RU2831087C2 - Compound, method and pharmaceutical composition for modulating dux4 expression - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: presented group of inventions can be used in medicine. There are disclosed modified oligonucleotides capable of inhibition of double homeobox 4 (DUX4) gene expression.

EFFECT: invention can be used in treating diseases associated with abnormal expression of said gene in an individual, in particular, in antisense gene therapy of facioscapulohumeral muscular dystrophy (FSHD), B-cell acute lymphocytic leukemia, differentiated round-cell sarcoma and fetal rhabdomyosarcoma.

29 cl, 6 dwg, 5 tbl, 12 ex

Description

PREREQUISITES FOR THE CREATION OF THE INVENTION

Field of technology to which the invention relates

[0001]

The present invention relates to a compound that reduces the expression of DUX4 mRNA and protein in animals, a method for using the compound and a pharmaceutical composition containing the compound. The method of the present invention can be used for the therapeutic treatment, prevention or alleviation of diseases associated with DUX4, such as facioscapulohumeral muscular dystrophy (FSHD).

Description of the prior art

[0002]

Facioscapulohumeral muscular dystrophy (FSHD) is a muscular dystrophy that occurs with a frequency of approximately 1 in 20,000 worldwide and 1 in 7,500 in Europe, and is the third most common muscular dystrophy after Duchenne muscular dystrophy and myotonic dystrophy. The initial symptom is weakness of the facial muscles, upper limbs, scapula, and pelvic girdle. The pelvic girdle and lower limbs are also affected as the disease progresses, and about 20% of patients will be wheelchair-bound by age 40 (even in middle age there may only be mild facial involvement). Complications of pain, neurological deafness, and retinopathy are also common. About 90% of patients develop symptoms by age 20. In severely affected patients (about 4%), muscle weakness is present from infancy.

[0003]

FSHD is divided into two types: FSHD1 and FSHD2, depending on the genes that cause the disease. FSHD1 accounts for about 95% of all FSHD patients. In patients with FSHD1, the D4Z4 repeat region on 4q35 is genetically shortened (1 to 10 D4Z4 repeats). This results in abnormal expression of DUX4, which is encoded in the D4Z4 repeat region (DUX4 is not expressed in healthy individuals). FSHD2 accounts for about 5% of all FSHD patients, and abnormal expression of DUX4 is due to a mutation in SMCHD1 (DNA methylase). DUX4 functions as a transcription factor and expresses a group of genes that are encoded downstream and cause muscle cell apoptosis or muscle atrophy. Abnormal expression of DUX4 is associated with the presence of an allele called 4qA, one of the two alleles 4qA and 4qB. The polyadenylation site present in 4qA is required for stabilization of DUX4 mRNA (Non-Patent Document 1, Non-Patent Document 2, Non-Patent Document 3).

[0004]

Antisense technology is emerging as an effective means to modulate the expression of specific gene products and may thus prove uniquely useful in some therapeutic, diagnostic and research applications for DUX4 modulation.

[0005]

A method for suppressing the expression of the DUX4 gene using an adeno-associated virus encoding DUX4 siRNA has been reported (Patent Document 1). However, the production of adeno-associated virus is cumbersome, and the delivery of adeno-associated virus to the desired systemic muscles is difficult.

[0006]

A method for silencing the expression of the DUX4 gene using a lentivirus encoding DUX4 shRNA has been reported (Patent Document 2). However, the production of the lentivirus is cumbersome, and the delivery of the lentivirus to the desired systemic muscles is difficult. In addition, the in vitro gene silencing has 21% and 44% residual activity in the cells of the quadriceps and trapezius muscles, and thus is insufficient.

[0007]

A compound in which multiple antisense oligonucleotides targeting DUX4 are linked has been reported (Patent Document 2). However, it is not a modified oligonucleotide, and its inhibitory effect is insufficient.

[0008]

An antisense oligonucleotide compound that binds to the splice site of DUX4 mRNA has been reported (Patent Document 3). However, since these compounds are selective for pre-mRNA containing an intron, their inhibitory effect on mature mRNA is weak, and due to knockdown by lipofection, they are difficult to administer to a living organism.

[0009]

Current treatment methods for FSHD include rehabilitation (stretching and exercise) as symptomatic treatment, NSAIDs, respiratory care, etc. However, their effects are insufficient and the burden on the patient is high. Therefore, the present document aims to provide a compound, composition, and method for treating FSHD.

[Prior Art]

[Patent documents]

[0010]

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

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

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

[Non-patent documents]

[0011]

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

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

[Non-patent document 3] Sacconi et al., Biochim. Biophys. Acta 2015, p607.

The entire contents of these publications are incorporated herein by reference.

ESSENCE OF THE INVENTION

[Problems solved by the invention]

[0012]

The aim of the present invention is to provide a compound, a method and a pharmaceutical composition for inhibiting the expression of DUX4 and for treating, i.e. therapeutically treating, preventing, delaying or alleviating DUX4-related diseases and/or their symptoms. The compound and pharmaceutical composition described herein also inhibit mutant DUX4, such as SNP and DUX4 splice variants.

[0013]

In some embodiments, the invention provides a method for reducing DUX4 expression in animals (including humans), wherein the method comprises administering to the animal a compound comprising a modified oligonucleotide targeting DUX4, or a pharmaceutical composition comprising the compound, as described herein.

[0014]

In some embodiments, the invention provides a method for knocking down a nuclear ribonuclease (such as RNase H) by administering to an animal a compound or pharmaceutical composition comprising a modified oligonucleotide targeting DUX4. Furthermore, a method for inhibiting transcription of DUX4 mRNA and translation of DUX4 protein by administering a compound comprising a modified oligonucleotide is provided. The modified oligonucleotide is preferably distributed in muscle, and is particularly preferred in skeletal muscle.

[0015]

In some embodiments, the invention provides a method of treating an animal suffering from FSHD. In some embodiments, the method of the present invention further comprises administering to the animal a therapeutically effective amount of a compound or pharmaceutical composition comprising a modified oligonucleotide targeting DUX4 described herein. In some embodiments, the method of the present invention comprises identifying an animal suffering from FSHD1 and/or FSHD2.

[0016]

In some embodiments of the invention, a method of treating, i.e. therapeutically treating, preventing, delaying or alleviating muscle atrophy and muscle weakness is provided. It is intended to alleviate or delay the worsening of poor facial expressions, sleep with open eyes, difficulty in raising the upper limbs, and winged scapulae. In addition, it is preferable to prevent muscle weakness of the lumbar girdle and lower limbs, as well as to prevent complications of neurological deafness and retinopathy.

[0017]

In some embodiments, the DUX4 mRNA has the sequence indicated by GenBank accession number NM_001293798.2 (incorporated herein as SEQ ID NO: 1 in the Sequence Listing). The splice variant of the DUX4 mRNA with SEQ ID NO: 1 in the Sequence Listing is also referred to as DUX4-FL1 or mature DUX4 mRNA. In some embodiments, the DUX4 mRNA has the sequence indicated by GenBank accession number NM_001306068.2 (incorporated herein as SEQ ID NO: 5 in the Sequence Listing). The splice variant of the DUX4 mRNA with SEQ ID NO: 5 in the Sequence Listing is also referred to as DUX4-FL2. In some embodiments, DUX4 has the sequence set forth in GenBank accession number NM_001363820.1 (incorporated herein as SEQ ID NO: 6 in the sequence listing). The mRNA splice variant of DUX4 with SEQ ID NO: 6 in the sequence listing is also referred to as DUX4-s. In some embodiments, DUX4 is a SNP of the splice variant set forth above.

[Problem Solving Tools]

[0018]

The present invention relates to, but is not limited to, the non-limiting enumerated embodiments described in the following aspects [1]-[27].

[0019]

Aspect [1] A modified oligonucleotide consisting of 12-30 residues, comprising a nucleobase sequence that comprises at least 8 immediately adjacent nucleobase sequences and is complementary to a portion of equal length at positions 126-147, 232-248, 1306-1325 or 1480-1495 from the 5'-end of the nucleobase sequence of the mature DUX4 mRNA in SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide has at least 90% complementarity with the portion of equal length in the nucleobase sequence of the mature DUX4 mRNA in SEQ ID NO: 1, when at least 8 immediately adjacent nucleobase sequences comprise a nucleobase sequence that is complementary to a portion of equal length at positions 1480-1495 from the 5'-end of the nucleobase sequence of SEQ ID NO: 1, said modified oligonucleotide consists of a nucleobase sequence having at the 3'-end a base complementary to the base at position 1480 from the 5'-end of the nucleobase of SEQ ID NO: 1.

[0020]

Aspect [2] A modified oligonucleotide according to aspect [1], wherein one or more modified nucleotides of the modified oligonucleotide each comprise a modified sugar.

Aspect [3] A modified oligonucleotide according to aspect [2], wherein the modified sugar is selected from the group consisting of a bicyclic sugar, a modified 2ʼ-O-methoxyethyl-modified sugar and a 2ʼ-O-methyl-modified sugar.

Aspect [4] A modified oligonucleotide according to aspect [3], wherein the bicyclic sugar is selected from the group consisting of LNA, GuNA, ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Oxz] and ALNA [Trz].

[0021]

Aspect [5] A modified oligonucleotide consisting of 12-30 residues, wherein the modified oligonucleotide comprises a nucleobase sequence that comprises at least 8 immediately adjacent nucleobase sequences and is complementary to an equal-length portion at positions 1472-1495 from the 5'-end of the nucleobase sequence of the mature DUX4 mRNA in SEQ ID NO: 1, said nucleobase sequence of the modified oligonucleotide is at least 90% complementary to an equal-length portion in the nucleobase sequence of the mature DUX4 mRNA in SEQ ID NO: 1, and said modified oligonucleotide comprises at least one nucleoside that comprises a modified sugar selected from GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz] and ALNA [Trz].

[0022]

Aspect [6] A modified oligonucleotide according to aspect [5], further comprising a 2ʼ-O-methoxyethyl-modified sugar and/or a 2ʼ-O-methyl-modified sugar.

[0023]

Aspect [7] A modified oligonucleotide according to any one of aspects [1]-[6], wherein at least one modified nucleotide of the modified oligonucleotide comprises a modified nucleobase.

Aspect [8] A modified oligonucleotide according to aspect [7], wherein the modified nucleobase is 5-methylcytosine.

[0024]

Aspect [9] A modified oligonucleotide according to any one of aspects [1]-[8], wherein at least one internucleoside linkage is a modified internucleoside linkage.

Aspect [10] A modified oligonucleotide according to aspect [9], wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

[0025]

Aspect [11] A modified oligonucleotide according to any one of aspects [1]-[10], comprising: 1) a gap segment, 2) a 5'-wing segment and 3) a 3'-wing segment, wherein the gap segment is located between the 5'-wing segment and the 3'-wing segment, all nucleosides of the 5'-wing segment and the 3'-wing segment each comprise at least one modified sugar, and the gap segment comprises only nucleosides that do not contain a modified sugar, or comprises one or two nucleosides each of which comprises a modified sugar and comprises other nucleosides that do not contain a modified sugar.

[0026]

Aspect [12] A modified oligonucleotide according to any of aspects [1]-[11], consisting of a nucleic base sequence that is complementary to

the nucleobase sequences at positions 128-143 from the 5'-end of the nucleobase sequence of the mature DUX4 mRNA in SEQ ID NO: 1,

nucleic acid base sequences at positions 232-247 from the 5'-end,

nucleic acid base sequences at positions 233-248 from the 5'-end,

nucleic acid base sequences at positions 1309-1323 from the 5'-end or

nucleic acid base sequences at positions 1480-1495 from the 5'-end.

[0027]

Aspect [13] A modified oligonucleotide according to any of aspects [1]-[12], consisting of a sequence of bases

gtggcgatgc ccgggt (SEQ ID NO: 75),

gagattcccg cnggtg (SEQ ID NO: 78: n is 5-methylcytosine),

ngagattcccgccggt (SEQ ID NO:2: n is 5-methylcytosine),

gnagttctccgcggt (SEQ ID NO:3: n is 5-methylcytosine) or

gnntagacagcgtngg (SEQ ID NO: 4: n is 5-methylcytosine).

[0028]

Aspect [14] A modified oligonucleotide according to aspect [13], represented by the following formula:

GlsMlsMlsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMlsGlsGl,

where nucleic bases are represented by the following symbols:

A=adenine, T=thymine, G=guanine, C=cytosine and M=5-methylcytosine;

Sugar components are represented by the following symbols:

l=LNA and d=2ʼ-deoxyribose;

and internucleoside linkages are represented by the following symbol:

s=phosphorothioate.

[0029]

Aspect [15] A modified oligonucleotide according to aspect [13], represented by the following formula:

GmsMmsMmsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMmsGmsGm,

where nucleic bases are represented by the following symbols:

A=adenine, T=thymine, G=guanine, C=cytosine and M=5-methylcytosine;

Sugar components are represented by the following symbols:

m=ALNA [Ms] and d=2ʼ-deoxyribose;

and internucleoside linkages are represented by the following symbol:

s=phosphorothioate.

[0030]

Aspect [16] A modified oligonucleotide according to aspect [13], represented by the following formula:

GmsMmsAmsGdsTdsTdsCdsTdsCdsCdsGdsCdsGmsGmsTm,

where nucleic bases are represented by the following symbols:

A=adenine, T=thymine, G=guanine, C=cytosine and M=5-methylcytosine;

sugar components are represented by the following symbols: m=ALNA [Ms], and d=2ʼ-deoxyribose;

and internucleoside linkages are represented by the following symbol:

s=phosphorothioate.

[0031]

Aspect [17] A modified oligonucleotide according to aspect [13], represented by the following formula:

MlsGlsAlsGdsAdsTdsTdsCdsCdsCdsGdsCdsCdsGlsGlsTl,

where nucleic bases are represented by the following symbols:

A=adenine, T=thymine, G=guanine, C=cytosine and M=5-methylcytosine;

Sugar components are represented by the following symbols:

l=LNA and d=2ʼ-deoxyribose;

and internucleoside linkages are represented by the following symbol:

s=phosphorothioate.

[0032]

Aspect [18] A modified oligonucleotide according to aspect [14], represented by the following formula, or a salt thereof:

.

[0033]

Aspect [19] A modified oligonucleotide according to aspect [15], represented by the following formula, or a salt thereof:

.

[0034]

Aspect [20] A modified oligonucleotide according to aspect [16], represented by the following formula, or a salt thereof:

.

[0035]

Aspect [21] A modified oligonucleotide according to aspect [17], represented by the following formula, or a salt thereof:

.

[0036]

Aspect [22] A pharmaceutical composition comprising a modified oligonucleotide according to any one of aspects [1]-[21] or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0037]

Aspect [23] A pharmaceutical composition according to aspect [22] for the therapeutic treatment, prevention or delay of the development of a disease associated with DUX4.

[0038]

Aspect [24] The pharmaceutical composition according to aspect [23], wherein the disease associated with DUX4 is facioscapulohumeral muscular dystrophy.

[0039]

Aspect [25] A method for therapeutically treating, preventing or slowing the progression of a disease associated with DUX4 in a subject, comprising administering to a subject in need thereof an effective amount of a modified oligonucleotide according to any of aspects [1]-[21].

