12 Alkoxy, substituted C1-C 12 Alkoxy, OJ j SJ j SOJ j SO2J j NJ j J k N3, CN, C(=O)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 ,

[0469] q i and q j or q l and q k both are =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.

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

[0471] In certain embodiments, as nucleosides having a bicyclic sugar, the following can be cited

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

[0473] (B) β-D-Methyleneoxy (4’-CH2-O-2’) BNA,

[0474] (C) Ethyleneoxy (4’-(CH2)2-O-2’) BNA,

[0475] (D) Aminooxy (4’-CH2-O-N(R)-2’) BNA,

[0476] (E) Oxyamino (4’-CH2-N(R)-O-2’) BNA,

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

[0478] (G) Methylene-thio (4’-CH2-S-2’) BNA,

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

[0480] (I) Methyl carbocyclic (4’-CH2-CH(CH3)-2’) BNA,

[0481] (J) Propylene carbocyclic (4’-(CH2)3-2’) BNA, and

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

[0483]

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

[0485] In a certain embodiment (LNA), examples of the nucleoside having a bicyclic sugar include the following general formula:

[0486]

[0487] [In the formula,

[0488] B is a nucleic acid base,

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

[0490]

[0491] represents a nucleotide.

[0492] In a certain embodiment (GuNA), the nucleoside containing a bicyclic sugar has the following general formula:

[0493]

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

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

[0496] In a certain embodiment (ALNA[mU]), the nucleoside containing a bicyclic sugar has the following general formula (I):

[0497]

[0498] [wherein,

[0499] B is a nucleic acid base,

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

[0501] R5 and R6 are each independently a hydrogen atom, a protecting group for a hydroxyl group, a phosphoryl group that may be substituted, a covalent bond to a phosphorus moiety or a support, etc.,

[0502] m is 1 or 2,

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

[0504]

[0505] The group represented,

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

[0507]

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

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

[0510] The nucleosides represented (for example, refer to Japanese Patent Application No. 2018-212424). Typical specific examples are nucleosides in which one of R7 and R8 is a hydrogen atom and the other is an unsubstituted methyl group.

[0511] In a certain embodiment (ALNA[ipU]), the nucleoside containing a bicyclic sugar is a nucleoside having the general formula (I) defined in the above-mentioned ALNA[mU], in which

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

[0513]

[0514] the group represented,

[0515] In one of R7 and R8 is a hydrogen atom and the other is an isopropyl group which may be substituted with one or more substituents (for example, refer to Japanese Patent Application No. 2018-212424). Typical specific examples are nucleosides in which one of R7 and R8 is a hydrogen atom and the other is an unsubstituted isopropyl group.

[0516] In a certain embodiment (ALNA[Trz]), the nucleoside containing a bicyclic sugar is a nucleoside having the above general formula (I), in which X is the following formula (II-2):

[0517]

[0518] the group represented,

[0519] A is a triazolyl group which may be substituted with one or more substituents (for example, refer to Japanese Patent Application No. 2018-212424). Typical specific examples of ALNA[Trz] are nucleosides in which A is a triazolyl group which may have one or more methyl groups, and more specifically, 1,5-dimethyl-1,2,4-triazol-3-yl.

[0520] A certain embodiment (ALNA[Oxz]) is a nucleoside having the general formula (I) defined in the above-mentioned ALNA[mU], in which

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

[0522]

[0523] the group represented,

[0524] A is a diazolyl group which may be substituted with one or more substituents (for example, refer to Japanese Patent Application No. 2018-212424). Typical specific examples are diazolyl groups which may have one or more methyl groups, and more specifically, 5-methyl-1,2,4- Nucleosides or nucleotides of 1,2,4-oxadiazol-3-yl.

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

[0526]

[0527] a group represented by

[0528] M is a sulfonyl group which may be substituted by a methyl group, and the methyl group may be substituted by 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 by an unsubstituted methyl group.

[0529] In certain embodiments, the nucleoside is modified by substitution of the ribose ring with a sugar surrogate. Examples of such modifications include substitution of the ribose ring with a surrogate ring system (also referred to as a DNA analogue), such as a morpholino ring, a cyclohexenyl ring, a cyclohexyl ring or a tetrahydropyranyl ring, for example a ring having one of the following formulas, but are not limited thereto.

[0530]

[0531] In certain embodiments, a sugar surrogate having the following formula is selected:

[0532]

[0533] wherein

[0534] Bx is a heterocyclic base moiety,

[0535] T3 and T4 are each independently a nucleoside internucleoside linking group obtained by linking a tetrahydropyran nucleoside analogue to an oligomeric compound, or one of T3 and T4 is a nucleoside internucleoside linking group obtained by linking a tetrahydropyran nucleoside analogue to an oligomeric compound or an 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,

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

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

[0538] In certain embodiments, q1, q2, q3, q4, q5, q6, and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is not H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is methyl. In certain embodiments, a THP nucleoside can be provided in which one of R1 and R2 is F. In certain embodiments, R1 is fluorine and R2 is H; R1 is methoxy and R2 is H; and R1 is methoxyethoxy and R2 is H.

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

[0540] In certain embodiments, the sugar substitute comprises a ring having more than 5 atoms and more than 1 heteroatom. For example, nucleosides containing a morpholino sugar moiety and their use in oligomeric compounds have been reported (see, for example, Braaschet al., Biochemistry, 2002, 41, 4503-4510; and U.S. Patents 5698685; 5166315; 5185444; and 5034506).

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

[0542]

[0543] In certain embodiments, for example, the morpholino group can be modified by attaching or varying various substituents from the above morpholino structure. Such sugar substitutes are referred to herein as "modified morpholino groups".

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

[0545] Certain modified cyclohexenyl nucleosides have the following formula:

[0546]

[0547] In the formula,

[0548] Bx is a heterocyclic base moiety,

[0549] T3 and T4 are each independently a internucleoside linker obtained by linking a cyclohexenyl nucleoside analogue to an oligonucleotide compound, or one of T3 and T4 is a internucleoside linker obtained by linking a tetrahydropyranyl nucleoside analogue 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,

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

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

[0552] Methods for preparing modified sugars are well known to those skilled in the art. Some representative U.S. patents teaching the preparation of such modified sugars include U.S.: 4981957; 5118800; 5319080; 5359044; 5393878; 5446137; 5466786; 5514785; 5519134; 5567811; 5576427; 5591722; 5597909; 5610300; 5627053; 5639873; 5646265; 5670633; 5700920; 5792847 and 6600032, as well as WO2005 / 121371, but are not limited to these, and these are hereby incorporated by reference in their entirety into this specification.

[0553] In nucleotides having a modified sugar moiety, the nucleic acid base moiety (natural, modified or a combination thereof) is maintained between hybridization to an appropriate nucleic acid target.

[0554] Modified nucleic acid bases

[0555] Modifications or substitutions of nucleic acid bases (or bases) can be structurally recognized by naturally occurring or synthetic unmodified nucleic acid bases and are further functionally interchangeable with such unmodified nucleic acid bases. Both natural and modified nucleic acid bases can be associated with hydrogen bonds. Such nucleic acid base modifications can confer nuclease stability, binding affinity, or some other beneficial biological property on the modified oligonucleotide. Modified nucleic acid bases include, for example, synthetic and natural nucleic acid bases such as 5-methylcytosine (5-me-C). Certain nucleic acid base substitutions containing 5-methylcytosine substitutions are particularly useful for increasing the binding affinity of the modified oligonucleotide to the target nucleic acid. For example, 5-methylcytosine substitutions show an increase in the duplex stability of nucleic acids by 0.6 to 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).

[0556] As further modified nucleic acid bases, there may be mentioned 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-hydroxy 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-azadenine, 7-deazaguanine and 7-deazadenine, 3-deazaguanine and 3-deazadenine.

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

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

[0559] Certain modified oligonucleotide motifs

[0560] In certain embodiments, to impart properties such as enhanced inhibitory activity, increased binding affinity to the target nucleic acid, or resistance to degradation by endogenous nucleases to the modified oligonucleotide, the modified oligonucleotide targeting DUX4 nucleic acid has chemical modification subunits arranged in a pattern or motif.

[0561] To impart increased resistance to nuclease-mediated degradation, increased cellular uptake, increased binding affinity to the target nucleic acid, and / or increased inhibitory activity, chimeric modified oligonucleotides typically comprise at least one modified region. The second region of the chimeric modified oligonucleotide can optionally function as a substrate for the intracellular endonuclease RNase H that cleaves the RNA strand of the RNA:DNA duplex.

[0562] A modified oligonucleotide having a gapmer moiety is a chimeric modified oligonucleotide. In a gapmer, an internal region having multiple nucleotides that support RNase H cleavage is located between outer regions that have multiple nucleotides with a nucleoside chemistry different from that of the internal region. In the case of a modified oligonucleotide having a gapmer moiety, the gap segment generally functions as a substrate for cleavage by an endonuclease, but the flanking segments contain modified nucleosides. In some embodiments, the gapmer regions can be distinguished by the type of sugar moiety that each contains a different region. 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 (for such 2'-modified nucleosides, which can include 2'-MOE and 2'-O-CH3), and bicyclic sugar-modified nucleosides (such bicyclic sugar-modified nucleosides can include bicyclic sugar-modified nucleosides having LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Trz], and / or ALNA[Oxz]). The flanking-gap-flanking moiety is often denoted as "X-Y-Z," where "X" represents the length of the 5' flanking region, "Y" represents the length of the gap region, and "Z" represents the length of the 3' flanking region. As used in this specification, a gapmer denoted as "X-Y-Z" has the following spatial configuration: the gap segment is located at positions directly adjacent to the 5' flanking segment and the 3' flanking segment, respectively. Thus, there are no intervening nucleotides between the 5' flanking segment and the gap segment, or between the gap segment and the 3' flanking segment. Any of the modified oligonucleotides of this specification can have a gapmer moiety. In some embodiments, X and Z are the same, and in other embodiments, they are different. In a preferred embodiment, Y is 8 to 16 nucleotides. X, Y, or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleotides. Thus, examples of gapmers include 2-10-3, 2-14-2, 2-15-2, 2-16-2, 3-6-7, 3-7-5, 3-8-3, 3-8-4, 3-8-5, 3-9-2, 3-9-3, 3-9-4, 3-9-5, 3-9-8, 3-10-2, 3-10-3, 3-10-4, 3-11-3, 3-12-3, 3-14-3, 4-7-4, 4-7-5, 4-8-3, 4-8-4, 4-9-3, 4-10-3, 5-6-4, 5-6-5, 5-7-3, 5-7-4, 5-8-3, 5

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

[0564] In certain embodiments, the modified oligonucleotides targeting DUX4 nucleic acid have a 3-10-3 gapmer motif. In certain embodiments, the modified oligonucleotides targeting DUX4 nucleic acid have a 3-9-3 gapmer motif. In certain embodiments, the modified oligonucleotides targeting DUX4 nucleic acid have a 3-9-4 gapmer motif. In certain embodiments, the modified oligonucleotides targeting DUX4 nucleic acid have a 3-8-5 gapmer motif.

[0565] In certain embodiments, these gapmer modified oligonucleotides comprise at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleic acid bases of any of the nucleic acid base sequences of the exemplary modified oligonucleotides described in this specification (e.g., at least 8 consecutive nucleic acid bases of the nucleic acid base sequences recited in any one of SEQ ID NOs: 2, 3, 4, 7-64, 69-97, or 102-112 of the Sequence Listing).

[0566] In certain embodiments, the present 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 nucleic acid bases. In certain embodiments, the chemically modified oligonucleotides comprise one or more modified internucleoside linkages. In certain embodiments, the chemical modifications (sugar modification, nucleic acid base modification, and / or linkage modification) define a pattern or motif. In certain embodiments, the patterns of chemical modifications of the sugar moiety, internucleoside linkage, and nucleic acid base are each independent of one another. Thus, the oligonucleotide can be characterized by its sugar modification motif, internucleoside linkage motif, and / or nucleic acid base modification motif (when used in this specification, the nucleic acid base modification motif is independent of the sequence of the nucleic acid bases and describes the chemical modification of the nucleic acid bases).

[0567] A certain sugar motif

[0568] In certain embodiments, the 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 comprise any of the sugar modifications discussed in this specification and / or other known sugar modifications.

[0569] In certain embodiments, the oligonucleotide comprises or consists of a region having a gapmer sugar modification motif, which region comprises two outer regions, the "flanking segments", and one inner region, the "gap segment". The three regions (5' flanking segment, gap segment, and 3' flanking segment) of the gapmer motif form a continuous sequence of nucleosides, where at least several of the sugar moieties of the nucleosides of each flanking segment are different from at least several of the sugar moieties of the nucleosides of the gap segment. In particular, the sugar moieties of the nucleosides of each flanking segment that are closest to the gap segment (the most 3' nucleoside of the 5' flanking segment and the most 5' nucleoside of the 3' flanking segment) are different from the sugar moieties of the adjacent nucleosides of the gap segment, thus defining the boundary between the flanking segment and the gap segment. In certain embodiments, the sugar moieties within the gap segment are identical to each other. In certain embodiments, the gap segment comprises 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 flanking segments are identical to each other (symmetric gapmer). In certain embodiments, the sugar modification motif of the 5' flanking segment is different from the sugar modification motif of the 3' flanking segment (asymmetric gapmer).

[0570] A certain 5' flanking segment

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

[0572] In certain embodiments, the 5'-flanking fragment of the spacer contains at least 1 bicyclic nucleoside. In certain embodiments, the 5'-flanking fragment of the spacer contains at least 2 bicyclic nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer contains at least 3 bicyclic nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer contains at least 4 bicyclic nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer contains at least 1 constrained ethyl nucleoside. In certain embodiments, the 5'-flanking fragment of the spacer contains at least 1 nucleoside containing LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Trz], and / or ALNA[Oxz]. In certain embodiments, each nucleoside of the 5'-flanking fragment of the spacer is a bicyclic nucleoside. In certain embodiments, each nucleoside of the 5'-flanking fragment of the spacer is a constrained ethyl nucleoside. In certain embodiments, each nucleoside of the 5'-flanking fragment of the spacer is a nucleoside containing LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Trz], and / or ALNA[Oxz].

[0573] In certain embodiments, the 5'-flanking fragment of the spacer contains at least 1 non-bicyclic modified nucleoside. In certain embodiments, the 5'-flanking fragment of the spacer contains at least 1 2'-substituted nucleoside. In certain embodiments, the 5'-flanking fragment of the spacer contains at least 1, for example, 3, 4, or 5 2'-MOE nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer contains at least 1 2'-OMe nucleoside. In certain embodiments, each nucleoside of the 5'-flanking fragment of the spacer is a non-bicyclic modified nucleoside. In certain embodiments, each nucleoside of the 5'-flanking fragment of the spacer is a 2'-substituted nucleoside. In certain embodiments, each nucleoside of the 5'-flanking fragment of the spacer is a 2'-MOE nucleoside. In certain embodiments, each nucleoside of the 5'-flanking fragment of the spacer is a 2'-OMe nucleoside.

[0574] In certain embodiments, the 5'-flanking fragment of the spacer contains at least 1 bicyclic nucleoside and at least 1 non-bicyclic modified nucleoside. In certain embodiments, the 5'-flanking fragment of the spacer contains at least 1 bicyclic nucleoside and at least 1 2'-substituted nucleoside. In certain embodiments, the 5'-flanking fragment of the spacer contains at least 1 bicyclic nucleoside and at least 1 2'-MOE nucleoside. In certain embodiments, the 5'-flanking fragment of the spacer contains at least 1 bicyclic nucleoside and at least 1 2'-OMe nucleoside. In certain embodiments, the 5'-flanking fragment of the spacer contains at least 1 bicyclic nucleoside and at least 1 2'-deoxynucleoside.

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

[0576] In certain embodiments, the 5'-flanking fragment of the spacer body 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'-flanking fragment of the spacer body 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'-flanking fragment of the spacer body 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'-flanking fragment of the spacer body 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'-flanking fragment of the spacer body 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.

[0577] In certain embodiments, the 5'-flanking fragment of the spacer body contains 2 LNA-containing nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer body contains 3 LNA-containing nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer body contains 4 LNA-containing nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer body contains 3 ALNA[Ms]-containing nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer body contains 2 GuNA-containing nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer body contains 3 GuNA-containing nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer body contains 3 ALNA[mU]-containing nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer body contains 3 ALNA[ipU]-containing nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer body contains 2 LNA-containing nucleosides and 1 GuNA-containing nucleoside. In certain embodiments, the 5'-flanking fragment of the spacer body contains 3 ALNA[Trz]-containing nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer body contains 3 ALNA[Ms]-containing nucleosides and 1 2'-OMe nucleoside. In certain embodiments, the 5'-flanking fragment of the spacer body contains 3 ALNA[Ms]-containing nucleosides and 2 2'-OMe nucleosides.

[0578] In certain embodiments, the 5'-flanking fragment of the spacer body contains 3 constrained ethyl nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer body contains 2 bicyclic nucleosides and 2 non-bicyclic modified nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer body contains 2 constrained ethyl nucleosides and 2 2'-OMe nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer body contains 2 bicyclic nucleosides and 2 non-bicyclic modified nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer body contains 2 constrained ethyl nucleosides and 2 2'-OMe nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer body contains 2 constrained ethyl nucleosides and 3 2'-OMe nucleosides.