[0040]

Aspect [26] Use of a modified oligonucleotide according to any of aspects [1]-[21] in the manufacture of a medicinal product for the therapeutic treatment, prevention or slowing down of the progression of a disease associated with DUX4.

[0041]

Aspect [27] Use of a modified oligonucleotide according to any of aspects [1]-[21] for the therapeutic treatment, prevention or slowing down of the progression of a disease associated with DUX4.

[Invention Effect]

[0042]

According to the present invention, a modified oligonucleotide can be provided that is effective for treating diseases such as fascioscapulohumeral muscular dystrophy caused by abnormal expression of the DUX4 gene.

BRIEF DESCRIPTION OF THE FIGURES

A more complete understanding of the invention and the many advantages attendant thereon will be readily obtained as the same becomes more fully understood by reference to the following detailed description when considered in conjunction with the accompanying drawings, in which:

[0043]

[Fig. 1] Figure showing the effects of suppression of DUX4 gene expression by introducing various DUX4-modified oligonucleotides.

[Fig. 2] Figure showing the effects of suppression of DUX4 gene expression by introducing various DUX4-modified oligonucleotides.

[Fig. 3] Figure showing the dose-dependent effects of suppression of DUX4 gene expression by administration of DUX4-modified oligonucleotides.

[Fig. 4] Figures showing the effects of suppression of DUX4 gene expression by introducing various DUX4-modified oligonucleotides.

[Fig. 5] Figures showing the effects of creatine kinase suppression in DUX4 Tg mice and DUX4 mRNA suppression by administration of DUX4-modified oligonucleotide (Compound No. 3).

[Fig. 6] Figures showing the effects of creatine kinase suppression in DUX4 Tg mice and DUX4 mRNA suppression by administration of DUX4-modified oligonucleotides (Compound No. 123, Compound No. 247, and Compound No. 113).

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Embodiments are now described with reference to the accompanying drawings, wherein in different drawings the same reference numerals designate corresponding or identical elements.

[0044]

It should be understood that both the above summary and the following detailed description are illustrative and explanatory only and do not limit the present invention as claimed. In the present specification, unless specifically stated otherwise, the use of the singular includes the plural. In the present specification, unless specifically stated otherwise, the use of the term "or" means "and/or". In addition, the use of the term "including" and its other forms, such as "includes" and "included", is not limiting. In addition, unless specifically stated otherwise, the term "element" and the like includes an element that covers one unit and an element that covers more than one subunit.

[0045]

The section headings used in this document are for organizational purposes only and are not to 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, reports, books, and articles, are expressly incorporated herein by reference with respect to the portions of the document discussed herein and in their entirety.

[0046]

(Definitions)

Unless otherwise defined, the nomenclature used in accordance with analytical chemistry, synthetic organic chemistry, medicinal chemistry and pharmaceutical chemistry, as well as the methods and techniques described herein, are well known in the art and widely used. As used herein, standard methods may be used for chemical synthesis and chemical analysis. Where applicable, all patents, patent applications, published patent applications and other publications cited in the description of the present invention, as well as GenBank accession numbers and corresponding sequence information, as well as other data available through databases such as the National Center for Biotechnology Information (NCBI), are incorporated by reference with respect to portions of documents discussed herein and in their entirety.

In addition, the present disclosure is provided together with a sequence listing in electronic format. However, the sequence listing information provided in electronic format is incorporated herein by reference in its entirety.

[0047]

Unless otherwise specified, the following terms have the following meanings.

[0048]

The term "nucleic acid" refers to a compound consisting of a monomeric nucleotide. Examples of nucleic acids include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acid, double-stranded nucleic acid, short interfering ribonucleic acid (siRNA), and microRNA (miRNA). A nucleic acid may also include combinations of these elements in a single molecule.

[0049]

The term "nucleobase" means a heterocyclic moiety that can form a base pair with another nucleic acid. Nucleobases include "modified nucleobases" and "unmodified nucleobases."

[0050]

The term "nucleobase sequence" means the order of immediately adjacent nucleobases, independent of any sugar linkage or modification of the nucleobases.

[0051]

The term "nucleoside" means a nucleic base linked to a sugar. In some embodiments, the nucleoside is linked to a phosphate group.

[0052]

The term "nucleotide" means a nucleoside having a phosphate group or the like covalently linked to a sugar moiety of the nucleoside. Natural nucleotides contain ribose or deoxyribose sugar moieties and are covalently linked by phosphodiester bonds through phosphate groups.

[0053]

The term "oligomeric compound" or "oligomer" refers to a polymer of linked monomeric subunits that is capable of hybridizing to at least one region of a nucleic acid molecule.

[0054]

The term "oligonucleotide" means a polymer of linked nucleosides in which the nucleosides and internucleoside linkages may be independently modified or unmodified.

[0055]

The term "unmodified nucleotide" means a nucleotide consisting of a natural nucleobase, a sugar moiety, and an internucleoside linkage. In some embodiments, the unmodified nucleotide is, but is not limited to, an RNA nucleotide (i.e., β-D-ribonucleoside) or a DNA nucleotide (i.e., β-D-deoxyribonucleoside).

[0056]

The term "modified nucleotide" means a nucleotide that independently contains a modified sugar moiety, a modified internucleoside linkage, or a modified nucleobase. "Modified nucleoside" means a nucleoside that independently has a modified sugar moiety or a modified nucleobase.

[0057]

The term "internucleoside linkage" refers to the chemical bond between nucleosides.

[0058]

The term "linked nucleosides" means adjacent nucleosides that are linked or connected by an internucleoside linkage.

[0059]

The term "natural internucleoside linkage" refers to a 3'-5'-phosphodiester linkage.

[0060]

The term "modified internucleoside linkage" refers to the substitution or any change of a natural internucleoside linkage (i.e., a phosphodiester internucleoside linkage). For example, there is a phosphorothioate internucleoside linkage, but this is not limited to it.

[0061]

The term "phosphorothioate internucleoside linkage" refers to an internucleoside linkage in which the phosphodiester linkage is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. The phosphorothioate linkage is one example of a modified internucleoside linkage.

[0062]

The term "modified nucleobase" refers to any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. Examples include, but are not limited to, 5-methylcytosine. "Unmodified nucleobases" refer to the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0063]

The term "modified oligonucleotide" means an oligonucleotide that contains at least one of a modified nucleoside and/or a modified internucleoside linkage.

The term "salt" is a general term for compounds in which one or more dissociable hydrogen ions contained in an acid are replaced by cations such as metal ions and ammonium ions, and examples of salts of the modified oligonucleotide include, but are not limited to, salts (e.g., sodium salt and magnesium salt) formed with inorganic ions (e.g., sodium ions and magnesium ions) on the thio (S) groups of phosphorothioate linkages or functional groups (in the case of, for example, an amino group) in the modified nitrogenous base.

[0064]

The term "sugar" or "sugar moiety" means a natural or modified sugar moiety.

[0065]

The term "natural sugar moiety" refers to the sugar found in DNA (2'-H) or RNA (2'-OH).

[0066]

The term "modified sugar" refers to the substitution or replacement of a natural sugar moiety. Examples of modified sugars include substituted sugar moiety and sugar moiety substitute.

[0067]

The term "substituted sugar moiety" refers to a furanosyl other than the natural sugar of RNA or DNA.

[0068]

"2ʼ-O-Methoxyethyl" (also 2ʼ-MOE and 2ʼ-O(CH ₂ ) ₂ -OCH ₃ ) refers to the O-methoxyethyl modification at the 2ʼ-position of the furanosyl ring. A 2ʼ-O-methoxyethyl-modified sugar is a modified sugar.

[0069]

"2ʼ-O-Methoxyethyl nucleotide" means a nucleotide containing a 2ʼ-O-methoxyethyl-modified sugar moiety.

[0070]

"2ʼ-O-Methyl" (also 2ʼ-OMe and 2ʼ-OCH ₃ ) refers to the O-methyl modification at the 2ʼ-position of the furanosyl ring. A 2ʼ-O-methyl-modified sugar is a modified sugar.

[0071]

"2ʼ-O-Methylnucleotide" means a nucleotide that contains a 2ʼ-O-methyl-modified sugar moiety.

[0072]

The term "sugar substitute" for the expression "sugar moiety substitute" is intended to mean substitution of the sugar unit (furanose ring) only. The sugar substitute may replace the natural sugar moiety of a nucleoside and the resulting nucleoside subunits may be linked to each other and/or to other nucleosides to form an oligomeric compound that may hybridize to a complementary oligomeric compound. Such structures include rings (e.g., 4, 6, or 7-membered rings) containing a different number of atoms than furanosyl; substitution of the oxygen in furanosyl with non-oxygen atoms (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a substitution of the oxygen. Such structures may also contain substitutions corresponding to those described with respect to the substituted sugar moiety (e.g., a 6-membered carbocyclic bicyclic sugar substitute optionally containing additional substituents). Sugar substitutes also include more complex sugar substitutes (e.g., acyclic peptide nucleic acids). Examples of sugar substitutes include, but are not limited to, morpholino, cyclohexenyl, and cyclohexitol.

[0073]

The term "bicyclic sugar" means a furanosyl ring modified by linking two different carbon atoms present in the same ring. Preferably, the "bicyclic sugar" means a modified sugar in which the 2' and 4' positions of the furanosyl ring are modified by linking via a bridge. "Bicyclic nucleic acid" refers to a nucleoside or nucleotide in which the furanose moiety of the nucleoside or nucleotide contains a "bicyclic sugar".

[0074]

"LNA" means a nucleoside or nucleotide, commonly referred to as 2',4'-locked nucleic acid, and examples thereof include a nucleoside or nucleotide represented by the general formula::

[wherein B represents a nucleobase; and X and Y each independently represent a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group which may be substituted, a phosphorus moiety covalently attached to a base, or the like] (see WO 98/39352). Typical specific examples thereof include a nucleoside or nucleotide represented by the following formula:

.

[0075]

"GuNA" is a nucleoside or nucleotide represented by the following formula:

[wherein B is a nucleobase; _R3 , _R4 , _R5 and _R6 each independently represent a hydrogen atom or a _C1-6 alkyl group which may be substituted with one or more substituents; _R7 and _R8 each independently represent a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group which may be substituted, a phosphorus moiety covalently attached to a base, or the like; and _R9 , _R10 and _R11 each independently represent a hydrogen atom, a _C1-6 alkyl group which may be substituted with one or more substituents, or a protecting group for an amino group]. (See, for example, WO2014/046212 and WO2017/047816).

[0076]

"ALNA [mU]" is a nucleoside or nucleotide represented by the following general formula (I):

[wherein B represents a nucleobase; _R1 , _R2 , _R3 and _R4 each independently represent a hydrogen atom or a _C1-6 alkyl group which may be substituted with one or more substituents; _R5 and _R6 each independently represent a hydrogen atom, a protecting group for a hydroxyl group or a phosphate group which may be substituted, a phosphorus moiety covalently attached to the base, or the like; m represents 1 or 2; X represents a group represented by the following formula (II-1):

;

symbol:

,

described in the formula (II-1) denotes the point of attachment to the 2ʼ-amino group; one of R ₇ and R ₈ is a hydrogen atom, and the other is a methyl group which may be substituted with one or more substituents] (see, for example, Japanese Patent Application No. 2018-212424). A typical specific example is a nucleoside or nucleotide in which one of R ₇ and R ₈ is a hydrogen atom and the other is an unsubstituted methyl group.

[0077]

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

,

wherein one of R ₇ and R ₈ is a hydrogen atom and the other is an isopropyl group which may be substituted with one or more substituents (see, for example, Japanese Patent Application No. 2018-212424). A typical specific example is a nucleoside or nucleotide in which one of R ₇ and R ₈ is a hydrogen atom and the other is an unsubstituted isopropyl group.

[0078]

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

,

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

[0079]

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

,

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

[0080]

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

,

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

[0081]

"5-Methylcytosine" means cytosine modified with a methyl group attached at the 5-position. 5-Methylcytosine is a modified nucleobase.

[0082]

The term "single-stranded oligonucleotide" means an oligonucleotide that does not hybridize with the complementary strand.

[0083]

"DUX4" stands for a nucleic acid or protein transcription factor that is also called dual homeobox 4. DUX4 has, for example, various splice variants transcribed from the DUX4 gene, or its single nucleotide substitutions (SNPs), and they can be both variants and/or SNPs.

[0084]

Many splice variants of DUX4 mRNA have been reported. Human DUX4 (SEQ ID NO: 6 in the sequence listing) consists of a short exon 1 (exon 1), exon 2, and exon 3 due to an atypical splice donor site in exon 1, and encodes a non-toxic short DUX4 protein. Human DUX4-FL consists of exon 1, exon 2, and exon 3, and encodes the full-length DUX4 protein. DUX4-FL includes DUX4-FL1 (SEQ ID NO: 1 in the sequence listing), which is a mature mRNA lacking intron 1, and DUX4-FL2 (SEQ ID NO: 5 in the sequence listing), which contains intron 1, and both encode the full-length DUX4 protein and cause FSHD when expressed in muscle (see the above-mentioned Non-Patent Document 2).

[0085]

DUX4 mRNA is also expressed in the normal testis, and 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 expressed (see the above-mentioned Non-Patent Document 1).

[0086]

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

[0087]

The DUX4 mRNA targeted by the modified oligonucleotide of the present invention is, for example, preferably human DUX4, more preferably DUX4-FL, and still more preferably DUX4-FL1 described in SEQ ID NO: 1 in the sequence listing. Further, as the target site of the modified oligonucleotide of the present invention with respect to DUX4, exon 1, intron 1, exon 2, intron 2, and exon 3 are preferable.

[0088]

It is known that the DUX4 gene is expressed by fusion with other genes due to chromosomal abnormalities such as translocation. Other genes such as 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)) are reported to be genes causing B-cell acute lymphocytic leukemia, differentiated round cell sarcoma, fetal rhabdomyosarcoma, and the like. The modified oligonucleotides of the present invention also include compounds targeting these fusion genes.

[0089]

The term "DUX4 expression" refers to the level of mRNA transcribed from the gene encoding DUX4 or the level of protein translated from the mRNA. DUX4 expression can be determined using techniques known in the art, such as Northern or Western blotting, PCR.

[0090]

The term "DUX4 nucleic acid" means any nucleic acid encoding DUX4. For example, in some embodiments, a 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 some embodiments, DNA and RNA sequences of genes obtained by fusion of the DUX4 gene and other genes are included.

[0091]

The term "DUX4 mRNA" refers to the mRNA encoding the DUX4 protein.

[0092]

The expression "immediately adjacent nucleobases" or "consecutive nucleobases" means nucleobases that are immediately adjacent to each other.

[0093]

The term "complementarity" means the ability to form pairs between the nucleobases of a first nucleic acid and a second nucleic acid.

[0094]

The term "fully complementary (also referred to as complementarity)" or "100% complementary (also referred to as complementarity)" means that all nucleobases in a nucleobase sequence of a first nucleic acid have complementary nucleobases with a second nucleobase sequence of a second nucleic acid. In some embodiments, the first nucleic acid is a modified oligonucleotide and the target nucleic acid is a second nucleic acid.

[0095]

The term "hybridization" means annealing of a complementary nucleic acid molecule. In some embodiments, examples of complementary nucleic acid molecules include a modified oligonucleotide and a target nucleic acid.