[0579] In certain embodiments, the 5'-flanking fragment of the spacer is composed of 1 nucleoside containing ALNA[Ms]. In certain embodiments, the 5'-flanking fragment of the spacer is composed of 2 linked nucleosides containing ALNA[Ms]. In certain embodiments, the 5'-flanking fragment of the spacer is composed of 3 linked nucleosides containing ALNA[Ms]. In certain embodiments, the 5'-flanking fragment of the spacer is composed of 4 linked nucleosides containing ALNA[Ms]. In certain embodiments, the 5'-flanking fragment of the spacer is composed of 5 linked nucleosides containing ALNA[Ms]. In certain embodiments, the 5'-flanking fragment of the spacer is composed of 1, 2 or 3 linked nucleosides containing ALNA[Ms] and 1 2'-OMe nucleoside. In certain embodiments, the 5'-flanking fragment of the spacer is composed of 1, 2 or 3 linked nucleosides containing ALNA[Ms] and 2 2'-OMe nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer is composed of 1, 2 or 3 linked nucleosides containing ALNA[Ms] and 3 2'-OMe nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer is composed of 3 linked nucleosides containing ALNA[Ms] and 1 2'-MOE nucleoside. In certain embodiments, the 5'-flanking fragment of the spacer is composed of 2 linked nucleosides containing ALNA[Ms] and 2 2'-MOE nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer is composed of 3 linked nucleosides containing LNA and 2 2'-MOE nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer is composed of 2 linked nucleosides containing LNA and 2 2'-MOE nucleosides.

[0580] In certain embodiments, the 5'-flanking fragment of the spacer is composed of 1, 2 or 3 linked nucleosides containing ALNA[Ms] and contains 1 5-methylcytosine. In certain embodiments, the 5'-flanking fragment of the spacer is composed of 1, 2 or 3 linked nucleosides containing ALNA[Ms] and contains 2 5-methylcytosines. In certain embodiments, the 5'-flanking fragment of the spacer is composed of 1, 2 or 3 linked nucleosides containing ALNA[Ms] and contains 3 5-methylcytosines.

[0581] A certain 3'-flanking fragment

[0582] In certain embodiments, the 3' flanking fragment of the spacer body consists of from 1 to 8 linked nucleosides. In certain embodiments, the 3' flanking fragment of the spacer body consists of from 2 to 5 linked nucleosides. In certain embodiments, the 3' flanking fragment of the spacer body consists of from 3 to 5 linked nucleosides. In certain embodiments, the 3' flanking fragment of the spacer body consists of 4 or 5 linked nucleosides. In certain embodiments, the 3' flanking fragment of the spacer body consists of from 1 to 4 linked nucleosides. In certain embodiments, the 3' flanking fragment of the spacer body consists of from 1 to 3 linked nucleosides. In certain embodiments, the 3' flanking fragment of the spacer body consists of 1 or 2 linked nucleosides. In certain embodiments, the 3' flanking fragment of the spacer body consists of from 2 to 4 linked nucleosides. In certain embodiments, the 3' flanking fragment of the spacer body consists of 2 or 3 linked nucleosides. In certain embodiments, the 3' flanking fragment of the spacer body consists of 3 or 4 linked nucleosides. In certain embodiments, the 3' flanking fragment of the spacer body consists of 1 nucleoside. In certain embodiments, the 3' flanking fragment of the spacer body consists of 2 linked nucleosides. In certain embodiments, the 3' flanking fragment of the spacer body consists of 3 linked nucleosides. In certain embodiments, the 3' flanking fragment of the spacer body consists of 4 linked nucleosides. In certain embodiments, the 3' flanking fragment of the spacer body consists of 5 linked nucleosides. In certain embodiments, the 3' flanking fragment of the spacer body consists of 6 linked nucleosides. In certain embodiments, the 3' flanking fragment of the spacer body consists of 7 linked nucleosides. In certain embodiments, the 3' flanking fragment of the spacer body consists of 8 linked nucleosides.

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

[0584] In certain embodiments, the 3'-flanking fragment of the spacer body comprises at least 1 non-bicyclic modified nucleoside. In certain embodiments, the 3'-flanking fragment of the spacer body comprises at least 1 2'-substituted nucleoside. In certain embodiments, the 3'-flanking fragment of the spacer body comprises at least 1 2'-MOE nucleoside. In certain embodiments, the 3'-flanking fragment of the spacer body comprises at least 1 2'-OMe nucleoside. In certain embodiments, each nucleoside of the 3'-flanking fragment of the spacer body is a non-bicyclic modified nucleoside. In certain embodiments, each nucleoside of the 3'-flanking fragment of the spacer body is a 2'-substituted nucleoside. In certain embodiments, each nucleoside of the 3'-flanking fragment of the spacer body is a 2'-MOE nucleoside. In certain embodiments, each nucleoside of the 3'-flanking fragment of the spacer body is a 2'-OMe nucleoside.

[0585] In certain embodiments, the 3'-flanking fragment of the spacer body comprises at least 1 bicyclic nucleoside and at least 1 non-bicyclic modified nucleoside. In certain embodiments, the 3'-flanking fragment of the spacer body comprises at least 1 bicyclic nucleoside and at least 1 2'-substituted nucleoside. In certain embodiments, the 3'-flanking fragment of the spacer body comprises at least 1 bicyclic nucleoside and at least 1 2'-MOE nucleoside. In certain embodiments, the 3'-flanking fragment of the spacer body comprises at least 1 bicyclic nucleoside and at least 1 2'-OMe nucleoside. In certain embodiments, the 3'-flanking fragment of the spacer body comprises at least 1 bicyclic nucleoside and at least 1 2'-deoxynucleoside.

[0586] In certain embodiments, the 3' flanking fragment of the spacer contains at least one constrained ethyl nucleoside and at least one non-bicyclic modified nucleoside. In certain embodiments, the 3' flanking fragment of the spacer contains at least one constrained ethyl nucleoside and at least one 2'-substituted nucleoside. In certain embodiments, the 3' flanking fragment of the spacer contains at least one constrained ethyl nucleoside and at least one 2'-MOE nucleoside. In certain embodiments, the 3' flanking fragment of the spacer contains at least one constrained ethyl nucleoside and at least one 2'-OMe nucleoside. In certain embodiments, the 3' flanking fragment of the spacer contains at least one constrained ethyl nucleoside and at least one 2'-deoxynucleoside.

[0587] In certain embodiments, the 3' flanking fragment of the spacer body comprises at least 1 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' flanking fragment of the spacer body comprises at least 2 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' flanking fragment of the spacer body comprises at least 3 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' flanking fragment of the spacer body comprises at least 4 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' flanking fragment of the spacer body comprises at least 5 modified nucleosides selected from 2'-MOE nucleosides, 2'-OMe nucleosides, LNA-containing nucleosides, GuNA-containing nucleosides, ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, ALNA[ipU]-containing nucleosides, ALNA[Trz]-containing nucleosides, and / or ALNA[Oxz]-containing nucleosides. In certain embodiments, the 3' flanking fragment of the spacer body comprises at least 6 modified nucleosides selected from 2'-MOE nucleosides, 2'-OMe nucleosides, LNA-containing nucleosides, GuNA-containing nucleosides, ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, ALNA[ipU]-containing nucleosides, ALNA[Trz]-containing nucleosides, and / or ALNA[Oxz]-containing nucleosides.In certain embodiments, the 3'-flanking fragment of the spacer body comprises at least 7 modified nucleosides selected from 2'-MOE nucleosides, 2'-OMe nucleosides, LNA-containing nucleosides, GuNA-containing nucleosides, ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, ALNA[ipU]-containing nucleosides, ALNA[Trz]-containing nucleosides, and / or ALNA[Oxz]-containing nucleosides. In certain embodiments, the 3'-flanking fragment of the spacer body comprises at least 8 modified nucleosides selected from 2'-MOE nucleosides, 2'-OMe nucleosides, LNA-containing nucleosides, GuNA-containing nucleosides, ALNA[Ms]-containing nucleosides, ALNA[mU]-containing nucleosides, ALNA[ipU]-containing nucleosides, ALNA[Trz]-containing nucleosides, and / or ALNA[Oxz]-containing nucleosides.

[0588] In certain embodiments, the 3' flanking fragment of the spacer contains 2 LNA-containing nucleosides. In certain embodiments, the 3' flanking fragment of the spacer contains 3 LNA-containing nucleosides. In certain embodiments, the 3' flanking fragment of the spacer contains 4 LNA-containing nucleosides. In certain embodiments, the 3' flanking fragment of the spacer contains 3 ALNA[Ms]-containing nucleosides. In certain embodiments, the 3' flanking fragment of the spacer contains 2 GuNA-containing nucleosides. In certain embodiments, the 3' flanking fragment of the spacer contains 3 GuNA-containing nucleosides. In certain embodiments, the 3' flanking fragment of the spacer contains 3 ALNA[mU]-containing nucleosides. In certain embodiments, the 3' flanking fragment of the spacer contains 3 ALNA[ipU]-containing nucleosides. In certain embodiments, the 3' flanking of the spacer contains 2 LNA-containing nucleosides and 1 GuNA-containing nucleoside. In certain embodiments, the 3' flanking fragment of the spacer contains 3 ALNA[Trz]-containing nucleosides. In certain embodiments, the 3' flanking fragment of the spacer contains 3 ALNA[Ms]-containing nucleosides and 1 2'-OMe nucleoside. In certain embodiments, the 3' flanking fragment of the spacer contains 3 ALNA[Ms]-containing nucleosides and 2 2'-OMe-containing nucleosides. In certain embodiments, the 3' flanking fragment of the spacer contains 3 2'-MOE nucleosides. In certain embodiments, the 3' flanking fragment of the spacer contains 4 2'-MOE nucleosides. In certain embodiments, the 3' flanking fragment of the spacer contains 5 2'-MOE. In certain embodiments, the 3' flanking fragment of the spacer contains 3 ALNA[Ms]-containing nucleosides and 1 2'-MOE-containing nucleoside. In certain embodiments, the 3' flanking fragment of the spacer contains 3 ALNA[Ms]-containing nucleosides and 2 2'-MOE nucleosides. In certain embodiments, the 3' flanking fragment of the spacer contains 2 ALNA[Ms]-containing nucleosides and 2 2'-MOE nucleosides. In certain embodiments, the 3' flanking fragment of the spacer contains 2 ALNA[Ms]-containing nucleosides and 3 2'-MOE nucleosides. In certain embodiments, the 3' flanking fragment of the spacer contains 3 ALNA[Ms]-containing nucleosides and 5 2'-MOE nucleosides. In certain embodiments, the 3' flanking fragment of the spacer contains 1 ALNA[Ms]-containing nucleoside and 3 2'-MOE nucleosides. In certain embodiments, the 3' flanking fragment of the spacer contains 2 LNA-containing nucleosides and 2 2'-MOE nucleosides.

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

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

[0591] In certain embodiments, the 3'-flanking fragment of the spacer is composed of 1, 2, or 3 linked nucleosides containing ALNA[Ms], and contains 1 5-methylcytosine. In certain embodiments, the 3'-flanking fragment of the spacer is composed of 1, 2, or 3 linked nucleosides containing ALNA[Ms], and contains 2 5-methylcytosines. In certain embodiments, the 3'-flanking fragment of the spacer is composed of 1, 2, or 3 linked nucleosides containing ALNA[Ms], and contains 3 5-methylcytosines. In certain embodiments, the 3'-flanking fragment of the spacer is composed of 1, 2, 3, 4, or 5 linked 2'-MOE nucleosides, and contains 1 5-methylcytosine.

[0592] In certain embodiments, the 5'-flanking fragment of the spacer is composed of 1 nucleoside containing ALNA[Ms], and the 3'-flanking fragment is composed of 1 nucleoside containing ALNA[Ms]. In certain embodiments, the 5'-flanking fragment of the spacer is composed of 2 linked nucleosides containing ALNA[Ms], and the 3'-flanking fragment is composed of 2 linked nucleosides containing ALNA[Ms]. In certain embodiments of the fragment, the 5'-flanking fragment of the spacer is composed of 3 linked nucleosides containing ALNA[Ms], and the 3'-flanking fragment is composed of 3 linked nucleosides containing ALNA[Ms]. In certain embodiments, the 5'-flanking fragment of the spacer is composed of 4 linked nucleosides containing ALNA[Ms], and the 3'-flanking fragment is composed of 4 linked nucleosides containing ALNA[Ms]. In certain embodiments, the 5'-flanking fragment of the spacer is composed of 5 linked nucleosides containing ALNA[Ms], and the 3'-flanking fragment is composed of 5 linked nucleosides containing ALNA[Ms].

[0593] In certain embodiments, the 5'-flanking fragment of the spacer is composed of 1, 2, or 3 linked nucleosides containing ALNA[Ms] and 1 2'-OMe nucleoside, and the 3'-flanking fragment is composed of 1, 2, or 3 linked nucleosides containing ALNA[Ms] and 1 2'-OMe nucleoside. In certain embodiments, the 5'-flanking fragment of the spacer is composed of 1, 2, or 3 linked nucleosides containing ALNA[Ms] and 2 2'-OMe nucleosides, and the 3'-flanking fragment is composed of 1, 2, or 3 linked nucleosides containing ALNA[Ms] and 2 linked 2'-OMe nucleosides. In certain embodiments, the 5'-flanking fragment of the spacer is composed of 1, 2, or 3 linked nucleosides containing ALNA[Ms] and 3 2'-OMe nucleosides, and the 3'-flanking fragment is composed of 1, 2, or 3 linked nucleosides containing ALNA[Ms] and 3 2'-OMe nucleosides.

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

[0595] In certain embodiments, the 5'-flanking segment of the spacer body consists of 1, 2, or 3 linked nucleosides containing ALNA[Ms], and contains 1 5-methylcytosine. Further, the 3'-flanking segment of the spacer body consists of 1, 2, or 3 linked nucleosides containing ALNA[Ms], and contains 1 5-methylcytosine. In certain embodiments, the 5'-flanking segment of the spacer body consists of 1, 2, or 3 linked nucleosides containing ALNA[Ms], and contains 2 5-methylcytosines. Further, the 3'-flank of the spacer body consists of 1, 2, or 3 linked nucleosides containing ALNA[Ms], and contains 1 5-methylcytosine. In certain embodiments, the 5'-flanking segment of the spacer body consists of 1, 2, or 3 linked nucleosides containing ALNA[Ms], and contains 2 5-methylcytosines. Further, the 3'-flanking segment of the spacer body consists of 1, 2, or 3 linked nucleosides containing ALNA[Ms], and contains 2 5-methylcytosines. In certain embodiments, the 5'-flanking segment of the spacer body consists of 1, 2, or 3 linked nucleosides containing ALNA[Ms], and contains 3 5-methylcytosines. Further, the 3'-flanking segment of the spacer body consists of 1, 2, or 3 linked nucleosides containing ALNA[Ms], and contains 3 5-methylcytosines. In certain embodiments, the 5'-flanking segment of the spacer body consists of 1, 2, or 3 linked nucleosides containing ALNA[Ms], and contains 1 5-methylcytosine. Further, the 3'-flanking segment of the spacer body consists of 1, 2, 3, 4, or 5 linked 2'-MOE nucleosides, and contains 1 5-methylcytosine. In certain embodiments, the 5'-flanking segment of the spacer body consists of 1, 2, or 3 linked nucleosides containing ALNA[Ms]. Further, the 3'-flanking segment of the spacer body consists of 1, 2, or 3 linked nucleosides containing ALNA[Ms] and 1, 2, or 3 linked 2'-MOE nucleosides, and contains 1 5-methylcytosine.

[0596] In certain embodiments, the spacer segment of the spacer body contains 10 consecutive nucleosides, contains a 2'-OMe nucleoside in any one of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, or 10th nucleosides, and the remaining nucleosides are deoxynucleosides.

[0597] Compositions and methods for formulating pharmaceutical compositions

[0598] To prepare 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 according to some judgment criteria including the administration route, the degree of the disease, or the dosage administered, but are not limited to these.

[0599] Modified oligonucleotides targeting DUX4 nucleic acids can be utilized in pharmaceutical compositions by combining them with a suitable pharmaceutically acceptable diluent or carrier. As a pharmaceutically acceptable diluent, phosphate buffered saline (PBS) can be mentioned. PBS is a diluent suitable for use in compositions for parenteral delivery. Thus, in one embodiment, a pharmaceutical composition comprising a modified oligonucleotide targeting DUX4 nucleic acids and a pharmaceutically acceptable diluent can be used in the methods described in this specification. In certain embodiments, the pharmaceutically acceptable diluent is PBS.

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

[0601] To form an active modified oligonucleotide, a prodrug can comprise additional nucleosides incorporated at one or both ends of the modified oligonucleotide, which is cleaved in vivo by endogenous nucleases.

[0602] Conjugated modified oligonucleotides

[0603] A 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. As exemplary conjugating groups, cholesterol moieties and lipid moieties can be mentioned. As further conjugating groups, carbohydrates, phospholipids, biotin, phenazines, folic acid, phenanthridines, anthraquinones, acridines, fluoresceins, rhodamines, coumarins, and pigments can be mentioned.

[0604] For example, to enhance properties such as nuclease stability, a modified oligonucleotide can also be modified in such a way as to have one or more stabilizing groups generally bonded to one end or both ends of the modified oligonucleotide. Stabilizing groups include capping structures. These terminal modifications protect modified oligonucleotides with terminal nucleic acids from exonuclease degradation and can contribute to intracellular delivery and / or localization. A cap can be present at the 5' end (5' cap) or the 3' end (3' cap), or can be present at both ends. Capping structures are well known in the art and, for example, inverted deoxyabasic caps can be mentioned. As further 3' and 5' stabilizing groups that can be used to cap one or both ends of a modified oligonucleotide to confer nuclease stability, the stabilizing groups disclosed in WO03 / 004602 can be mentioned.