[0096]

The term "specifically hybridizable" refers to a modified oligonucleotide that has sufficient complementarity between the modified oligonucleotide and the target nucleic acid to produce the desired effect while exhibiting minimal or no effect on non-target nucleic acid under conditions where specific binding is desired, i.e., under physiological conditions for in vivo assays and therapeutic treatments.

[0097]

The term "mismatch" or "non-complementary nucleobase" refers to the case where a nucleobase of a first nucleic acid fails to pair with the corresponding nucleobase of a second nucleic acid or a target nucleic acid.

[0098]

The term "targeting" or "selecting a target" refers to the process of designing and selecting a modified oligonucleotide that specifically hybridizes to a target nucleic acid and produces the desired effect.

[0099]

The term "target nucleic acid", "target RNA" and "target RNA transcript" refer to a nucleic acid that can be targeted by a modified oligonucleotide. In some embodiments, the target nucleic acid comprises a region of a DUX4 nucleic acid.

[0100]

The term "target segment" refers to the nucleotide sequence of the target nucleic acid that is targeted by the modified oligonucleotide. The "5' target site" refers to the 5'-most nucleotide of the target segment. The "3' target site" refers to the 3'-most nucleotide of the target segment.

[0101]

The term "active target region" or "target region" means a region that is targeted by one or more active modified oligonucleotides. "Active modified oligonucleotide" means a modified oligonucleotide that reduces a target nucleic acid level or protein level.

[0102]

The term "antisense inhibition" means that, compared to the level of the target nucleic acid or the level of the target protein in the absence of the modified oligonucleotide, the level of the target nucleic acid or the level of the protein in the presence of a modified oligonucleotide complementary to the target nucleic acid is reduced.

[0103]

The term "siRNA" means a double-stranded RNA oligonucleotide having a nucleobase sequence that allows hybridization to an appropriate region or segment of a target nucleic acid.

[0104]

The term "shRNA" refers to a single-stranded hairpin RNA oligonucleotide having a nucleobase sequence that allows hybridization to an appropriate region or segment of a target nucleic acid.

[0105]

The term "snoRNA" refers to a single-stranded oligonucleotide that is a non-coding RNA present in the nucleolus, has a nucleobase sequence that allows hybridization to an appropriate region or segment of the target RNA nucleic acid, and directs chemical modification by methylation or pseudouridylation of the target RNA nucleic acid.

[0106]

The term "siRNA" refers to a single-stranded or double-stranded RNA oligonucleotide that is a non-coding RNA that modulates the expression of other genes and has a nucleobase sequence that allows hybridization to an appropriate region or segment of a target nucleic acid.

[0107]

The term "cap structure" or "terminal cap fragment" means a chemical modification incorporated into either end of a modified oligonucleotide.

[0108]

The term "chemically distinct region" refers to a region of a modified oligonucleotide that is chemically distinct in some way from another region of the same modified oligonucleotide. For example, a region containing a 2'-O-methoxyethyl nucleotide is chemically distinct from a region containing a nucleotide that is not 2'-O-methoxyethyl modified.

[0109]

The term "chimeric modified oligonucleotide" means a modified oligonucleotide having at least two chemically distinct regions.

[0110]

The term "motif" refers to a set of chemically distinct regions within a modified oligonucleotide.

[0111]

The term "gapmer" means a chimeric modified oligonucleotide in which an internal region having multiple nucleosides that support RNase H cleavage is located between external regions containing one or more nucleosides, and the nucleosides forming the internal region are chemically distinct from the nucleoside or nucleosides forming the external regions. The internal region may be referred to as a "gap segment" and the external regions may be referred to as "wing segments". A wing segment located at a position 5' from a gap segment may be referred to as a "5' wing segment", and a wing segment located at a position 3' from a gap segment may be referred to as a "3' wing segment".

[0112]

The term "immediately adjacent" means that there are no intervening elements between immediately adjacent elements.

[0113]

The term "nuclear ribonuclease" refers to a ribonuclease found in the nucleus. Examples of nuclear ribonucleases include, but are not limited to, RNase H, including RNase H1 and RNase H2, and double-stranded RNase Drosha, and other double-stranded RNases.

[0114]

In some embodiments, a gapmer is a modified oligonucleotide having a 5'-wing segment, a 3'-wing segment, and a gap segment, wherein the 5'- and 3'-wing segments each contain 1-8 nucleosides and the gap segment contains 6 or more nucleosides and is located between and immediately adjacent to two wing segments, wherein all of the nucleosides of the gap segment are nucleosides that do not contain a modified sugar, or one or two nucleosides of the gap segment are nucleosides that each contain a modified sugar and the other nucleosides of the gap segment are nucleosides that do not contain a modified sugar.

[0115]

The term "agent" refers to an active substance that can exert a therapeutic effect when administered to an animal. "First agent" means a therapeutic compound of the present invention. For example, the first agent can be a modified oligonucleotide targeting DUX4. "Second agent" means a second therapeutic compound of the present invention (e.g., a second modified oligonucleotide targeting DUX4) and/or a therapeutic compound that does not target DUX4.

[0116]

The term "pharmaceutically acceptable salt" means a physiologically and pharmaceutically acceptable salt of a modified oligonucleotide, i.e., a salt that retains the desired biological activity of the modified oligonucleotide and does not impart an undesirable toxic effect to the modified oligonucleotide.

[0117]

The term "diluent" means an ingredient in a composition that does not have pharmacological activity but is pharmaceutically necessary or desirable. For example, a diluent in an injection composition may be a liquid such as saline.

[0118]

The term "DUX4-related disease" refers to a disease caused by abnormal expression of DUX4 mRNA or DUX4 protein, or fusion gene mRNA or protein due to DUX4 gene translocation. Examples include, but are not limited to, fascioscapulohumeral muscular dystrophy, B-cell acute lymphocytic leukemia, differentiated round cell sarcoma, fetal rhabdomyosarcoma, and the like.

[0119]

"Facioscapulohumeral muscular dystrophy" or "FSHD" refers to a muscular dystrophy that results from weakness of the facial, scapular, and shoulder muscles. It is believed that in humans, along with a shortened genomic repeat (D4Z4), which is located primarily near the telomere (end of the chromosome) of chromosome 4, the resulting genomic structure is altered, and the DUX4 gene, which is not naturally expressed in muscle (precursor) cells, is ectopically expressed and causes cell death (FSHD1). Additionally, some patients with FSHD have been found to have a gene mutation in SMCHD1, one of the genomic structural regulators that suppress gene expression (FSHD2).

[0120]

FSHD is a type of muscular dystrophy associated with progressive muscle weakness and loss of muscle fibers. Unlike Duchenne muscular dystrophy and Becker muscular dystrophy, which primarily affect the lower body, FSHD occurs in the upper body, primarily in the facial, scapular, and shoulder muscles. However, it can also occur in the pelvis, lower back, and lower extremities. Symptoms of FSHD often appear between the ages of 10 and 26, but they often develop much later. In some cases, they may not occur at all. Symptoms are usually mild, and the rate of deterioration is also very slow. Frequently seen are facial weakness and drooping eyelids, inability to whistle, decreased facial expression, melancholy or angry expression, difficulty pronouncing words, weakness of the scapular muscles (causing deformities such as prominent scapula (winged scapula) and sloping shoulders), weakness of the lower limbs, hearing loss and possible heart disease, etc.

[0121]

The term "active pharmaceutical agent" means a substance (or substances) in a pharmaceutical composition that provides a therapeutic effect when administered to an animal. For example, in some embodiments, a modified oligonucleotide targeting DUX4 is an active pharmaceutical agent.

[0122]

The term "concomitant administration" refers to the co-administration of two agents by any route in which the pharmacological effects of both agents are produced in the patient at the same time. Concomitant administration does not require that both agents be administered in the same pharmaceutical composition, in the same dosage form, or by the same route of administration. The pharmacological effects of both agents need not be produced at the same time. The pharmacological effects need only overlap for a specified period of time and need not be the same.

[0123]

The term "administration" means the administration of an agent to an animal and includes, but is not limited to, administration by a health care professional and self-administration.

[0124]

The term "improvement" refers to a decrease in at least one indicator, sign, or symptom associated with a disease, disorder, or condition. The significance of the indicator may be determined by a subjective or objective measure known to one skilled in the art.

[0125]

The term "animal" refers to a human or non-human animal, including but not limited to mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including but not limited to monkeys and chimpanzees.

[0126]

The term "co-administration" means the administration to an individual of two or more agents. The two or more agents may be in a single pharmaceutical composition or may be in separate pharmaceutical compositions. Each of the two or more agents may be administered by the same or different routes of administration. Co-administration includes concurrent or sequential administration.

[0127]

The term "dose" means a certain amount of a pharmaceutical agent administered at one time or over a certain period of time. In some embodiments, the dose may be administered as one, two or more boluses, tablets or injections. For example, in some embodiments where subcutaneous administration is desired, the desired dose is needed in a volume that is not easily covered by a single injection, and thus two or more injections may be used to achieve the desired dose. In some embodiments, the pharmaceutical agent is administered by infusion over a long period of time or continuously. The dose may be described as an amount of the pharmaceutical agent per hour, day, week or month.

[0128]

The term "effective amount" or "therapeutically effective amount" means an amount of an active pharmaceutical agent that is sufficient to achieve the desired physiological result in an individual in need of the agent. An effective amount may vary from person to person depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated, the composition of the formulation, an assessment of the individual's health status, and other relevant factors.

[0129]

The phrase "identification of an animal affected by facioscapulohumeral muscular dystrophy (FSHD)" means identification of an animal diagnosed with an FSHD disorder or condition or identification of an animal susceptible to an FSHD disorder or condition. For example, an individual with a family history of FSHD may be predisposed to FSHD disorders or conditions.

Such identification can be accomplished using any method, including examination of the individual's medical history and standard clinical tests or assessments. Both FSHD1 and FSHD2 are known to be associated with pathogenesis, and both are included in the discussion.

[0130]

The term "individual" means a human or non-human animal selected for treatment or therapy.

[0131]

The term myotonia refers to abnormally slow muscle relaxation following voluntary contraction or electrical stimulation.

[0132]

The term "parenteral administration" means administration by injection or infusion. Examples of parenteral administration include subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration such as intrathecal or intraventricular administration. Administration may be continuous or long-term, short-term or intermittent.

[0133]

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

[0134]

The term "prevention" means delaying or preventing the onset or progression of a disease, disorder, adverse health condition, or one or more symptoms associated with the disease, disorder, or adverse health condition for a period of time ranging from minutes to an indefinite period. "Prevention" also means reducing the risk of developing the disease, disorder, or adverse health condition.

[0135]

The term "therapeutic treatment" means the alleviation or elimination of a disease, disorder or adverse health condition, or of one or more symptoms associated with a disease, disorder or adverse health condition, or the partial or complete removal of one or more causes of the disease, disorder or adverse health condition.

[0136]

"Treatment" is intended to include prevention or therapeutic treatment as described above. For example, "treatment" includes administration of a pharmaceutical composition of the present invention to cause a change in or alleviation of a disease, disorder, or undesirable health condition.

[0137]

The term "prodrug" means a therapeutic agent that is produced in an inactive form but that is converted to an active form in the body or its cells by an endogenous enzyme or other chemicals or conditions.

[0138]

The term "side effects" means physiological reactions that can be attributed to a response that differs from the desired effects. In some embodiments, side effects include injection site reactions, liver function test abnormalities, kidney function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, elevated alanine aminotransferase (ALT), aspartate aminotransferase (AST), or γ-glutamyl transpeptidase (γ-GTP) in the blood may indicate liver toxicity or impaired liver function. For example, elevated bilirubin may indicate liver toxicity or impaired liver function. In addition, elevated urine protein and elevated blood creatinine and blood urea nitrogen (UN) levels may indicate renal toxicity or impaired renal function.

[0139]

The term "subcutaneous injection" means injection directly under the skin.

[0140]

The term "therapeutically effective amount" means the amount of an agent that provides a therapeutic effect to an individual.

[0141]

(Specific embodiments)

Implementation options

In some specific embodiments described below, there are provided, but not limited to, compounds for inhibiting DUX4 expression, methods of using the compounds, and pharmaceutical compositions comprising the compounds.

[0142]

In some embodiments, the invention provides a method for reducing the expression of DUX4 in an animal, comprising administering to the animal a compound comprising a modified oligonucleotide targeting DUX4.

[0143]

In some embodiments of the invention, a method of administering a modified oligonucleotide is provided to prevent the accumulation of pathogenic DUX4 transcription factors by inhibiting transcription of the DUX4 gene, or inhibiting translation of DUX4 mRNA, or directing cleavage of DUX4 mRNA.

[0144]

In some embodiments of the invention, a method of treating an animal suffering from facioscapulohumeral muscular dystrophy is provided, wherein the method comprises the following steps: a) identifying an animal suffering from facioscapulohumeral muscular dystrophy; and b) administering to the animal a therapeutically effective amount of a compound comprising a modified oligonucleotide targeting DUX4.

[0145]

In some embodiments, the invention provides a method for alleviating myotonia in a subject in need thereof. The method comprises administering to the subject a modified oligonucleotide complementary to DUX4 mRNA. The modified oligonucleotide activates ribonuclease or nuclear ribonuclease upon binding to DUX4 mRNA, thereby reducing myotonia. In some embodiments, the subject has or is suspected of having facioscapulohumeral muscular dystrophy, or has or is suspected of having high expression of DUX4 mRNA, has or is suspected of having a reduced number of D4Z4 repeats on human chromosome 4, or has or is suspected of having a SMCHD1 (DNA methylase) mutation.

[0146]

In some embodiments, the modified oligonucleotide used in the method of the present invention is chimeric. In some embodiments, the modified oligonucleotide used in the method of the present invention is a gapmer.

[0147]

In some embodiments of the method of the present invention described herein, the administration is subcutaneous. In some embodiments, the administration is intravenous or intramuscular.

[0148]

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

[0149]

In some embodiments of the method of the present invention described herein, DUX4 mRNA is cleaved by the nuclear ribonuclease RNase H1.

[0150]

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

[0151]

In some embodiments, the DUX4 mRNA has the sequence set forth in GenBank Accession Number NM_001293798.2 (incorporated herein as SEQ ID NO: 1 in the Sequence Listing). The splice variant of SEQ ID NO: 1 in the Sequence Listing is also referred to as DUX4-FL1 or mature DUX4 mRNA. In some embodiments, the DUX4 mRNA has the sequence set forth in GenBank Accession Number NM_001306068.2 (incorporated herein as SEQ ID NO: 5 in the Sequence Listing). The splice variant of SEQ ID NO: 5 in the Sequence Listing is also referred to as DUX4-FL2. In some embodiments, DUX4 has the sequence identified by GenBank accession number NM_001363820.1 (incorporated herein as SEQ ID NO: 6 in the sequence listing). The splice variant of SEQ ID NO: 6 is also referred to as DUX4-s in the sequence listing. In some embodiments, DUX4 mRNA has the SNP of the splice variant described above.