[0605] Cell culture and treatment with modified oligonucleotides

[0606] The effect of a modified oligonucleotide on the level, activity, or expression of DUX4 nucleic acid can be tested in vitro in a variety of cell types. The cell types used in such assays can be obtained 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 using commercially available reagents (e.g., Invitrogen Life Technologies, Carlsbad, CA) according to the supplier's instructions. Exemplary cell types include C2C12 cells, HepG2 cells, Hep3B cells, primary hepatocytes, A549 cells, GM04281 fibroblasts, and LLC-MK2 cells, but are not limited to these. These cells can be transfected with a vector expressing human DUX4 mRNA. As the vector, it is preferably expressed as a fusion protein with a reporter gene such as luciferase or GFP, and examples include psiCHECK-2 vector (Promega).

[0607] In vitro assays of modified oligonucleotides

[0608] The method of treating cells with a modified oligonucleotide is described in this specification and can be appropriately changed according to the type of modified oligonucleotide.

[0609] Typically, cells are treated with a modified oligonucleotide when they reach a confluence of approximately 60-80% in culture.

[0610] The modified oligonucleotide is introduced into cells, for example, by lipofection.

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

[0612] As other reagents for introducing modified oligonucleotides into cultured cells, LIPOFE CTAMINE2000 (registered trademark) (Invitrogen, Carlsbad, CA) can be mentioned. In OPTI-MEM (registered trademark) 1 serum-reduced medium (Invitrogen, Carlsbad, CA), the modified oligonucleotide is mixed with LIPOFECTAMINE2000 (registered trademark) to obtain the desired concentration of the modified oligonucleotide and typically a LIPOFECTIN (registered trademark) concentration of 2 to 12 μg / mL relative to every 100 nM of the modified oligonucleotide.

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

[0614] As another method for introducing modified oligonucleotides into cultured cells, electroporation can be mentioned.

[0615] Modified oligonucleotides can be introduced into cells even without using LIPOFECTIN or the like. The method of inhibiting the expression of the target gene of the modified oligonucleotide at this time is called the Gymnosis method.

[0616] Cells are treated with the modified oligonucleotide by a conventional method. Typically, the cells are collected 16 to 48 hours after the modified oligonucleotide treatment, and at this time, the RNA or protein level of the target nucleic acid is measured by methods known in the art and described in this specification. Usually, when the treatment is repeatedly performed multiple times, data showing the average value of the repeated treatments is presented.

[0617] The concentration of the modified oligonucleotide used varies depending on the cell system. The method for determining the optimal concentration of the modified oligonucleotide for a specific cell system is well-known in the art. When using LIP OFECTAMINE2000 (registered trademark), LIPOFECTIN, or Cytofectin for transfection, typically, the modified oligonucleotide is used at a concentration of 1 nM to 300 nM. When using electroporation for transfection, the modified oligonucleotide is used at a higher concentration of 625 to 20000 nM. The concentration of the modified oligonucleotide (IC50) that inhibits 50% of the gene expression can be calculated from the inhibition rate of gene expression at each concentration.

[0618] RNA isolation

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

[0620] Analysis of inhibition of target level or expression

[0621] Inhibition of DUX4 nucleic acid level or expression can be analyzed by various methods known in the art. For example, the target nucleic acid level can be quantified 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 routine in the art. Quantitative real-time PCR can be conveniently achieved using a commercially available ABI PRISM (registered trademark) 7600, 7700, or 7900 sequence detection system according to the manufacturer's instructions available from PE-Applied Biosystems, Foster City, CA.

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

[0623] Quantification of the target RNA level can be achieved by quantitative real-time PCR using an ABI PRISM (registered trademark) 7600, 7700, or 7900 sequence detection system (PE-Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. Methods for quantitative real-time PCR are well known in the art.

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

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

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

[0627] Analysis at the protein level

[0628] The antisense inhibition of the DUX4 nucleic acid is evaluated by measuring the level of the DUX4 protein. The level of the DUX4 protein can be investigated or quantified by various methods well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein analysis, protein activity analysis (such as caspase activity analysis), 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 can be prepared by conventional monoclonal or polyclonal antibody production methods well known in the art.

[0629] Analysis of gene expression

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

[0631] In vivo experiments of modified oligonucleotides

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

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

[0634] A certain antisense mechanism

[0635] FSHD is caused by the abnormal expression of the DUX4 gene (especially the spliced variant of DUX4-FL) in muscle. On the other hand, DUX4 is also expressed in healthy individuals, such as in the testis. Among certain spliced variants of DUX4 expressed in the testis and the like, in addition to DUX4-FL, spliced variants of exon 1, exon 2, exon 6, exon 7, and spliced variants of exon 1, exon 2, exon 4, exon 5, exon 6, exon 7 are also expressed (Non-Patent Document 1 above).

[0636] A certain biomarker

[0637] The expression of genes such as MBD3L2, ZS CAN4, TRIM43, DEFB103, ZNF217, etc. is regulated at least in part by the cumulative level of DUX4 protein (Non-Patent Document 2 above). In addition, creatine kinase in the blood can be measured as a biomarker of muscle disorder.

[0638] A certain indication

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

[0640] Accordingly, the present specification provides a method of ameliorating symptoms associated with FSHD in a subject in need thereof. In certain embodiments, a method of reducing the incidence of one or more symptoms associated with FSHD is provided. In certain embodiments, a method of reducing the severity of symptoms associated with FSHD is provided. In certain embodiments, as symptoms associated with FSHD, muscle rigidity, myotonia, facial muscle weakness, ptosis, inability to whistle, reduced facial expression changes, depressive or angry facial expressions, dysarthria, scapular muscle weakness (such as winged scapula, drooping shoulders, etc.), lower limb weakness, hearing loss, and heart disease can be cited.

[0641] In certain embodiments, the method of the present invention comprises administering to an individual in need thereof a therapeutically effective amount of a compound that targets DUX4 nucleic acid.

[0642] 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.

[0643] In certain embodiments, a pharmaceutical composition comprising a modified oligonucleotide that targets DUX4 can be used to prepare a medicament for treating patients suffering from or prone to DUX4-related diseases such as FSHD.

[0644] In certain embodiments, the method described in the present specification comprises administering a compound comprising a modified oligonucleotide having a continuous nucleic acid base portion as described in the present specification and having a sequence set forth in SEQ ID NO: 2, 3, 4, 7-64, 69-97, or 102-112 of the sequence listing.

[0645] Administration

[0646] In certain embodiments, the compounds and pharmaceutical compositions described in the present specification are administered parenterally.

[0647] In certain embodiments, non-oral administration is effected by infusion. The infusion can be continuous or long-term, or short-term or intermittent. In certain embodiments, the medicament being infused is delivered using a pump. In certain embodiments, non-oral administration is effected by injection (e.g., bolus injection). The injectate can be delivered using a syringe.

[0648] As non-oral administration, examples thereof include subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration or intracranial administration, such as intramedullary or intraventricular administration. The administration can be continuous or long-term, or short-term or intermittent.

[0649] In certain embodiments, delivery of the compounds of the pharmaceutical compositions described in this specification results in at least 70% down-regulation of the levels of the target mRNA and / or target protein. In certain embodiments, delivery of the compounds or compositions described in this specification continues for at least 1 day, at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, at least 20 days, at least 21 days, at least 28 days, at least 30 days, at least 35 days, at least 40 days, at least 45 days, at least 50 days, at least 55 days, at least 60 days, at least 65 days, at least 70 days, at least 75 days, at least 76 days, at least 77 days, at least 78 days, 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, at least 1 year to effect 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% down-regulation of the levels of the target mRNA and / or target protein.

[0650] In certain embodiments, the modified oligonucleotide is delivered by injection or infusion once a day, once every 3 days, once a week, once every 2 weeks, once every 3 weeks, once a month, once every 2 months, once every 3 months, once every 6 months, twice a year or once a year.

[0651] Certain combination therapies

[0652] 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 a second agent is designed to treat the same FSHD as the first agent described in this specification. In certain embodiments, such a second agent is designed to treat a disease, disorder, or condition different from the first agent described in this specification. In certain embodiments, such a second agent is designed to treat an undesirable side effect of one or more pharmaceutical compositions described in this specification. In certain embodiments, the second agent is co-administered with the first agent to treat an undesirable effect of the first agent. In certain embodiments, the second agent is co-administered with the first agent to achieve an additive effect. In certain embodiments, the second agent is co-administered with the first agent to achieve a synergistic effect.

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

[0654] Certain compounds

[0655] In certain embodiments, the compounds disclosed in this specification can be used to synthesize oligomers by the phosphoramidite method using commercially available DNA·RNA synthesis amidites (including LNA). The artificial nucleic acid GuNA can be used to synthesize 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 used to synthesize oligomers by the methods described in Japanese Patent Application No. 2018-212424.

[0656] In certain embodiments, the compounds disclosed in this specification enjoy the benefit of improved one or more in vitro and / or in vivo properties compared to appropriate comparator compounds.

[0657] In certain embodiments, it is Compound No. 1 having the sequence (5’ to 3’) ngagattcccgccggt (n is 5-methylcytosine, incorporated into this specification as SEQ ID NO: 2), that is, a gapmer in which the 5’ flank and 3’ flank are each composed of three LNA-containing nucleosides, and the internucleoside bond is a phosphorothioate bond.