[0152]

In some embodiments, the modified oligonucleotide is a modified oligonucleotide that is 12-30 residues long and has a nucleobase sequence comprising a modified oligonucleotide that is a nucleobase sequence that includes at least 8 immediately adjacent nucleobase sequences that are complementary to an equal length portion at positions 126-147, 232-248, 1306-1325, or 1472-1495 from the 5'-end of the nucleobase of the mature DUX4 mRNA in SEQ ID NO: 1 in the sequence listing. In some embodiments, the modified oligonucleotide is a modified oligonucleotide that is 12-30 residues long and has a nucleobase sequence comprising a modified oligonucleotide that is a nucleobase sequence that includes at least 9, 10, 11, or 12 immediately adjacent nucleobase sequences that are complementary to an equal-length portion at positions 126-147, 232-248, 1306-1325, or 1472-1495 from the 5'-end of the nucleobase of the mature DUX4 mRNA in SEQ ID NO: 1 in the sequence listing.

The modified oligonucleotide may consist of at least 8 immediately adjacent nucleobase sequences that are complementary to a portion of equal length at positions 126-147, 232-248, 1306-1325 or 1472-1495 from the 5'-end of the nucleobase sequence of the mature DUX4 mRNA in SEQ ID NO: 1 in the sequence listing, and may have, in addition to this nucleobase sequence, an additional sequence on the 5'-end side and/or the 3'-end side.

[0153]

In some embodiments, the modified oligonucleotide is a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 immediately adjacent nucleobase sequences that are complementary to the equal length portion at positions 126-147 from the 5'-end of the mature DUX4 mRNA nucleobase in SEQ ID NO: 1 in the sequence listing, and is a modified oligonucleotide of 30 or fewer residues.

[0154]

In some embodiments, the modified oligonucleotide is a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 immediately adjacent nucleobase sequences that are complementary to the equal-length portion at positions 232-248 from the 5'-end of the mature DUX4 mRNA nucleobase in SEQ ID NO: 1 in the sequence listing, and is a modified oligonucleotide of 30 or fewer residues.

[0155]

In some embodiments, the modified oligonucleotide is a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 immediately adjacent nucleobase sequences that are complementary to the equal-length portion at positions 1306-1325 from the 5'-end of the mature DUX4 mRNA nucleobase in SEQ ID NO: 1 in the sequence listing, and is a modified oligonucleotide of 30 or fewer residues.

[0156]

In some embodiments, the modified oligonucleotide is a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 immediately adjacent nucleobase sequences that are complementary to the equal-length portion at positions 1472-1495 from the 5'-end of the mature DUX4 mRNA nucleobase in SEQ ID NO: 1 in the sequence listing, and is a modified oligonucleotide of 30 or fewer residues.

[0157]

In some embodiments, the modified oligonucleotide is a modified oligonucleotide that consists of 12-30 residues and comprises a nucleobase sequence that is complementary to the equal-length portion at positions 126-147 from the 5'-end of the nucleobase of the mature DUX4 mRNA in 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. Furthermore, in some embodiments, the modified oligonucleotide comprises 12-29 residues, 12-28 residues, 12-27 residues, 12-26 residues, 12-25 residues, 12-24 residues, 12-23 residues, 12-22 residues, 12-21 residues, 12-20 residues, 12-19 residues, 12-18 residues, 12-17 residues, 12-16 residues, 12-15 residues, 12-14 residues, 13-29 residues, 13-28 residues, 13-27 residues, 13-26 residues, 13-25 residues, 13-24 residues, 13-23 residues, 13-22 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 or 14-15 residues.

[0158]

In some embodiments, the modified oligonucleotide is a modified oligonucleotide that consists of 12-30 residues and comprises a nucleobase sequence that is complementary to the equal-length portion at positions 232-248 from the 5'-end of the nucleobase of the mature DUX4 mRNA in 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. Furthermore, in some embodiments, the modified oligonucleotide comprises 12-29 residues, 12-28 residues, 12-27 residues, 12-26 residues, 12-25 residues, 12-24 residues, 12-23 residues, 12-22 residues, 12-21 residues, 12-20 residues, 12-19 residues, 12-18 residues, 12-17 residues, 12-16 residues, 12-15 residues, 12-14 residues, 13-29 residues, 13-28 residues, 13-27 residues, 13-26 residues, 13-25 residues, 13-24 residues, 13-23 residues, 13-22 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 or 14-15 residues.

[0159]

In some embodiments, the modified oligonucleotide is a modified oligonucleotide that is 12-30 residues long and comprises a nucleobase sequence that is complementary to the equal-length portion at positions 1306-1325 from the 5'-end of the nucleobase of the mature DUX4 mRNA in 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. Furthermore, in some embodiments, the modified oligonucleotide comprises 12-29 residues, 12-28 residues, 12-27 residues, 12-26 residues, 12-25 residues, 12-24 residues, 12-23 residues, 12-22 residues, 12-21 residues, 12-20 residues, 12-19 residues, 12-18 residues, 12-17 residues, 12-16 residues, 12-15 residues, 12-14 residues, 13-29 residues, 13-28 residues, 13-27 residues, 13-26 residues, 13-25 residues, 13-24 residues, 13-23 residues, 13-22 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 or 14-15 residues.

[0160]

In some embodiments, the modified oligonucleotide is a modified oligonucleotide that consists of 12-30 residues and comprises a nucleobase sequence that is complementary to the equal-length portion at positions 1472-1495 from the 5'-end of the nucleobase of the mature DUX4 mRNA in 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. Furthermore, in some embodiments, the modified oligonucleotide consists 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 or 14-15 residues.

[0161]

In some embodiments, the modified oligonucleotide is a modified oligonucleotide that is 12-30 residues long and has a nucleobase sequence comprising a modified oligonucleotide that is a nucleobase sequence that includes at least 8 immediately adjacent nucleobase sequences that are complementary to an equal length portion at positions 128-143, 233-248, 1309-1323, or 1480-1495 from the 5'-end of the nucleobase of the mature DUX4 mRNA in SEQ ID NO: 1 in the sequence listing. In some embodiments, the modified oligonucleotide is a modified oligonucleotide that is 12-30 residues long and has a nucleobase sequence comprising a modified oligonucleotide that is a nucleobase sequence that includes at least 9, 10, 11, or 12 immediately adjacent nucleobase sequences that are complementary to an equal-length portion at positions 128-143, 233-248, 1309-1323, or 1480-1495 from the 5'-end of the nucleobase of the mature DUX4 mRNA in SEQ ID NO: 1 in the sequence listing.

[0162]

In some embodiments, the modified oligonucleotide is a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15 or 16 immediately adjacent nucleobase sequences that are complementary to the equal length portion at positions 128-143 from the 5'-end of the mature DUX4 mRNA nucleobase in SEQ ID NO: 1 in the sequence listing, and is a modified oligonucleotide of 30 or fewer residues.

[0163]

In some embodiments, the modified oligonucleotide is a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15 or 16 immediately adjacent nucleobase sequences that are complementary to the equal length portion at positions 233-248 from the 5'-end of the mature DUX4 mRNA nucleobase in SEQ ID NO: 1 in the sequence listing, and is a modified oligonucleotide of 30 or fewer residues.

[0164]

In some embodiments, the modified oligonucleotide is a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14 or 15 immediately adjacent nucleobase sequences that are complementary to the equal length portion at positions 1309-1323 from the 5'-end of the mature DUX4 mRNA nucleobase in SEQ ID NO: 1 in the sequence listing, and is a modified oligonucleotide of 30 or fewer residues.

[0165]

In some embodiments, the modified oligonucleotide is a modified oligonucleotide of 30 or fewer residues that includes at least 8, 9, 10, 11, 12, 13, 14, 15 or 16 immediately adjacent nucleobase sequences that are complementary to an equal-length portion at positions 1480-1495 from the 5'-end of the nucleobase of the mature DUX4 mRNA in SEQ ID NO: 1 in the sequence listing, and consists of a nucleobase sequence having at the 3'-end a base complementary to the base at position 1480 from the 5'-end of the nucleobase sequence of SEQ ID NO: 1.

[0166]

In some embodiments, the modified oligonucleotide is a modified oligonucleotide that consists of 12-30 residues and comprises a nucleobase sequence that is complementary to the equal-length portion at positions 128-143 from the 5'-end of the nucleobase of the mature DUX4 mRNA in 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. Furthermore, in some embodiments, the modified oligonucleotide comprises 12-29 residues, 12-28 residues, 12-27 residues, 12-26 residues, 12-25 residues, 12-24 residues, 12-23 residues, 12-22 residues, 12-21 residues, 12-20 residues, 12-19 residues, 12-18 residues, 12-17 residues, 12-16 residues, 12-15 residues, 12-14 residues, 13-29 residues, 13-28 residues, 13-27 residues, 13-26 residues, 13-25 residues, 13-24 residues, 13-23 residues, 13-22 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 or 14-15 residues.

[0167]

In some embodiments, the modified oligonucleotide is a modified oligonucleotide that consists of 12-30 residues and comprises a nucleobase sequence that is complementary to the equal-length portion at positions 233-248 from the 5'-end of the nucleobase of the mature DUX4 mRNA in 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. Furthermore, in some embodiments, the modified oligonucleotide comprises 12-29 residues, 12-28 residues, 12-27 residues, 12-26 residues, 12-25 residues, 12-24 residues, 12-23 residues, 12-22 residues, 12-21 residues, 12-20 residues, 12-19 residues, 12-18 residues, 12-17 residues, 12-16 residues, 12-15 residues, 12-14 residues, 13-29 residues, 13-28 residues, 13-27 residues, 13-26 residues, 13-25 residues, 13-24 residues, 13-23 residues, 13-22 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 or 14-15 residues.

[0168]

In some embodiments, the modified oligonucleotide is a modified oligonucleotide that is 12-30 residues long and comprises a nucleobase sequence that is complementary to the equal-length portion at positions 1309-1323 from the 5'-end of the nucleobase of the mature DUX4 mRNA in 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. Furthermore, in some embodiments, the modified oligonucleotide comprises 12-29 residues, 12-28 residues, 12-27 residues, 12-26 residues, 12-25 residues, 12-24 residues, 12-23 residues, 12-22 residues, 12-21 residues, 12-20 residues, 12-19 residues, 12-18 residues, 12-17 residues, 12-16 residues, 12-15 residues, 12-14 residues, 13-29 residues, 13-28 residues, 13-27 residues, 13-26 residues, 13-25 residues, 13-24 residues, 13-23 residues, 13-22 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 or 14-15 residues.

[0169]

In some embodiments, the modified oligonucleotide is a modified oligonucleotide of 12-30 residues and is a nucleobase sequence that is complementary to the equal-length portion at positions 1480-1495 from the 5′-end of the mature DUX4 mRNA nucleobase of SEQ ID NO: 1 in the sequence listing, and consists of a nucleobase sequence that has at the 3′-end a base complementary to the base at position 1480 from the 5′-end of the nucleobase of SEQ ID NO: 1, 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 length. Furthermore, in some embodiments, the modified oligonucleotide consists 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 or 14-15 residues.

[0170]

In some embodiments, a modified oligonucleotide provided herein targets any of the following regions in 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 from 5ʼ-end. In some embodiments, the modified oligonucleotide provided herein targets any of the following regions in SEQ ID NO: 1 in the sequence listing: positions 128-143, 232-247, 233-248, 1309-1323, and 1480-1495 from the 5′ end.

[0171]

In some embodiments, a modified oligonucleotide provided herein has a nucleobase sequence that includes a complementary region comprising at least 8 immediately adjacent nucleobases complementary to a target region. In some embodiments, a modified oligonucleotide provided herein has a nucleobase sequence that includes a complementary region comprising at least 8 immediately adjacent nucleobases complementary to a target region, and the target region is targeted to 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 in SEQ ID NO: 1 in the sequence listing. In some embodiments, a modified oligonucleotide provided herein has a nucleobase sequence that includes a complementary region comprising at least 8 immediately adjacent nucleobases complementary to a target region, and the target region is targeted to positions 128-143, 232-247, 233-248, 1309-1323, or 1480-1495 from the 5′ end of SEQ ID NO: 1 in the sequence listing.

[0172]

In some embodiments, a modified oligonucleotide provided herein has a nucleobase sequence that includes a complementary region comprising at least 10 immediately adjacent nucleobases complementary to a target region. In some embodiments, a modified oligonucleotide provided herein has a nucleobase sequence that includes a complementary region comprising at least 10 immediately adjacent nucleobases complementary to a target region, and the target region is targeted to 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 in SEQ ID NO: 1 in the sequence listing. In some embodiments, a modified oligonucleotide provided herein has a nucleobase sequence that includes a complementary region comprising at least 10 immediately adjacent nucleobases complementary to a target region, and the target region is targeted to positions 128-143, 232-247, 233-248, 1309-1323, or 1480-1495 from the 5′ end of SEQ ID NO: 1 in the sequence listing.

[0173]

In some embodiments, a modified oligonucleotide provided herein has a nucleobase sequence that includes a complementary region comprising at least 12 immediately adjacent nucleobases complementary to a target region. In some embodiments, a modified oligonucleotide provided herein has a nucleobase sequence that includes a complementary region comprising at least 12 immediately adjacent nucleobases complementary to a target region, and the target region is targeted to 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 in SEQ ID NO: 1 in the sequence listing. In some embodiments, a modified oligonucleotide provided herein has a nucleobase sequence that includes a complementary region comprising at least 12 immediately adjacent nucleobases complementary to a target region, and the target region is targeted to positions 128-143, 232-247, 233-248, 1309-1323, or 1480-1495 from the 5′ end of SEQ ID NO: 1 in the sequence listing.

[0174]

In some embodiments, a modified oligonucleotide provided herein has a nucleobase sequence that includes a complementary region comprising at least 14 immediately adjacent nucleobases complementary to a target region. In some embodiments, a modified oligonucleotide provided herein has a nucleobase sequence that includes a complementary region comprising at least 14 immediately adjacent nucleobases complementary to a target region, and the target region is targeted to 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 in SEQ ID NO: 1 in the sequence listing. In some embodiments, a modified oligonucleotide provided herein has a nucleobase sequence that includes a complementary region comprising at least 14 immediately adjacent nucleobases complementary to a target region, and the target region is targeted to positions 128-143, 232-247, 233-248, 1309-1323, or 1480-1495 from the 5′ end of SEQ ID NO: 1 in the sequence listing.

[0175]

In some embodiments, the modified oligonucleotide is a modified oligonucleotide that comprises a nucleobase sequence that is complementary to an equal length portion at positions 126-141, 126-143, 127-142, 127-143, 127-144, 127-146, 128-143, 128-144, 128-147, 232-245, 232-247, 233-246, 233-247, 233-248, 234-247, 234-248, 1304-1323, 1306-1321, 1306-1324, 1307-1323, 1307-1324, 1307-1325, 1307-1326, 1308-1323, 1308-1324, 1308-1322, 1308-1325, 1309-1323, 1309-1324, 1309-1325, 1309-1322, 1310-1323, 1310-1324, 1472-1485, 1472-1486, 1472-1487, 1472-1488, 1473-1487, 1473-1488, 1473-1489, 1474-1488, 1474-1489, 1475-1490, 1476-1490, 1476-1491, 1476-1495, 1477-1495, 1478-1496, 1479-1495, 1479-1496, 1479-1498, 1480-1494, 1480-1495, 1480-1496, 1480-1497 or 1480-1499 from the 5'-nucleobase end of the mature DUX4 mRNA in SEQ ID NO: 1 in the sequence listing. Furthermore, in some embodiments of the invention, the modified oligonucleotide is a modified oligonucleotide that comprises a nucleobase sequence that is complementary to an equal length portion at positions 128-143, 232-247, 233-248, 1309-1323, or 1480-1495 from the 5′-end of the nucleobase of the mature DUX4 mRNA in SEQ ID NO: 1 in the sequence listing.

[0176]

In some embodiments, the modified oligonucleotide is a modified oligonucleotide comprising a nucleobase sequence represented by any of SEQ ID NOs: 2-4, 7-64, 69-97, and 102-109 in the sequence listing. Furthermore, in some embodiments, the modified oligonucleotide is a modified oligonucleotide comprising a nucleobase sequence represented by any of SEQ ID NOs: 2-4, 75, and 78 in the sequence listing.

[0177]

In some embodiments, the modified oligonucleotide is a modified oligonucleotide comprising a nucleobase sequence represented by any of SEQ ID NOs: 2-4, 7-64, 69-97, and 102-109 in the sequence listing. Furthermore, in some embodiments, the modified oligonucleotide is a modified oligonucleotide comprising a nucleobase sequence represented by any of SEQ ID NOs: 2-4, 75, and 78 in the sequence listing.

[0178]

In some embodiments of the invention, the modified oligonucleotide has a nucleobase sequence comprising at least 8 immediately adjacent nucleotides in a nucleobase sequence represented by any of SEQ ID NO: 2, 3, 4, 75 or 78 in the sequence listing.

[0179]

In some embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 10 immediately adjacent nucleotides in a nucleobase sequence represented by any of SEQ ID NOs: 2, 3, 4, 75 or 78 in the sequence listing.

[0180]

In some embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 12 immediately adjacent nucleotides in a nucleobase sequence represented by any of SEQ ID NOs: 2, 3, 4, 75 or 78 in the sequence listing.

[0181]

In some embodiments, the modified oligonucleotide has a nucleobase sequence comprising at least 14 immediately adjacent nucleotides in a nucleobase sequence represented by any of SEQ ID NOs: 2, 3, 4, 75 or 78 in the sequence listing.

[0182]

In some embodiments of the invention, the modified oligonucleotide has a nucleobase sequence comprising a nucleobase consisting of a nucleobase sequence represented by any one of SEQ ID NO: 2, 3, 4, 75 or 78 in the sequence listing.

[0183]

In some embodiments of the invention, the modified oligonucleotide has a nucleobase sequence consisting of a nucleobase sequence represented by any one of SEQ ID NO: 2, 3, 4, 75 or 78 in the sequence listing.

[0184]

In some embodiments of the invention, the animal is a human.

[0185]

In some embodiments of the invention, administration comprises parenteral administration.

[0186]

In some embodiments, the compound is a single-stranded modified oligonucleotide.

[0187]

In some embodiments, the nucleobase sequence of the modified oligonucleotide is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to an equal-length portion of any of the regions of SEQ ID NO: 1 in the sequence listing, as determined over the entire modified oligonucleotide. In some embodiments, the nucleobase sequence of the modified oligonucleotide is 100% complementary to an equal-length portion of any of the regions of SEQ ID NO: 1 in the sequence listing, as determined over the entire modified oligonucleotide.

[0188]

In some embodiments, at least one internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage. In some embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage.

[0189]

In some embodiments, at least one nucleoside of the modified oligonucleotide comprises a modified sugar. In some embodiments, the at least one modified sugar is a bicyclic sugar. In some embodiments, the at least one modified sugar comprises a 2′-O-methoxyethyl, 2′-O-methyl, and/or 4′-(CH ₂ ) _n -O-2′ (where n is 1 or 2) linkage.

[0190]

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

[0191]

In some embodiments, at least one nucleoside of the modified oligonucleotide comprises a modified nucleobase. In some embodiments, the modified nucleobase is 5-methylcytosine.

[0192]

In some embodiments, the modified oligonucleotide is a gapmer comprising: a) a gap segment consisting of linked deoxynucleosides, b) a 5′-wing segment consisting of linked nucleosides, and c) a 3′-wing segment consisting of linked nucleosides. The gap segment is located between the 5′-wing segment and the 3′-wing segment, and the nucleoside of the wing segments each comprises a modified sugar, such as a 2′-O-methyl-modified sugar, a 2′-O-methoxyethyl-modified sugar, or a bicyclic sugar.

[0193]

In some embodiments, the modified oligonucleotide is a gapmer comprising: a) a gap segment that comprises one or two nucleosides that each comprise a modified sugar, such as a 2'-O-methyl-modified sugar or a 2'-O-methoxyethyl-modified sugar, and comprises other nucleosides that do not comprise a modified sugar, b) a 5'-wing segment that comprises linked nucleosides, and c) a 3'-wing segment that comprises linked nucleosides. The gap segment is located between the 5'-wing segment and the 3'-wing segment, and a nucleoside of the wing segments each comprises a modified sugar, such as a 2'-O-methyl-modified sugar, a 2'-O-methoxyethyl sugar, or a bicyclic sugar.

[0194]

In some embodiments, the modified oligonucleotide is a gapmer comprising: a) a gap segment that comprises 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleosides and wherein all of the nucleosides are nucleosides that do not contain a modified sugar or one or two nucleosides each contain a modified sugar such as a 2′-O-methyl-modified sugar or a 2′-O-methoxyethyl-modified sugar and the other nucleosides do not contain a modified sugar, b) 5′-wing segments comprising 2, 3, 4, 5, 6, or 7 nucleosides, and c) 3′-wing segments comprising 2, 3, 4, 5, 6, 7, or 8 nucleosides.

In this case, the gap segment is located between the 5'-wing segment and the 3'-wing segment; the nucleosides of the wing segments each include a 2'-O-methyl-modified sugar, a 2'-O-methoxyethyl sugar, or a bicyclic sugar; the internucleoside linkages of the modified oligonucleotide include a phosphorothioate linkage; and some or all of the cytosine in the modified oligonucleotide may be 5'-methylcytosine.

[0195]

In some embodiments, the modified oligonucleotide is a gapmer and the number of nucleosides forming the gapmer is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

[0196]

In some embodiments, the modified oligonucleotide is a gapmer and includes a 2'-O-methyl, 2'-O-methoxyethyl, and/or 4'-(CH ₂ ) _n -O-2' (where n is 1 or 2) bridge in the sugar moiety of the nucleoside.

[0197]

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

[0198]

In some embodiments, the modified oligonucleotide is a gapmer that is a modified oligonucleotide that consists of 12-30 residues and has a nucleobase sequence that comprises a modified oligonucleotide that is a nucleobase sequence that includes at least 8 immediately adjacent nucleobase sequences that are complementary to an equal-length portion at positions 126-147, 232-248, 1306-1325, or 1472-1495 from the 5'-end of the nucleobase of the mature DUX4 mRNA in SEQ ID NO: 1 in the sequence listing. In some embodiments, the modified oligonucleotide is a modified oligonucleotide that is 12-30 residues long and has a nucleobase sequence comprising a modified oligonucleotide that is a nucleobase sequence that includes at least 9, 10, 11, or 12 immediately adjacent nucleobase sequences that are complementary to an equal-length portion at positions 126-147, 232-248, 1306-1325, or 1472-1495 from the 5'-end of the nucleobase of the mature DUX4 mRNA in SEQ ID NO: 1 in the sequence listing.

[0199]

In some embodiments, the modified oligonucleotide is a gapmer and is a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 immediately adjacent nucleobase sequences that are complementary to the equal-length portion at positions 126-147 from the 5'-end of the nucleobase of the mature DUX4 mRNA in SEQ ID NO: 1 in the sequence listing, and is a modified oligonucleotide of 30 or fewer residues.

[0200]

In some embodiments, the modified oligonucleotide is a gapmer that is a nucleobase sequence comprising at least 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 immediately adjacent nucleobase sequences that are complementary to the equal-length portion at positions 232-248 from the 5'-end of the mature DUX4 mRNA nucleobase in SEQ ID NO: 1 in the sequence listing, and is a modified oligonucleotide of 30 or fewer residues.

[0201]

In some embodiments, the modified oligonucleotide is a gapmer, which is a nucleobase sequence that includes at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 immediately adjacent nucleobase sequences that are complementary to the equal-length portion at positions 1306-1325 from the 5'-end of the mature DUX4 mRNA nucleobase in SEQ ID NO: 1 in the sequence listing, and is a modified oligonucleotide of 30 or fewer residues.

[0202]

In some embodiments, the modified oligonucleotide is a gapmer, which is a nucleobase sequence that includes at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 immediately adjacent nucleobase sequences that are complementary to the equal-length portion at positions 1472-1495 from the 5'-end of the mature DUX4 mRNA nucleobase in SEQ ID NO: 1 in the sequence listing, and is a modified oligonucleotide of 30 or fewer residues.

[0203]

In some embodiments, the modified oligonucleotide is a modified oligonucleotide gapmer of 12-30 residues and comprises a nucleobase sequence that is complementary to the equal-length portion at positions 126-147 from the 5'-end of the nucleobase of the mature DUX4 mRNA in 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. Furthermore, in some embodiments, the modified oligonucleotide comprises 12-29 residues, 12-28 residues, 12-27 residues, 12-26 residues, 12-25 residues, 12-24 residues, 12-23 residues, 12-22 residues, 12-21 residues, 12-20 residues, 12-19 residues, 12-18 residues, 12-17 residues, 12-16 residues, 12-15 residues, 12-14 residues, 13-29 residues, 13-28 residues, 13-27 residues, 13-26 residues, 13-25 residues, 13-24 residues, 13-23 residues, 13-22 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 or 14-15 residues.

[0204]

In some embodiments, the modified oligonucleotide is a modified oligonucleotide gapmer of 12-30 residues and comprises a nucleobase sequence that is complementary to the equal-length portion at positions 233-248 from the 5'-end of the nucleobase of the mature DUX4 mRNA in 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. Furthermore, in some embodiments, the modified oligonucleotide comprises 12-29 residues, 12-28 residues, 12-27 residues, 12-26 residues, 12-25 residues, 12-24 residues, 12-23 residues, 12-22 residues, 12-21 residues, 12-20 residues, 12-19 residues, 12-18 residues, 12-17 residues, 12-16 residues, 12-15 residues, 12-14 residues, 13-29 residues, 13-28 residues, 13-27 residues, 13-26 residues, 13-25 residues, 13-24 residues, 13-23 residues, 13-22 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 or 14-15 residues.

[0205]

In some embodiments, the modified oligonucleotide is a modified oligonucleotide gapmer of 12-30 residues and comprises a nucleobase sequence that is complementary to the equal-length portion at positions 1309-1323 from the 5'-end of the nucleobase of the mature DUX4 mRNA in 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. Furthermore, in some embodiments, the modified oligonucleotide comprises 12-29 residues, 12-28 residues, 12-27 residues, 12-26 residues, 12-25 residues, 12-24 residues, 12-23 residues, 12-22 residues, 12-21 residues, 12-20 residues, 12-19 residues, 12-18 residues, 12-17 residues, 12-16 residues, 12-15 residues, 12-14 residues, 13-29 residues, 13-28 residues, 13-27 residues, 13-26 residues, 13-25 residues, 13-24 residues, 13-23 residues, 13-22 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 or 14-15 residues.

[0206]

In some embodiments, the modified oligonucleotide is a modified oligonucleotide gapmer of 12-30 residues and comprises a nucleobase sequence that is complementary to the equal-length portion at positions 1472-1495 from the 5'-end of the nucleobase of the mature DUX4 mRNA in 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. Furthermore, in some embodiments, the modified oligonucleotide comprises 12-29 residues, 12-28 residues, 12-27 residues, 12-26 residues, 12-25 residues, 12-24 residues, 12-23 residues, 12-22 residues, 12-21 residues, 12-20 residues, 12-19 residues, 12-18 residues, 12-17 residues, 12-16 residues, 12-15 residues, 12-14 residues, 13-29 residues, 13-28 residues, 13-27 residues, 13-26 residues, 13-25 residues, 13-24 residues, 13-23 residues, 13-22 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 or 14-15 residues. In this case, the oligonucleotide included in the 5'-wing segment and/or the 3'-wing segment comprises at least one nucleoside that comprises at least one modified sugar selected from GuNA, ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Oxz] and ALNA [Trz], and may further comprise a 2'-O-methoxyethyl-modified sugar and/or a 2'-O-methyl-modified sugar.

[0207]

In some embodiments, the modified oligonucleotide is a modified oligonucleotide gapmer of 12-30 residues and is a nucleobase sequence that is complementary to an equal-length portion at positions 1480-1495 from the 5′-end of the mature DUX4 mRNA nucleobase in SEQ ID NO: 1 in the sequence listing and consists of a nucleobase sequence that has at the 3′-end a base complementary to the base at position 1480 from the 5′-end of the nucleobase of SEQ ID NO: 1 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 a portion of equal length. Furthermore, in some embodiments, the modified oligonucleotide consists 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 or 14-15 residues.

[0208]

In some embodiments, a modified oligonucleotide provided herein is a gapmer and targets any of the following regions in 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 from 5ʼ-end. In some embodiments, the modified oligonucleotide provided herein is a gapmer and targets any of the following regions in SEQ ID NO: 1 in the sequence listing: positions 128-143, 232-247, 233-248, 1309-1323, and 1480-1495 from the 5′ end.

[0209]

In some embodiments, a modified oligonucleotide provided herein is a gapmer and has a nucleobase sequence that includes a complementary region comprising at least 8 immediately adjacent nucleobases complementary to a target region. In some embodiments, a modified oligonucleotide provided herein has a nucleobase sequence that includes a complementary region comprising at least 8 immediately adjacent nucleobases complementary to a target region, and the target region is targeted to 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 in SEQ ID NO: 1 in the sequence listing. In some embodiments, a modified oligonucleotide provided herein is a gapmer and has a nucleobase sequence that includes a complementary region comprising at least 8 immediately adjacent nucleobases complementary to a target region, and the target region is targeted to positions 128-143, 232-247, 233-248, 1309-1323, or 1480-1495 from the 5′ end of SEQ ID NO: 1 in the sequence listing.

[0210]

In some embodiments, a modified oligonucleotide provided herein is a gapmer and has a nucleobase sequence that includes a complementary region comprising at least 10 immediately adjacent nucleobases complementary to a target region. In some embodiments, a modified oligonucleotide provided herein has a nucleobase sequence that includes a complementary region comprising at least 10 immediately adjacent nucleobases complementary to a target region, and the target region is targeted to 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 in SEQ ID NO: 1 in the sequence listing. In some embodiments, a modified oligonucleotide provided herein is a gapmer and has a nucleobase sequence that includes a complementary region comprising at least 10 immediately adjacent nucleobases complementary to a target region, and the target region is targeted to positions 128-143, 232-247, 233-248, 1309-1323, or 1480-1495 from the 5′ end of SEQ ID NO: 1 in the sequence listing.

[0211]

In some embodiments, a modified oligonucleotide provided herein is a gapmer and has a nucleobase sequence that includes a complementary region comprising at least 12 immediately adjacent nucleobases complementary to a target region. In some embodiments, a modified oligonucleotide provided herein has a nucleobase sequence that includes a complementary region comprising at least 12 immediately adjacent nucleobases complementary to a target region, and the target region is targeted to 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 in SEQ ID NO: 1 in the sequence listing. In some embodiments, a modified oligonucleotide provided herein is a gapmer and has a nucleobase sequence that includes a complementary region comprising at least 12 immediately adjacent nucleobases complementary to a target region, and the target region is targeted to positions 128-143, 232-247, 233-248, 1309-1323, or 1480-1495 from the 5′ end of SEQ ID NO: 1 in the sequence listing.