[0658] In certain embodiments, it is Compound No. 2 of a compound having the sequence (5' to 3') gnagttctccgcggt (n is 5-methylcytosine, incorporated herein by reference as SEQ ID NO: 3 of the Sequence Listing), i.e., a gapmer in which the 5' flank and the 3' flank are each composed of 3 nucleosides containing ALNA[Ms], and the internucleoside bond is a phosphorothioate bond.

[0659] In certain embodiments, it is Compound No. 3 of a compound having the sequence (5' to 3') gnntagacagcgtngg (n is 5-methylcytosine, incorporated herein by reference as SEQ ID NO: 4 of the Sequence Listing), i.e., a gapmer in which the 5' flank and the 3' flank are each composed of 3 nucleosides containing LNA, and the internucleoside bond is a phosphorothioate bond.

[0660] In certain embodiments, it is Compound No. 123 of a compound having the sequence (5' to 3') gnntagacagcgtngg (n is 5-methylcytosine, incorporated herein by reference as SEQ ID NO: 4 of the Sequence Listing), i.e., a gapmer in which the 5' flank and the 3' flank are each composed of 3 nucleosides containing ALNA[Ms], and the internucleoside bond is a phosphorothioate bond.

[0661] Non-limiting disclosure and incorporation by reference

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

[0663] The sequence listing attached to this application specifies each sequence in the form of either "RNA" or "DNA" as needed, but in fact these sequences can be modified by any combination of chemical modifications. Those skilled in the art can easily understand that the designation such as "RNA" or "DNA" used to describe modified oligonucleotides is arbitrary depending on the circumstances. For example, an oligonucleotide containing a nucleoside with a 2'-OH sugar moiety and a thymine base can be described as a DNA with a modified sugar (2'-OH relative to the natural 2'-H of DNA) or an RNA with a modified base (thymine (methylated uracil) relative to the natural uracil of RNA).

[0664] Accordingly, the nucleic acid sequences provided in this specification, including those in the sequence listing, are not limited to these, and are intended to include nucleic acids comprising any combination of natural or modified RNA and / or DNA, including nucleic acids containing modified nucleic acid bases, but are not limited to these. Although not limited, as a further example, an oligomeric compound having a nucleic acid base sequence of "ATCGATCG" may or may not be modified, and any oligomeric compound having such a nucleic acid base sequence is included, including compounds containing RNA bases, such as a compound having a sequence of "AUCGAUCG", and compounds 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 having a methyl group at the 5-position), but are not limited to these.

[0665] Examples

[0666] Non-limiting disclosure and incorporation by reference

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

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

[0669] Structures and abbreviations of the various artificial nucleic acids

[0670]

[0671] Example 1

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

[0673] Using various imides (LNA imides were purchased from Chem Genes and Hongene Biotechnology Limited, 2'-OMe imides were 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), modified oligonucleotide compounds were synthesized using a DNA / RNA oligonucleotide automatic synthesizer nS-8II (manufactured by Gene Design Co., Ltd.) on a CPG or polystyrene support at a scale of 0.2 or 1.0 μmol. All imides were adjusted to 0.1 M acetonitrile solutions, the coupling time for non-natural nucleosides was 10 minutes, and the other steps were carried out under the standard conditions of the nS-8II. Activator 42 (Sigma-Aldrich) was used as the activator, and Sulfurizing Reagent II (Gren Research Corporation) was used for sulfurization. The synthesized oligonucleotides were added to 28% aqueous ammonia solution and reacted at 60 - 65 °C for 8 hours to cleave from the support and deprotect the base moieties. After concentrating and distilling off ammonia, reverse-phase HPLC purification was carried out.

[0674] Example 2

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

[0676] Using various imides, a modified oligonucleotide compound was synthesized on a polystyrene support on a scale of 20 - 50 μmol using a DNA / RNA oligonucleotide automatic synthesizer AKTAoligo pilot plus 10 (manufactured by GE Healthcare Japan Co., Ltd.). The DNA imide was adjusted to a 0.1 M acetonitrile solution, and the unnatural imide was adjusted to a 0.05 - 0.1 M acetonitrile solution. The coupling recycle time for the unnatural nucleoside was 20 minutes. When introducing the first base into the universal carrier, the coupling, sulfurization, and capping steps were each performed twice continuously. Other steps were carried out under the standard conditions of AKTA oligopilot plus 10. Activator 42 (Sigma-Aldrich) was used as the activator, and Sulfurizing Reagent II (Gren Research Corporation) was used for sulfurization. The synthesized oligonucleotide was subjected to decyanoethylation treatment 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 moiety. After concentrating and distilling off the ammonia, purification was carried out using an anion exchange column. The remaining salts contained after anion exchange were removed using a desalting column.

[0677] Example 3

[0678] Purity confirmation of modified oligonucleotide compounds

[0679] Purification and purity confirmation of the synthesized modified oligonucleotide compound were carried out by reverse-phase HPLC under the following conditions. The purity of any compound was 85% or more.

[0680] Reverse-phase HPLC (purification)

[0681] Mobile phase:

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

[0683] Solution B: Methanol

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

[0685] Column used:

[0686] Allocate Waters XBridge@Oligonucleotide BEH C18 OBDTM Prep Column, 2.5 μm, 10 mm * 50 mm

[0687] Flow rate:

[0688] Flow rate: 5 mL / min

[0689] Column temperature: 60 °C

[0690] Detection: UV (260 nm)

[0691] Reverse-phase HPLC (purity confirmation)

[0692] Mobile phase:

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

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

[0695] Column used:

[0696] Analytical Waters ACQUITY UPLC@Oligonucleotide BEH C18 Column, 1.7 μm, 2.1 mm * 50 mm

[0697] Flow rate: 0.2 mL / min

[0698] Column temperature: 60 °C

[0699] Detection: UV (260 nm)

[0700] Anion exchange purification

[0701] Mobile phase:

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

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

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

[0705] Flow rate: 7 mL / min

[0706] Column temperature: room temperature

[0707] Detection: UV (260 nm)

[0708] Desalting column

[0709] Mobile phase:

[0710] Solution A: 20% acetonitrile aqueous solution

[0711] Solution B: 20% acetonitrile aqueous solution

[0712] Column used:

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

[0714] Flow rate: 12 mL / min

[0715] Column temperature: room temperature

[0716] Example 4

[0717] Determination of the molecular weight of the modified oligonucleotide compound

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

[0719] Mobile phase:

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

[0721] Solution B: methanol

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

[0723] Column used:

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

[0725] Flow rate: 0.2 mL / min

[0726] Column temperature: 60 °C.

[0727] Detection: UV (260 nm)

[0728] Example 5

[0729] Molecular weight of synthesized modified oligonucleotide compounds

[0730] The synthesized modified oligonucleotide compounds are shown in Table 1 below. The compounds are represented by three letters for each nucleotide. Among them, the nucleotide at the 3'-end has no internucleoside bond, so it is represented by two letters.

[0731] 1) The first letter is in uppercase and represents the following nucleic acid bases:

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

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

[0734] l = LNA, g = GuNA, m = ALNA[Ms], u = ALNA[mU], p = ALNA[ipU], t = ALNA[Trz], e = 2'-MOE, o = 2'-OMe, d = 2'-deoxyribose,

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

[0736] s = phosphorothioate, p = phosphodiester.

[0737] As the target position, the 5' target site of the DUX4 mature mRNA of the modified oligonucleotide is shown (the position of Sequence Listing Serial Number 1 corresponding to the 3' end of the modified oligonucleotide).

[0738] [Table 1-1]

[0739]

[0740] [Table 1-2]

[0741] (Continued Table 1)

[0742]

[0743] [Table 1-3]

[0744] (Continued Table 1)

[0745]

[0746] [Table 1-4]

[0747] (Continued Table 1)

[0748]

[0749] [Table 1-5]

[0750] (Continued Table 1)

[0751]

[0752] [Table 1-6]

[0753] (Continued Table 1)

[0754]

[0755] Example 6

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

[0757] C2C12 cells were seeded at a density of 1.25×10 4 Cells / cm 2 onto a transfection reagent mixed with DUX4-modified oligonucleotides and Lipofectamine RNAi reagent, and cultured overnight in a CO2 incubator. The next day, cells were transfected with a reporter plasmid in which the DUX4 sequence was cloned into the multiple cloning site of the psiCHECK-2 vector (Promega) using Lipofectamine 2000 reagent, and cultured in a CO2 incubator for about 24 hours. Then, the Dual-Glo Luciferase Assay System was used to detect the luminescence values of firefly luciferase and Renilla luciferase in the cells using a microplate reader. To correct for the effects of transfection efficiency and cell number on the luminescence value generated by Renilla luciferase activity, the ratio to the luminescence value generated by firefly luciferase activity was calculated. The inhibition rate was calculated as a percentage from the reduction rate of Renilla / firefly when the modified oligonucleotide was added, and the IC 50 value was calculated from the concentration at around 50% and the inhibition rate at that time (Table 2). Compared with compounds (Compound Nos. 1 to 132) complementary to positions 232 to 248, 1306 to 1325, or 1472 to 1495 of the DUX4 mature mRNA, it was found that the inhibition rates of 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 significantly weaker.