[0212]

In some embodiments, a modified oligonucleotide provided herein is a gapmer and has a nucleobase sequence that includes a complementary region comprising at least 14 immediately adjacent nucleobases complementary to a target region. In some embodiments, a modified oligonucleotide provided herein has a nucleobase sequence that includes a complementary region comprising at least 14 immediately adjacent nucleobases complementary to a target region, and the target region is targeted to 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 in SEQ ID NO: 1 in the sequence listing. In some embodiments, a modified oligonucleotide provided herein is a gapmer and has a nucleobase sequence that includes a complementary region comprising at least 14 immediately adjacent nucleobases complementary to a target region, and the target region is targeted to positions 128-143, 232-247, 233-248, 1309-1323, or 1480-1495 from the 5′ end of SEQ ID NO: 1 in the sequence listing.

[0213]

In some embodiments, the modified oligonucleotide is a gapmer and is a modified oligonucleotide that consists of a nucleobase sequence that is complementary to an equal length portion at positions 126-141, 126-143, 127-142, 127-143, 127-144, 127-146, 128-143, 128-144, 128-147, 232-245, 232-247, 233-246, 233-247, 233-248, 234-247, 234-248, 1304-1323, 1306-1321, 1306-1324, 1307-1323, 1307-1324, 1307-1325, 1307-1326, 1308-1323, 1308-1324, 1308-1322, 1308-1325, 1309-1323, 1309-1324, 1309-1325, 1309-1322, 1310-1323, 1310-1324, 1472-1485, 1472-1486, 1472-1487, 1472-1488, 1473-1487, 1473-1488, 1473-1489, 1474-1488, 1474-1489, 1475-1490, 1476-1490, 1476-1491, 1476-1495, 1477-1495, 1478-1496, 1479-1495, 1479-1496, 1479-1498, 1480-1494, 1480-1495, 1480-1496, 1480-1497 or 1480-1499 from the 5'-nucleobase end of the mature DUX4 mRNA in SEQ ID NO: 1 in the sequence listing. In some embodiments, the modified oligonucleotide is a gapmer and is a modified oligonucleotide that consists of a nucleobase sequence that is complementary to an equal length portion at positions 128-143, 232-247, 233-248, 1309-1323, 1480-1495 from the 5'-end of the nucleobase of the mature DUX4 mRNA in SEQ ID NO: 1 in the sequence listing.

[0214]

In some embodiments, the modified oligonucleotide is a gapmer, and is a modified oligonucleotide consisting of a nucleobase sequence represented by any of SEQ ID NOs: 2-4, 7-64, 69-97, and 102-109 in the sequence listing. Furthermore, in some embodiments, the modified oligonucleotide is a gapmer, and is a modified oligonucleotide consisting of a nucleobase sequence represented by any of SEQ ID NOs: 2-4, 75, and 78 in the sequence listing.

[0215]

In some embodiments, the modified oligonucleotide is a gapmer, and is a modified oligonucleotide that is any one of Compounds Nos. 1-112, 114-132, and 137-246. In some embodiments, the modified oligonucleotide is a gapmer, and is a modified oligonucleotide that is any one of Compounds Nos. 1-3, 123, 157, 204, 221, and 231.

[0216]

In the present description, in symbols such as "Gls" denoting nucleotides, the letter abbreviation on the left denotes the nucleobase moiety, the letter abbreviation in the center denotes the sugar moiety, and the letter abbreviation on the right denotes the mode of internucleoside linkage.

[0217]

In some embodiments of the invention, the modified oligonucleotide is a gapmer and is represented by the formula:

GlsMlsMlsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMlsGlsGl,

where nucleic bases are represented by the following symbols:

A=adenine, T=thymine, G=guanine, C=cytosine and M=5-methylcytosine;

Sugar components are represented by the following symbols:

l=LNA and d=2ʼ-deoxyribose;

and internucleoside linkages are represented by the following symbol:

s=phosphorothioate.

[0218]

In some embodiments of the invention, the modified oligonucleotide is a gapmer and is represented by the formula:

GmsMmsMmsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMmsGmsGm,

where nucleic bases are represented by the following symbols:

A=adenine, T=thymine, G=guanine, C=cytosine and M=5-methylcytosine;

Sugar components are represented by the following symbols:

m=ALNA [Ms] and d=2ʼ-deoxyribose;

and internucleoside linkages are represented by the following symbol:

s=phosphorothioate.

[0219]

In some embodiments of the invention, the modified oligonucleotide is a gapmer and is represented by the formula:

GmsMmsAmsGdsTdsTdsCdsTdsCdsCdsGdsCdsGmsGmsTm,

where nucleic bases are represented by the following symbols:

A=adenine, T=thymine, G=guanine, C=cytosine and M=5-methylcytosine;

Sugar components are represented by the following symbols:

m=ALNA [Ms] and d=2ʼ-deoxyribose;

and internucleoside linkages are represented by the following symbol:

s=phosphorothioate.

[0220]

In some embodiments of the invention, the modified oligonucleotide is a gapmer and is represented by the formula:

MlsGlsAlsGdsAdsTdsTdsCdsCdsCdsGdsCdsCdsGlsGlsTl,

where nucleic bases are represented by the following symbols:

A=adenine, T=thymine, G=guanine, C=cytosine and M=5-methylcytosine;

Sugar components are represented by the following symbols:

l=LNA and d=2ʼ-deoxyribose;

and internucleoside linkages are represented by the following symbol:

s=phosphorothioate.

[0221]

In some embodiments, the invention provides a method for reducing DUX4 mRNA and/or DUX4 protein levels (e.g., intramuscular levels) in an animal suffering from a DUX4-associated disease, which comprises: administering to the animal a DUX4-effective amount of a modified oligonucleotide.

[0222]

In some embodiments, the invention provides a method of reducing DUX4 mRNA and/or DUX4 protein levels (e.g., intramuscular levels) in an animal suffering from FSHD, wherein the method comprises administering to the animal a DUX4-effective amount of a modified oligonucleotide. Further, in some embodiments, the invention provides a method of reducing muscle damage and/or improving motor function in an animal suffering from FSHD, wherein the method comprises administering to the animal a DUX4-effective amount of a modified oligonucleotide.

[0223]

In some embodiments, the invention provides a method of treating, i.e., therapeutically treating, preventing, alleviating, or ameliorating FSHD, wherein the method comprises administering to an animal an effective amount of a modified oligonucleotide with respect to DUX4.

[0224]

In some embodiments, the invention provides a method for preventing, alleviating or alleviating various symptoms of FSHD (e.g., facial weakness, ptosis of the eyelids, inability to whistle, decreased ability to change facial expression, melancholic or angry facial expression, difficulty pronouncing words, scapular weakness (deformities such as winged scapulae and sloping shoulders), lower limb weakness, hearing loss and heart disease), wherein the method comprises administering to the animal an effective amount of a modified oligonucleotide with respect to DUX4.

[0225]

In some embodiments, the invention provides a method of reducing levels (e.g., levels in B cells in the blood) of mRNA and/or protein generated by fusion of the DUX4 gene to the IGH gene in an animal with B-cell acute lymphocytic leukemia or the like, wherein the method comprises administering to the animal an effective amount of a modified oligonucleotide with respect to DUX4. Furthermore, in some embodiments, the invention provides a method of treating, i.e., therapeutically treating, preventing, ameliorating, or ameliorating B-cell acute lymphocytic leukemia or the like, wherein the method comprises administering to the animal an effective amount of a modified oligonucleotide with respect to DUX4.

[0226]

In some embodiments, the invention provides a method for reducing levels (e.g., levels in a sarcoma) of mRNA and/or protein formed by fusing a DUX4 gene to a CIC gene in an animal with differentiated round cell sarcoma or the like, wherein the method comprises administering to the animal an effective amount of a modified oligonucleotide with respect to DUX4. Furthermore, in some embodiments, the invention provides a method for treating, i.e., therapeutically treating, preventing, alleviating, or alleviating differentiated round cell sarcoma or the like, wherein the method comprises administering to the animal an effective amount of a modified oligonucleotide with respect to DUX4.

[0227]

In some embodiments, the invention provides a method for reducing levels (e.g., levels in a sarcoma) of mRNA and/or protein generated by fusing a DUX4 gene to an EWSR1 gene in an animal with fetal rhabdomyosarcoma or the like, wherein the method comprises administering to the animal an effective amount of a modified oligonucleotide with respect to DUX4. In addition, in some embodiments, the invention provides a method for treating, i.e., therapeutically treating, preventing, ameliorating, or alleviating fetal rhabdomyosarcoma or the like, wherein the method comprises administering to the animal an effective amount of a modified oligonucleotide with respect to DUX4.

[0228]

In some embodiments, the invention provides a method for the therapeutic treatment, prevention, amelioration or alleviation of a disease associated with DUX4 with reduced side effects, wherein the method comprises administering to an animal a modified oligonucleotide with respect to DUX4. In some embodiments, side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity (histopathological abnormalities: degenerative necrosis of hepatocytes, hepatocellular hypertrophy, etc.), renal toxicity (histopathological abnormalities, etc.), central nervous system abnormalities, myopathies and malaise. For example, elevated ALT, AST, γ-GTP, GLDH, ALP (alkaline phosphatase), or TBA (total bile acids) in the blood may indicate hepatotoxicity or liver dysfunction. For example, elevated bilirubin may indicate liver toxicity or liver dysfunction. Additionally, elevated levels of protein in the urine or elevated levels of creatinine or UN in the blood may indicate kidney toxicity or impaired kidney function.

[0229]

In some embodiments, the invention provides the use of a compound described herein or a pharmaceutical composition comprising the compound in the manufacture of a medicament for use in any of the treatment methods described herein. For example, in some embodiments, the invention provides the use of a compound described herein or a pharmaceutical composition comprising the compound in the manufacture of a medicament for treating, i.e., therapeutically treating, preventing, delaying, or ameliorating FSHD. In some embodiments, the invention provides the use of a compound described herein or a pharmaceutical composition comprising the compound in the manufacture of a medicament for inhibiting DUX4 expression and for treating, i.e., therapeutically treating, preventing, delaying, or ameliorating a DUX4-associated disease and/or its symptoms. In some embodiments, the invention provides the use of a compound described herein or a pharmaceutical composition comprising the compound in the manufacture of a medicament for reducing DUX4 expression in animals. In some embodiments, there is provided the use of a compound as described herein, or a pharmaceutical composition comprising the compound, in the manufacture of a medicament for ameliorating myotonia by preferentially reducing the level of DUX4-FL mRNA (e.g., the level in muscle) in an animal. In some embodiments, there is provided the use of a compound as described herein, or a pharmaceutical composition comprising the compound, in the manufacture of a medicament for treating an animal suffering from FSHD. In some embodiments, there is provided the use of a compound as described herein, or a pharmaceutical composition comprising the compound, in the manufacture of a medicament for treating one or more symptoms and outcomes associated with the development of FSHD, including muscle rigidity, myotonia, facial weakness, ptosis of the eyelids, inability to whistle, decreased ability to change facial expression, melancholic or angry expression, difficulty pronouncing words, scapular muscle weakness (deformities such as winged scapulae and sloping shoulders), lower limb weakness, hearing loss, and cardiac disease. In some embodiments, the invention provides the use of a compound described herein, or a pharmaceutical composition comprising the compound, in the manufacture of a medicament for preventing the expression of DUX4 protein by controlling the transcription of the DUX4 gene, the translation of DUX4 mRNA, and the cleavage of DUX4 mRNA.

[0230]

In some embodiments, the invention provides a kit for treating, i.e., therapeutically treating, preventing or ameliorating the condition of FSHD described herein, comprising: a) a compound described herein, and optionally b) an additional agent or therapy described herein. The kit may further include instructions or a label describing the use of the kit for treating, i.e., therapeutically treating, preventing or ameliorating the condition of FSHD.

[0231]

In some embodiments, the invention provides the use of a compound described herein, or a pharmaceutical composition comprising the compound, in the manufacture of a medicament for use in any of the treatment methods described herein. For example, in some embodiments, the invention provides the use of a compound described herein, or a pharmaceutical composition comprising the compound, in the manufacture of a medicament for treating, i.e., therapeutically treating, ameliorating, or preventing FSHD. In some embodiments, the invention provides the use of a compound described herein, or a pharmaceutical composition comprising the compound, in the manufacture of a medicament for inhibiting DUX4 expression and for treating, i.e., therapeutically treating, preventing, delaying, or ameliorating, a DUX4-associated disease and/or its symptoms. In some embodiments, the invention provides the use of a compound described herein, or a pharmaceutical composition comprising the compound, in the manufacture of a medicament for reducing DUX4 expression in animals. In some embodiments, the invention provides the use of a compound as described herein, or a pharmaceutical composition comprising the compound, in the manufacture of a medicament for ameliorating myotonia by preferentially reducing the level of DUX4-FL mRNA (e.g., the level in muscle) in an animal. In some embodiments, the invention provides the use of a compound as described herein, or a pharmaceutical composition comprising the compound, in the manufacture of a medicament for treating an animal suffering from FSHD. In some embodiments, the invention provides a compound as described herein, or a pharmaceutical composition comprising the compound, for treating one or more symptoms and outcomes associated with the development of FSHD, including muscle rigidity, myotonia, facial weakness, ptosis of the eyelids, inability to whistle, decreased ability to change facial expression, melancholic or angry expression, difficulty pronouncing words, scapular muscle weakness (deformities such as winged scapulae and sloping shoulders), lower limb weakness, hearing loss, and heart disease. In some embodiments of the invention, there is provided a modified oligonucleotide having a nucleobase sequence consisting of the nucleobase sequence of SEQ ID NO: 2-4, 7-64, 69-97 or 102-109 in the sequence listing, or a compound selected from compounds Nos. 1-112, 114-132 or 137-246.

[0232]

In some embodiments, the modified oligonucleotide has a nucleobase sequence that, when described in the 5″ to 3″ direction, includes a reverse complementary portion of a target segment of a target nucleic acid to which the modified oligonucleotide is directed. In some such embodiments, the modified oligonucleotide has a nucleobase sequence that, when described in the 5″ to 3″ direction, includes a reverse complementary portion of a target segment of a target nucleic acid to which the modified oligonucleotide is directed.

[0233]

In some embodiments, the modified oligonucleotide described herein that targets DUX4 is 12-30 nucleotides in length. In other words, in some embodiments, the modified oligonucleotide is a linked nucleobase that is 12-30 residues in length. In other embodiments, the modified oligonucleotide comprises a modified oligonucleotide consisting of a linked nucleobase 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 or 14-15 residues. In some such embodiments, the modified oligonucleotide comprises a modified oligonucleotide consisting of a linked nucleobase of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 residues in length or in a range defined by any two of the above values. In some embodiments, a modified oligonucleotide having any such length comprises at least 8 immediately adjacent nucleobases in a nucleobase sequence described by any immediately adjacent nucleobases (e.g., SEQ ID NOs: 2-4, 7-64, 69-97, or 102-109 in a sequence listing) of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 residues of the nucleobase sequence of any of the exemplary modified oligonucleotides described herein.

[0234]

It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce a mismatch without eliminating activity. For example, according to Woolf et al., (Proc. Natl. Acad. Sci. USA 89: 7305-7309, 1992), a series of antisense oligonucleotides of 13-25 nucleotides in length were tested for their ability to induce cleavage of target RNA in an oocyte injection model. A 25-nucleotide long antisense oligonucleotide with 8 or 11 mismatches near the ends of the antisense oligonucleotide was able to drive specific cleavage of the target mRNA, although to a lesser extent than an antisense oligonucleotide that did not contain mismatches. Similarly, target-specific cleavage was reported to be achieved using 13-base antisense oligonucleotides, including oligonucleotides with 1 or 3 mismatches.

[0235]

According to Gautschi et al., (J. Natl. Cancer Inst. 93: 463-471, March 2001) an oligonucleotide having 100% complementarity to bc1-2 mRNA and having 3 mismatches to bc1-xL mRNA demonstrated the ability to reduce the expression of both bc1-2 and bc1-xL in vitro and in vivo. In addition, this oligonucleotide was reported to exhibit potent antitumor activity in vivo.

[0236]

Target Nucleic Acid, Target Region, and NSequence

Examples of nucleotide sequence encoding DUX4 include, but are not limited to, the following sequences.

・ The sequence indicated by GenBank accession number NM_001293798.2 (incorporated herein as SEQ ID NO: 1 in the Sequence Listing). The splice variant of SEQ ID NO: 1 in the Sequence Listing is also referred to as DUX4-FL1 or mature DUX4 mRNA.

・ The sequence indicated by GenBank accession number NM_001306068.2 (incorporated herein as SEQ ID NO: 5 in the Sequence Listing). The splice variant of SEQ ID NO: 5 is also referred to as DUX4-FL2 in the Sequence Listing.

・ The sequence indicated by GenBank accession number NM_001363820.1 (incorporated herein as SEQ ID NO: 6 in the Sequence Listing). The splice variant of SEQ ID NO: 6 is also referred to as DUX4-s in the Sequence Listing.

・ SNPs of the above splicing variants.

The sequences presented in SEQ ID NO in the sequence listing in the examples contained herein are independent of any modifications to the sugar moiety, the internucleoside linkage or the nucleobase. Therefore, a modified oligonucleotide defined by SEQ ID NO in the sequence listing may independently include one or more modifications to the sugar moiety, the internucleoside linkage or the nucleobase. A modified oligonucleotide described by a compound number indicates a combination of a nucleobase sequence and a motif.

[0237]

In some embodiments, the target region is a structurally defined region of a target nucleic acid. For example, the target region may include one or more of the following: 3' UTR, 5' UTR, exon, intron, exon/intron junction, coding region, translation initiation region, translation termination region, or other defined regions of a nucleic acid. A structurally defined region for DUX4 can be obtained by accession number from a sequence database, such as NCBI, and such information is incorporated herein by reference. In some embodiments, the target region may include sequence from the 5' target site of one target segment in the target region to the 3' target site of another target segment in the target region.

[0238]

Targeting includes determining at least one target segment to which the modified oligonucleotide hybridizes such that a desired effect occurs. In some embodiments, the desired effect is a decrease in the level of a target nucleic acid mRNA. In some embodiments, the desired effect is a decrease in the level of a protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid.

[0239]

The target region may comprise one or more target segments. Multiple target segments in a target region may overlap. Alternatively, they may not overlap. In some embodiments, the target segments in a target region are separated by no more than about 300 nucleotides. In some embodiments, the target segments in a target region are separated by a number of nucleotides of a target nucleic acid, wherein this number is or is about, or is less than or about 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10, or in a range defined by any two of the preceding numbers. In some embodiments, the segments in a target region are separated by no more than or about no more than 5 nucleotides on a target nucleic acid. In some embodiments, the target segments are contiguous. A target region is provided that is defined by a range having a source nucleic acid that is either a 5' target site or a 3' target site as listed herein.

[0240]

A suitable target segment can be found in the 5' UTR, coding region, 3' UTR, intron, exon, or exon/intron junction. A target segment containing a start codon or stop codon is also a suitable target segment. A suitable target segment may specifically exclude some structurally defined region, such as a start codon or stop codon.

[0241]

Determining a suitable target segment may involve comparing the target nucleic acid sequence to other sequences throughout the genome. For example, the BLAST algorithm can be used to identify regions of similarity between different nucleic acids. This comparison can prevent the selection of a modified oligonucleotide sequence that may hybridize non-specifically to a sequence other than the selected target nucleic acid (i.e., a non-target sequence or off-target sequence).

[0242]

There may be variations in the activity (e.g., as determined by the percentage reduction in the level of the target nucleic acid) of the modified oligonucleotide in the active target region. In some embodiments, a decrease in the level of DUX4 mRNA is indicative of inhibition of DUX4 protein expression. A decrease in the level of DUX4 protein is also indicative of suppression of target mRNA expression. In addition, phenotypic changes, such as a decrease in myotonia and a decrease in myopathy, may be indicative of inhibition of DUX4 mRNA and/or protein expression.

[0243]

Hybridization

In some embodiments, hybridization occurs between a modified oligonucleotide described herein and a DUX4 nucleic acid. The most common mechanism of hybridization involves hydrogen bonds (e.g., Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds) between complementary nucleic acid bases of nucleic acid molecules. The strength of hybridization can be represented by the melting temperature Tm. Tm is the temperature at which 50% of the hybridized double-stranded nucleic acid dissociates into a single-stranded nucleic acid. Tm varies depending on the salt state of the solution, the length of the nucleic acid, the base sequence, and the like, but a higher Tm indicates stronger hybridization.

[0244]

Hybridization can occur under various conditions. The stringent conditions depend on the sequence and are determined by the nature and composition of the nucleic acid molecules to be hybridized.

[0245]

A method for determining whether a sequence specifically hybridizes to a target nucleic acid is well known in the art (Sambrooke and Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., 2001). In some embodiments, a modified oligonucleotide provided herein is capable of specifically hybridizing to a DUX4 nucleic acid.

[0246]

Complementarity

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

[0247]

The modified oligonucleotide can hybridize to one or more segments of the DUX4 nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., loop structure, mismatch, or hairpin structure).

[0248]

In some embodiments, a modified oligonucleotide provided herein, or a specified portion thereof, is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a DUX4 nucleic acid, target region, target segment, or a specified portion thereof. In some embodiments, the modified oligonucleotide is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to a DUX4 nucleic acid, target region, target segment, or specified portion thereof, and comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 immediately adjacent nucleotides in the nucleobase sequence of any of the exemplified modified oligonucleotides. oligonucleotides described herein (e.g., at least 8 immediately adjacent nucleotides in a nucleobase sequence described in any of SEQ ID NOs: 2, 3, 4, 7-64, 69-97, or 102-109 in the sequence listing). The percentage of complementarity of a modified oligonucleotide to a target nucleic acid can be determined using a conventional method and measured over the entire modified oligonucleotide.

[0249]

For example, 18 of the 20 nucleobases of the modified oligonucleotide are complementary to the target region, and thus the modified oligonucleotide that appears to hybridize specifically corresponds to 90 percent complementarity. In this example, the remaining non-complementary nucleobases may be grouped or interspersed with the complementary nucleobases and need not be directly adjacent to each other or to the complementary nucleobases. The percentage complementarity of the modified oligonucleotide to a region of the target nucleic acid can be determined in the usual manner using the BLAST (Basic Local Alignment Search Tools) program and the PowerBLAST program known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). The percentage homology, sequence identity or complementarity can be determined, for example, with the Gap program (Wisconsin Sequence Analysis Package, version 8 for Unix, Genetics Computer Group, University Research Park, Madison WI) using default settings, using the Smith and Waterman algorithm (Adv. Appl. Math., 1981, 2, 482-489).

[0250]

The non-complementary nucleobase region may be at the 5'-end or the 3'-end of the modified oligonucleotide. Alternatively, the non-complementary nucleobase (or non-complementary nucleobases) may be at an internal position of the modified oligonucleotide. When two or more non-complementary nucleobases are present, they may either be directly adjacent to each other (i.e., linked) or not directly adjacent to each other. In one embodiment, the non-complementary nucleobase is located in a wing segment of the gapmer-modified oligonucleotide.

[0251]

In some embodiments of the invention, a modified oligonucleotide that is up to or equal to 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleobases in length includes no more than 3, no more than 2, or no more than 1 non-complementary nucleobase relative to a target nucleic acid, such as a DUX4 nucleic acid, or a specified portion thereof.

[0252]

Modified oligonucleotides provided herein also include those that are complementary to a portion of a target nucleic acid. As used herein, the term "portion" refers to a specified number of immediately adjacent (i.e., linked) nucleobases in a region or segment of a target nucleic acid. "Portion" may also refer to a specified number of immediately adjacent nucleobases of a modified oligonucleotide. In some embodiments, a modified oligonucleotide is complementary to at least a portion of 8 nucleobases of a target segment. In some embodiments, a modified oligonucleotide is complementary to at least a portion of 10 nucleobases of a target segment. In some embodiments, a modified oligonucleotide is complementary to at least a portion of 15 nucleobases of a target segment. Also considered is a modified oligonucleotide that is complementary to a portion of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or more nucleobases of the target segment, or a portion of nucleobases in a number in the range determined by any two of these values.

[0253]

Identity

The modified oligonucleotide provided herein may also have a certain percentage of identity to a particular nucleotide sequence of SEQ ID NO in the sequence listing or a compound represented by a particular compound number, or a portion thereof. As used herein, a modified oligonucleotide is identical to a sequence described herein when it has the same nucleobase pairing ability. For example, RNA that contains uracil in place of thymidine in the described DNA sequence is considered identical to the DNA sequence because both uracil and thymidine pair with adenine. Also contemplated are shortened and extended versions of a modified oligonucleotide described herein and a compound that has a non-identical base to the modified oligonucleotide provided herein. The non-identical bases may be contiguous or dispersed throughout the modified oligonucleotide. The percentage identity of a modified oligonucleotide is calculated according to the number of bases that have identical base pairs relative to the compared sequence.

[0254]

In some embodiments, a modified oligonucleotide or portion thereof is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to one or more examples of modified oligonucleotides described herein, or to SEQ ID NO in the sequence listing, or portions thereof.

[0255]

Modifications

A nucleoside is a combination of a base sugar. The nucleotide base portion (also known as the base) of a nucleoside is usually a heterocyclic base moiety. A nucleotide is a nucleoside that further includes a phosphate group covalently linked to the sugar moiety of the nucleoside. For a nucleoside that includes a pentofuranosyl sugar, the phosphate group may be linked to the 2', 3', or 5' hydroxyl group of the sugar. An oligonucleotide is formed by covalently linking adjacent nucleosides to each other to form a linear polymeric oligonucleotide. In the structure of an oligonucleotide, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.

[0256]

Modifications of the modified oligonucleotide include substitution or alteration of internucleoside linkages, sugar moieties, or nucleobases. The modified oligonucleotide is often preferred over the native form due to desirable properties such as, for example, increased cellular uptake, increased affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity.

[0257]

Modified internucleoside linkages

The natural internucleoside linkage of RNA and DNA is a 3'-5'-phosphodiester linkage. A modified oligonucleotide having one or more modified, i.e., non-natural, internucleoside linkages is often chosen over a modified oligonucleotide having natural internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, increased affinity for target nucleic acids, and increased stability in the presence of nucleases.

[0258]

An oligonucleotide having modified internucleoside linkages includes internucleoside linkages that retain a phosphorus atom and internucleoside linkages that lack a phosphorus atom. Typical phosphorus-containing internucleoside linkages include, but are not limited to, one or more of the following: phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates. Methods for preparing phosphorus-containing and phosphorus-free linkages are well known.

[0259]

In some embodiments, a modified oligonucleotide targeting a DUX4 nucleic acid comprises one or more modified internucleoside linkages. In some embodiments, the modified internucleoside linkages are phosphorothioate linkages. In some embodiments, each internucleoside linkage of the modified oligonucleotide is a phosphorothioate internucleoside linkage.

[0260]

Modified sugar moieties

As the modified oligonucleotide of the present invention, it is preferable to use a modified oligonucleotide in which at least one nucleoside contains a modified sugar. In the present invention, the term "modified sugar" refers to a sugar in which a sugar moiety is modified, and a modified oligonucleotide containing one or more such modified sugars has advantageous characteristics such as increased nuclease stability and increased binding affinity. It is preferable that at least one of the modified sugars contains a bicyclic sugar or a substituted sugar moiety.

[0261]

Examples of nucleosides having a modified sugar include, but are not limited to, nucleosides containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, _2' - _{OCH3 ,} 2'- _OCH2CH3 , 2'- _OCH2CH2F , and 2'-O( _CH2 ) _{2OCH3 substituents} . The substituent at position 2ʼ may also be selected from allyl, amino, azido, thio, O _{-allyl, OC1-C10 alkyl, OCF3, OCH2F, O(CH2)2SCH3 , O ( CH2 ) 2 - ON} ( _Rm )( _Rn ), O- _CH2 -C(=O)-N( _Rm )( _Rn ) and O- _CH2 -C(=O)-N( _Rl )-( _CH2 ) ₂ -N( _Rm )( _Rn ) (where _Rl , _Rm and _Rn each independently represent H or substituted or unsubstituted _C1 - _C10 alkyl).

[0262]

Examples of nucleosides having a bicyclic sugar include, but are not limited to, nucleosides that contain a bridge between the 4′ and 2′ atoms of the ribosyl ring. In some embodiments, an oligonucleotide provided herein includes one or more nucleosides having a bicyclic sugar wherein the bridge comprises one of the following formulas: 4′-(CH ₂ )-O-2′(LNA); 4′-(CH ₂ )-S-2′; 4′-(CH ₂ ) ₂ -O-2′(ENA); 4′-CH(CH ₃ )-O-2′ and 4′-CH(CH ₂ OCH ₃ )-O-2′ (and analogs thereof, see U.S. Patent No. 7,399,845); 4ʼ-C( _CH3 )( _CH3 )-O-2ʼ (and its analogues, see WO 2009/006478); 4ʼ- _CH2 -N( _OCH3 )-2ʼ (and its analogues; see WO 2008/150729); 4ʼ-CH2 _- ON( _CH3 )-2ʼ (and its analogues; see US 2004-0171570); 4ʼ-CH2 _- N(R)-O-2ʼ (where R is H, _C1 - _C12 alkyl or a protecting group) (see US Patent No. 7,427,672); 4ʼ-CH ₂ -C(H)(CH ₃ )-2ʼ (and its analogues; see Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4ʼ-CH ₂ -C(=CH ₂ )-2ʼ (and its analogues; see WO 2008/154401).

[0263]

Additional nucleosides containing a bicyclic sugar 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.; US Patent Nos. 7399845, 6770748, 6525191 and 6268490; US 2008-0039618; US 2007-0287831; US 2004-0171570; US 2009-0012281; WO 2010/036698; WO 2009/067647; WO 2009/067647; WO 2007/134181; WO 2005/021570; WO 2004/106356; WO 94/14226; WO 2009/006478; WO 2008/154401 and WO 2008/150729). Each of the above bicyclic sugar-containing nucleosides can be prepared with one or more stereochemical configurations of the sugar, including, for example, α-L-ribofuranose and β-D-ribofuranose.