[0758] Example 7

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

[0760] In the DUX4-modified oligonucleotide solution, at a density of 6×10 3 Cells / cm 2Inoculate C2C12 cells and culture them in a CO2 incubator for 2 nights. After 2 days, remove the medium containing the DUX4-modified oligonucleotide solution from the cells and wash them with fresh medium. Then, use Lipofectamine 2000 reagent to transfect the cells with a reporter plasmid in which the DUX4 sequence is cloned into the multiple cloning site of the psiCHECK-2 vector (Promega), and culture the cells in a CO2 incubator for about 24 hours. Then, use the Dual-Glo Luciferase Assay System to detect the luminescence values of firefly luciferase and Renilla luciferase in the cells with a microplate reader. To correct for the effects of transfection efficiency and cell number by the luminescence value generated by Renilla luciferase activity, calculate the ratio to the luminescence value generated by firefly luciferase activity. Calculate the inhibition rate as a percentage from the reduction rate of Renilla / firefly when adding the modified oligonucleotide, and calculate the IC 50 value. The results are shown in Table 2 below. Compared with the compounds (Compound Nos. 1 to 132) containing nucleic acid base sequences complementary to the equi-length portions within the regions of positions 232 to 248, 1306 to 1325, or 1472 to 1495 of the DUX4 mature mRNA, it can be seen that the inhibition rates of 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) are significantly weaker.

[0761] [Table 2-1]

[0762]

[0763] [Table 2-2]

[0764] (Continued Table 2)

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

[0766] [Table 2-3]

[0767] (Continued Table 2)

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

[0769] [Table 2-4]

[0770] (Continued Table 2)

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

[0772] Example 8

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

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

[0775] It should be noted that the compounds in Table 3 are expressed with each nucleotide represented by three letters. Among them, the nucleotide at the 3'-end has no internucleoside bond, so it is represented by two letters.

[0776] 1) The first letter is in uppercase and represents the following nucleic acid bases:

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

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

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

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

[0781] s = phosphorothioate, p = phosphodiester.

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

[0783] Compared with the compounds (Compound Nos. 137 - 247) containing nucleic acid base sequences complementary to the equi-length portions within the regions of positions 126 - 147, 232 - 248, 1306 - 1325, or 1472 - 1495 of the DUX4 mature mRNA, it can be seen that the inhibition rates of Compound Nos. 248 (complementary to positions 112 - 127 of SEQ ID NO: 1 in the sequence listing), 249 (complementary to positions 162 - 177 of SEQ ID NO: 1 in the sequence listing), 250 (complementary to positions 264 - 279 of SEQ ID NO: 1 in the sequence listing), and 251 (complementary to positions 1273 - 1288 of SEQ ID NO: 1 in the sequence listing) are significantly weaker.

[0784] [Table 3]

[0785] [Table 3-1]

[0786]

[0787] Continued Table 3 [Table 3-2]

[0788]

[0789] Example 9

[0790] In vivo DUX4 knockdown activity assay

[0791] Produce the adeno-associated virus vector AAV-DUX4 (SignaGen Laboratories, Cat.# SL100862) of DUX4 mature mRNA with the serial number 1 incorporated into an ordered list. Under isoflurane (Pfizer) anesthesia, administer AAV-DUX4 intramuscularly at 1E+10 VG / 50 μL to the tibialis anterior muscle of 8-week-old C57BL / 6J mice (male, Charles River Japan). Three days later, prepare modified oligonucleotides targeting DUX4 in saline at concentrations of 1, 3, 10, and 50 mg / 5 mL / kg, and administer them to 8-week-old C57BL / 6J mice (male, Charles River Japan) via the tail vein. After 72 hours, under cervical dislocation or isoflurane (Pfizer) anesthesia, collect whole blood from the inferior vena cava of the abdomen and sacrifice the mice. After sacrifice, excise the tibialis anterior muscle, immerse it in RNAlater Soln. (invitrogen), and freeze it at -80 °C. Add the homogenization buffer of the Maxwell RSC simplyRNA Tissue Kit (Promega) to the tissue, disrupt it using a multi-bead shaker, and purify the RNA according to the protocol described in the kit. Reverse transcribe 400 ng of the RNA, and perform quantitative PCR using the resulting cDNA. The knockdown activity of the modified oligonucleotides is expressed as the ratio of the amount of 18S rRNA relative to DUX4 as a relative value to the vehicle group. The results at 1, 3, 10, and 50 mg / 5 mL / kg are shown in Figures 1 - 4Compound No. 1 (sequence complementary to positions 233 - 248 of DUX4 mature mRNA), Compound No. 2 (sequence complementary to positions 1309 - 1323 of DUX4 mature mRNA), Compound No. 3 (sequence complementary to positions 1480 - 1495 of DUX4 mature mRNA), Compound No. 13 (sequence complementary to positions 234 - 247 of DUX4 mature mRNA), Compound No. 41 (sequence complementary to positions 1308 - 1323 of DUX4 mature mRNA), Compound No. 54 (sequence complementary to positions 1309 - 1323 of DUX4 mature mRNA), Compound No. 57 (sequence complementary to positions 1309 - 1323 of DUX4 mature mRNA), Compound No. 68 (sequence complementary to positions 1309 - 1323 of DUX4 mature mRNA), Compound No. 78 (sequence complementary to positions 1309 - 1324 of DUX4 mature mRNA), Compound No. 88 (sequence complementary to positions 1310 - 1323 of DUX4 mature mRNA), Compound No. 104 (sequence complementary to positions 1473 - 1488 of DUX4 mature mRNA), and Compound No. 122 (sequence complementary to positions 1480 - 1495 of DUX4 mature mRNA) can inhibit the expression of the DUX4 gene in muscle even when administered to an organism.

[0792] Example 10

[0793] Safety of modified oligonucleotides

[0794] 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. As a result, no hepatotoxicity (increase in ALT and AST in blood and abnormal findings in pathological histology), nephrotoxicity (increase in UN and creatinine in blood and abnormal findings in pathological histology), general symptom changes, death, etc. were observed.

[0795] Example 11

[0796] In vivo DUX4 knockdown activity assay in Tg mice

[0797] Male FLExDUX4-heteto / HSA-MCM-hetero: TG(DUX4-Tg) and FLExDUX4-wild / HSA-MCM-hetero: TG(MCM, control) at 9 weeks of age (male, introduced by The Jackson Laboratory into the Japanese Charles River strain) were used. The modified oligonucleotide targeting DUX4 was prepared with physiological saline so that the dosing solution for each dose became 5 mL / kg, and was administered intravenously into the tail vein once a week. One week after the end of the 4-week dosing, whole blood was collected from the abdominal vena cava under deep anesthesia with isoflurane, and the animals were euthanized. Plasma was separated with EDTA, and creatine kinase (CK) was measured. The muscles of the lower limbs were taken, the wet weight was measured, and gene expression analysis was performed.

[0798] As Figure 5 and Figure 6 shown, Compound No. 3 and Compound No. 123 inhibited the mRNA expression of DUX4. In addition, the blood level of CK, which is a marker of muscle disorder, was decreased. On the other hand, no clear effects on DUX4 mRNA expression and blood CK level were confirmed for Compound No. 113 and Compound No. 247.

[0799] Example 12

[0800] Repeated-dose toxicity test in mice

[0801] Using physiological saline, a DUX4-targeted modified oligonucleotide was prepared to be 100 mg / 5 mL / kg, and 6-week-old ICR mice (male, Charles River, Japan) were intravenously administered via the tail vein for 4 consecutive days. 72 hours after the final administration, blood was collected from the posterior vena cava under isoflurane anesthesia, and clinical biochemical examinations were performed. In addition, autopsy was performed after bleeding to death, and pathological histological examinations of the liver and kidneys were performed. In Compound No. 123, no deaths, changes in general condition, food intake, or body weight were confirmed after administration, and no hepatotoxicity (increase in ALT and AST in serum and abnormal findings in pathological histology) or nephrotoxicity (increase in UN and creatinine in serum and abnormal findings in pathological histology) was observed. On the other hand, for Compound No. 113 and Compound No. 247, although no deaths, changes in general condition, food intake, or body weight were confirmed after administration, definite hepatotoxicity (increase in ALT, AST, GLDH, ALP, bilirubin, and bile acid in serum, abnormal findings in pathological histology: degeneration and necrosis of hepatocytes, hepatocyte hypertrophy) and nephrotoxicity (increase in creatinine in serum) were confirmed in both. In addition, the concentrations of Compound No. 123 in the liver and kidneys under these test conditions were 323 and 251 μg / g, respectively. Compared with the concentrations of Compound No. 247 in the liver and kidneys, which were 111 and 185 μg / g, and the concentrations of Compound No. 113 in the liver and kidneys, which were 39.3 and 235 μg / g, although the tissue concentrations were equal to or higher, no hepatotoxicity or nephrotoxicity was confirmed.

[0802] [Table 4]

[0803] Table 4. Blood Biochemical Examinations of Continuous Administration Test in Mice

[0804] Test items Control group Compound No. 123 Control group Compound No. 247 Compound No. 113 AST (U / L) 53.8 60.2 38.0 1187.0 1355.8 ALT (U / L) 30.4 38.6 17.6 2300.2 1282.8 GLDH (U / L) 16.0 36.0 7.0 813.2 768.8 ALP (U / L) 340.2 345.2 204.2 1063.8 640.6 T - Bil (mg / dL) 0.086 0.052 0.106 1.208 0.298 D - Bil (mg / dL) 0.024 0.012 0.044 1.086 0.238 I - Bil (mg / dL) 0.062 0.040 0.062 0.122 0.060 TBA (μmol / L) 0.80 1.60 0.90 28.02 63.32 UN (mg / dL) 20.34 18.62 18.32 23.02 23.20 Cre (mg / dL) 0.056 0.054 0.062 0.126 0.152

[0805] [Table 5]

[0806] Table 5. Pathological Findings of Continuous Administration Test in Mice

[0807] Pathological findings Control group Compound No. 123 Control group Compound No. 247 Compound No. 113 Degeneration and necrosis of hepatocytes 0 / 5 0 / 5 0 / 5 3 / 5 4 / 5 Hepatocyte hypertrophy 0 / 5 0 / 5 0 / 5 4 / 5 5 / 5

[0808] Reference Example

[0809] The synthetic routes of nucleotides containing ALNA[Ms], nucleotides containing ALNA[mU], nucleotides containing ALNA[ipU], nucleotides containing ALNA[Trz], and nucleotides containing ALNA[Oxz] are shown below. It should be noted that starting compounds 1a, 1d, and 1g can be synthesized by the methods described in International Publication No. 2017 / 047816.

[0810] Synthesis of ALNA[Ms]-T

[0811]

[0812] Synthesis of ALNA[Ms]-mC

[0813]

[0814] Synthesis of ALNA[Ms]-G

[0815]

[0816] Synthesis of ALNA[Ms]-A

[0817]

[0818] Synthesis of ALNA[mU]-T

[0819]

[0820] Synthesis of ALNA[mU]-mC

[0821]

[0822] Synthesis of ALNA[mU]-G

[0823]

[0824] Synthesis of ALNA[mU]-A

[0825]

[0826] Synthesis of ALNA[ipU]-T

[0827]

[0828] Synthesis of ALNA[ipU]-mC

[0829]

[0830] Synthesis of ALNA[ipU]-G

[0831]

[0832] Synthesis of ALNA[ipU]-A

[0833]

[0834] Synthesis of ALNA[Trz]-T

[0835]

[0836] Synthesis of ALNA[Trz]-mC

[0837]

[0838] Synthesis of ALNA[Trz]-G

[0839]

[0840] Synthesis of ALNA[Trz]-A

[0841]

[0842] Synthesis of ALNA[Oxz]-T

[0843]

[0844] Synthesis of ALNA[Oxz]-mC

[0845]

[0846] Industrial Applicability

[0847] The modified oligonucleotides of the present invention can be used as compounds useful for the treatment, prevention, or delay of the progression of DUX4-related diseases.

[0848] [Sequence Listing Free Text]

[0849] SEQ ID NO: 1 in the sequence listing represents the base sequence of DUX4 mature mRNA.

[0850] SEQ ID NOs: 2 to 4 and 7 to 112 in the sequence listing represent the base sequences of modified oligonucleotides.

[0851] SEQ ID NOs: 5 to 6 in the sequence listing represent the base sequences of DUX4-FL2 and DUX4-s, respectively, as splicing variants of SEQ ID NO: 1.

Claims

1. A modified oligonucleotide consisting of 12 to 30 residues, comprising at least 8 consecutive nucleic acid base sequences that are complementary to the corresponding length portions starting from 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 nucleic acid base sequence of the modified oligonucleotide having at least 90% complementarity with the corresponding length portion of the nucleic acid base sequence of the mature mRNA of DUX4 of SEQ ID NO: 1, in the case where the at least 8 consecutive nucleic acid base sequences include a nucleic acid base sequence complementary to the corresponding length portion starting from position 1480 - 1495 from the 5'-end of the nucleic acid base sequence of SEQ ID NO: 1, the modified oligonucleotide consists of a nucleic acid base sequence having a complementary base to the base at position 1480 from the 5'-end of the nucleic acid base sequence of SEQ ID NO: 1 at the 3'-end.

2. The modified oligonucleotide according to claim 1, wherein One or more modified nucleotides in the modified oligonucleotide contain a modified sugar.

3. The modified oligonucleotide according to claim 2, wherein, The modified sugar is selected from bicyclic sugars, sugars modified by 2'-O-methoxyethyl, and sugars modified by 2'-O-methyl.

4. The modified oligonucleotide according to claim 3, wherein The bicyclic sugars are selected from LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], and ALNA[Trz].

5. A modified oligonucleotide consisting of 12 to 30 residues, comprising at least 8 consecutive nucleic acid base sequences that are complementary to the corresponding length portion starting from positions 1472 - 1495 from the 5'-end of the nucleic acid base sequence of the mature mRNA of DUX4 of SEQ ID NO: 1, the nucleic acid base sequence of the modified oligonucleotide having at least 90% complementarity with the corresponding length portion of the nucleic acid base sequence of the mature mRNA of DUX4 of SEQ ID NO: 1, the modified oligonucleotide containing at least one nucleoside comprising a modified sugar selected from GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz], and ALNA[Trz].

6. The modified oligonucleotide according to claim 5, wherein, The modified oligonucleotide further comprises a sugar modified by 2'-O-methoxyethyl and / or a sugar modified by 2'-O-methyl.

7. The modified oligonucleotide according to any one of claims 1 to 6, wherein, At least one modified nucleotide in the modified oligonucleotide contains a modified nucleic acid base.

8. The modified oligonucleotide according to claim 7, wherein, The modified nucleic acid base is 5-methylcytosine.

9. The modified oligonucleotide according to any one of claims 1 to 8, wherein At least one internucleoside bond is a modified internucleoside bond.

10. The modified oligonucleotide according to claim 9, wherein, The modified internucleoside bond is a phosphorothioate internucleoside bond.

11. The modified oligonucleotide according to any one of claims 1 to 10, wherein, The modified oligonucleotide comprises: 1) a spacer segment, 2) a 5'-flanking segment, and 3) a 3'-flanking segment; the spacer segment is located between the 5'-flanking segment and the 3'-flanking segment, the nucleosides of the 5'-flanking segment and the 3'-flanking segment each contain at least one modified sugar, the nucleosides of the spacer segment are either nucleosides without modified sugars only, or nucleosides containing one or two nucleosides with modified sugars and no other modified sugars.

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

1.

13. The modified oligonucleotide according to any one of claims 1 to 12, wherein, The modified oligonucleotide is composed of the base sequences of gtggcgatgc ccgggt, i.e., SEQ ID NO: 75, gagattcccg cnggtg, i.e., SEQ ID NO: 78, ngagattcccgccggt, i.e., SEQ ID NO: 2, gnagttctccgcggt, i.e., SEQ ID NO: 3, or gnntag acagcgtngg, i.e., SEQ ID NO: 4, where n represents 5-methylcytosine.

14. The modified oligonucleotide according to claim 13 is represented by the following formula: GlsMlsMlsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMlsGlsGl, In the formula, Each nucleic acid base is represented by the following notations: A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine, Each sugar moiety is represented by the following notations: l = LNA, d = 2'-deoxyribose, Each internucleoside bond is represented by the following notations: s = phosphorothioate.

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

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

17. The modified oligonucleotide according to claim 13 is represented by the following formula: MlsGlsAlsGdsAdsTdsTdsCdsCdsCdsGdsCdsCdsGlsGlsTl, In the formula, Each nucleic acid base is represented by the following notations: A = adenine, T = thymine, G = guanine, C = cytosine, M = 5-methylcytosine; Each sugar moiety is represented by the following notations: l = LNA, d = 2'-deoxyribose, The internucleoside linkages are represented by the following notations: s = phosphorothioate.

18. The modified oligonucleotide or a salt thereof according to claim 14 is represented by the following formula:

19. The modified oligonucleotide or a salt thereof according to claim 15 is represented by the following formula:

20. The modified oligonucleotide or a salt thereof according to claim 16 is represented by the following formula:

21. The modified oligonucleotide or a salt thereof according to claim 17 is represented by the following formula:

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 treating, preventing or delaying the progression of a DUX4-related disease in a subject, characterized in that, An effective amount of the modified oligonucleotide according to any one of claims 1 to 21 is administered to a subject in need thereof.

26. Use of the modified oligonucleotide according to any one of claims 1 to 21 for 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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