[0264]

GuNA, a nucleoside containing a bicyclic sugar, is described as an artificial nucleoside having a guanidine bridge (see WO 2014/046212 and WO 2017/047816). The bicyclic nucleosides ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Trz], and ALNA[Oxz] have been described as cross-linked artificial amino nucleic acids LNA (ALNA) (see Japanese Patent Application No. 2018-212424).

[0265]

In some embodiments, a nucleoside having a bicyclic sugar comprises a bridge between the 4′ and 2′ carbon atoms of the pentofuranosyl sugar moiety, and the bridge comprises from 1 to 4 linked groups each independently selected from, but not limited to, -[C(R _a )(R _b )] _n -, -C(R _a )=C(R _b )-, -C(R _a )=N-, -C(=NR _a )-, -C(= O)-, -C(=S)-, -N(R _a )-, -O-, -Si(R _a ) ₂ - and -S(=O) _x -, wherein: X is 0, 1, or 2; n is 1, 2, 3, or 4; R _a and R _b are each independently H, a protecting group, hydroxyl, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, an aromatic ring group, a substituted aromatic ring group, a heterocyclic group, a substituted heterocyclic group, a C ₅ -C ₇ alicyclic group, a substituted C ₅ -C ₇ alicyclic group, a halogen, OJ ₁ , NJ ₁ J ₂ , SJ ₁ , N ₃ , COOJ ₁ , acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O) ₂ -J ₁ ) or sulfoxyl (S(=O)-J ₁ ), J ₁ and J ₂ each independently represent H, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, an aromatic ring group, a substituted aromatic ring group, acyl (C(=O)-H), substituted acyl, a heterocyclic group, a substituted heterocyclic group, C ₁ -C ₁₂ aminoalkyl, substituted C ₁ -C ₁₂ aminoalkyl or a protecting group.

[0266]

In some embodiments, the bicyclic sugar moiety bridge is [C(R _a )(R _b )] _n -, -[CR _a ) (R _b )] _n -O-, -C(R _a R _b )-N(R)-O-, or -C(R _a R _b )-ON(R)-. In some embodiments, the bridge is 4ʼ-CH ₂ -2ʼ, 4ʼ-(CH ₂ ) ₂ -2ʼ, 4ʼ-(CH ₂ ) ₃ -2ʼ, 4ʼ-CH ₂ -O-2ʼ (the nucleoside having a bicyclic sugar in this case is also referred to as LNA), 4ʼ-(CH ₂ ) ₂ -O-2ʼ, 4ʼ-CH ₂ -ON(R)-2ʼ, and 4ʼ-CH ₂ -N(R)-O-2ʼ-, wherein each R is independently H, a protecting group, or C ₁ -C ₁₂ alkyl.

[0267]

In some embodiments, the bicyclic sugar moiety bridge 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]), or 5-methyl-2,4-oxadiazol-3-yl (ALNA [Oxz]) (Japanese Patent Application No. 2018-212424).

[0268]

In some embodiments, a nucleoside containing a bicyclic sugar is further characterized by an isomeric steric configuration. For example, a nucleoside containing a 4'-(CH ₂ )-O-2' bridge can exist in an α-L-steric configuration or a β-D-steric configuration.

[0269]

In some embodiments, nucleosides containing a bicyclic sugar include nucleosides containing a 4′-2′ bridge, and examples of such bridges include, but are not limited to, α-L-4′-(CH ₂ )-O-2′, β-D-4′-CH ₂ -O-2′,4′-(CH ₂ ) ₂ -O-2′, 4′-CH ₂ -ON(R)-2′, 4′-CH ₂ -N(R)-O-2′, 4′-CH(CH ₃ )-O-2′, 4′-CH ₂ -S-2′, 4′-CH ₂ -CH(CH ₃ )-2′, and 4′-(CH ₂ ) ₃ -2′ (wherein R is H, a protecting group, C ₁ -C ₁₂ alkyl, or urea or guanidine, which may be substituted by C ₁ -C ₁₂ alkyl).

[0270]

In some embodiments, the nucleoside having a bicyclic sugar has the following formula:

,

wherein Bx is a heterocyclic base moiety; T _a and T _b each independently represent a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group which may be substituted, a phosphorus moiety covalently attached to the base, or the like; and Z _a is C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, substituted C ₁ -C ₆ alkyl, substituted C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkynyl, acyl, substituted acyl, substituted amide, thiol, or substituted thiol.

[0271]

In some embodiments, the substituents are each independently monosubstituted or polysubstituted with a substituent independently selected from halogen, oxo, hydroxyl, _OJc , _NJcJd , _{SJc , N3} , OC(=X) _Jc , and _NJeC (=X) _NJcJd (where _Jc , _Jd, and _Je are each independently H _{, C1} - _C6 alkyl, or substituted _C1 - _C6 alkyl; and X is O or _NJc ).

[0272]

In some embodiments, the nucleoside having a bicyclic sugar has the following formula:

,

wherein Bx is a heterocyclic base moiety; T _a and T _b each independently represent a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group which may be substituted, a phosphorus moiety covalently attached to the base, or the like; and Z _b is C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, substituted C ₁ -C ₆ alkyl, substituted C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkynyl, or substituted acyl (C(=O)-).

[0273]

In some embodiments, the nucleoside having a bicyclic sugar has the following formula:

,

wherein Bx is a heterocyclic base moiety; T _a and T _b each independently represent a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group which may be substituted, a phosphorus moiety covalently attached to the base, or the like; R _d is C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, or substituted C ₂ -C ₆ alkynyl; and q _a , q _b , q _c and q _d each independently represent H, halogen, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl or substituted C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxyl, substituted C ₁ -C ₆ alkoxyl, acyl, substituted acyl, C ₁ -C ₆ aminoalkyl or substituted C ₁ -C ₆ aminoalkyl.

[0274]

In some embodiments, the nucleoside having a bicyclic sugar has the following formula:

,

wherein Bx is a heterocyclic base moiety; T _a and T _b each independently represent a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group which may be substituted, a phosphorus moiety covalently attached to the base, or the like; q _a , q _b , q _e and q _f each independently represent hydrogen, halogen, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, C ₁ -C ₁₂ alkoxy, substituted C ₁ -C ₁₂ alkoxy, OJ _j , SJ _j , SOJ _j , SO ₂ J _j , NJ _j J _k , N ₃ , CN, C(=O)OJ _j , C(=O)NJ _j J _k , C(=O)J _j , OC(=O)NJ _j J _k , N(H)C(=NH)NJ _j J _k , N(H)C(=O)-NJ _j J _k or N(H)C(=S)NJ _j J _k ; or q _e and q _f are both =C(q _g )(q _h ); and q _g and q _h are each independently H, halogen, C ₁ -C ₁₂ alkyl or substituted C ₁ -C ₁₂ alkyl.

[0275]

The synthesis and preparation of adenine, cytosine, guanine, 5-methylcytosine, thymine and the bicyclic nucleoside uracil (also called LNA), which have a 4ʼ-CH2- _O -2ʼ bridge, have been described together with their oligomerization and nucleic acid recognition properties (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). The synthesis of a nucleoside containing a bicyclic sugar is also described in WO 98/39352 and WO 99/14226.

[0276]

Various analogues of bicyclic nucleosides having 4'-2' bridging groups have also been prepared, such as 4'- _CH2 -O-2' (the bicyclic nucleoside in this case is also referred to as LNA) and 4'- _CH2 -S-2' (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 has also been described (Wengel et al., WO 99/14226). In addition, the synthesis of 2'-amino-BNA (the bicyclic nucleoside in this case is also referred to as ALNA), i.e., a conformationally constrained oligonucleotide analogue with high affinity, has been described in this area (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2'-amino- and 2'-methylamino-BNA have been prepared, and thermal stability of duplexes with complementary RNA and DNA strands has been previously reported.

[0277]

In some embodiments, the nucleoside having a bicyclic sugar has the following formula:

,

wherein Bx is a heterocyclic base moiety; T _a and T _b each independently represent a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group which may be substituted, a phosphorus moiety covalently attached to the base, or the like; q _i , q _j , q _k and q _l each independently represent H, halogen, C ₁ -C ₁₂ alkyl, substituted C ₁ -C ₁₂ alkyl, C ₂ -C ₁₂ alkenyl, substituted C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, substituted C ₂ -C ₁₂ alkynyl, C ₁ -C ₁₂ alkoxyl, substituted C ₁ -C ₁₂ alkoxyl, OJ _j , SJ _j , SOJ _j , SO ₂ J _j , NJ _j J _k , N ₃ , CN, C(=O)OJ _j , C(=O)NJ _j J _k , C(=O)J _j , OC(=O)NJ _j J _k , N(H)C(=NH)NJ _j J _k , N(H)C(=O)NJ _j J _k or N(H)C(=S)NJ _j J _k ; and both q _i and q _j or q _l and q _k are =C(q _g )(q _h ), where q _g and q _h are each independently H, halogen, C ₁ -C ₁₂ alkyl or substituted C ₁ -C ₁₂ alkyl.

[0278]

One carbocyclic bicyclic nucleoside having a 4ʼ-(CH ₂ ) ₃ -2ʼ bridge and an alkenyl analog bridge 4ʼ-CH=CH-CH ₂ -2ʼ 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 carbocyclic bicyclic nucleosides, as well as their oligomerization and biochemical studies, have also been described (Srivastava et al., J. Am. Chem. Soc. 2007, 129 (26), 8362–8379).

[0279]

In some embodiments, examples of nucleosides containing a bicyclic sugar include, but are not limited to,

(A) α-L-methyleneoxy (4ʼ-CH ₂ -O-2ʼ)BNA,

(B) β-D-methyleneoxy (4ʼ-CH ₂ -O-2ʼ)BNA,

(C) ethyleneoxy (4ʼ-(CH ₂ ) ₂ -O-2ʼ) BNA,

(D) aminooxy (4ʼ-CH ₂ -ON(R)-2ʼ) BNA,

(E) oxyamino (4ʼ-CH ₂ -N(R)-O-2ʼ) BNA,

(F) methyl (methyleneoxy) (4ʼ-CH(CH ₃ )-O-2ʼ) BNA (also called ethyl-limited or cEt),

(G) methylene-thio(4ʼ-CH ₂ -S-2ʼ)BNA,

(H) methylene-amino (4ʼ-CH ₂ -N(R)-2ʼ) BNA,

(I) methylcarbocyclic (4ʼ-CH ₂ -CH(CH ₃ )-2ʼ) BNA,

(J) propylenecarbocyclic (4ʼ-(CH ₂ ) ₃ -2ʼ) BNA and

(K) BNA vinyl as below:

[0280]

,

wherein Bx is a basic moiety; and R, independently, is a protecting group, C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy.

[0281]

In some embodiments, the (LNA) nucleoside having a bicyclic sugar is a nucleoside represented by the following general formula:

,

[wherein B represents a nucleobase; and X and Y each independently represent a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group which may be substituted, a phosphorus moiety, or a covalent attachment to a base, or the like] (see WO 98/39352). Typical specific examples thereof include nucleotides represented by the following formula:

.

[0282]

In some embodiments, the (GuNA) nucleoside having a bicycle is a nucleoside represented by the following general formula:

,

[wherein B is a nucleobase; _R3 , _R4 , _R5 and _R6 each independently represent a hydrogen atom or a _C1-6 alkyl group which may be substituted with one or more substituents; _R7 and _R8 each independently represent a hydrogen atom, a protecting group for a hydroxyl group, a phosphate group which may be substituted, a phosphorus moiety covalently attached to a base, or the like; and _R9 , _R10 and _R11 each independently represent a hydrogen atom, a _C1-6 alkyl group which may be substituted with one or more substituents, or a protecting group for an amino group] (see, for example, WO2014/046212, and WO2017/047816).

[0283]

In some embodiments (ALNA [mU]), the bicyclic sugar-containing nucleoside is a nucleoside represented by the following general formula (I):

,

[wherein B represents a nucleobase; _R1 , _R2 , _R3 and _R4 each independently represent a hydrogen atom or a _C1-6 alkyl group which may be substituted with one or more substituents; _R5 and _R6 each independently represent a hydrogen atom, a protecting group for a hydroxyl group or a phosphate group which may be substituted, a phosphorus moiety covalently attached to the base, or the like; m represents 1 or 2; and X represents a group represented by the following formula (II-1):

;

symbol:

,

described in the formula (II-1) represents a point of attachment to the 2ʼ-amino group; one of R ₇ and R ₈ represents a hydrogen atom, and the other is a methyl group which may be substituted with one or more substituents] (see, for example, Japanese Patent Application No. 2018-212424). A typical specific example is a nucleoside in which one of R ₇ and R ₈ represents a hydrogen atom and the other is an unsubstituted methyl group.

[0284]

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

,

wherein one of R ₇ and R ₈ is a hydrogen atom and the other is an isopropyl group which may be substituted with one or more substituents (see, for example, Japanese Patent Application No. 2018-212424). A typical specific example is a nucleoside in which one of R ₇ and R ₈ is a hydrogen atom and the other is an unsubstituted isopropyl group.

[0285]

In some embodiments (ALNA [Trz]), the nucleoside having a bicycle is a nucleoside having the above general formula (I), and in the formula X is a group represented by the following formula (II-2):

,

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

[0286]

In some embodiments, (ALNA [Oxz]) is a nucleoside having the general formula (I) defined above ALNA [mU], and in the formula X is a group represented by the following formula (II-2):

,

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

[0287]

In some embodiments (ALNA [Ms]), the nucleoside having a bicycle is a nucleoside having the above general formula (I), and in the formula X is a group represented by the following formula (II-3):

,

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

[0288]

In some embodiments, the nucleoside is modified by replacing the ribosyl ring with a sugar substitute. Examples of such modifications include, but are not limited to, replacing the ribosyl ring with a substituent ring system (sometimes referred to as a DNA analog), such as a morpholine ring, a cyclohexenyl ring, a cyclohexyl ring, or a tetrahydropyranyl ring, and such as having one of the following formulas:

,

[0289]

In some embodiments of the invention, a sugar substitute is selected that has the following formula:

,

wherein Bx is a heterocyclic base moiety; _T3 and _T4 each independently represent an internucleoside linking group linking a tetrahydropyran nucleoside analogue to an oligomeric compound; or one of _T3 and _T4 is an internucleoside linking group 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 group of a conjugate, or a 5'- or 3'-terminal group; q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ and q ₇ each independently represent H, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl or substituted C ₂ -C ₆ alkynyl; and one of R ₁ and R ₂ is hydrogen and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ ₁ J ₂ , SJ ₁ , N ₃ , OC(=X)J ₁ , OC(=X)NJ ₁ J ₂ , NJ ₃ C(=X)NJ ₁ J ₂ and CN (where X is O, S or NJ ₁ ; and J ₁ , J ₂ and J ₃ each independently represent H or C ₁ -C ₆ alkyl).

[0290]

In some embodiments, q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ and q ₇ are each H. In some embodiments, at least one of q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ and q ₇ is other than H. In some embodiments, at least one of q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ and q ₇ is methyl. In some embodiments, the invention provides a THP nucleoside wherein one of R ₁ and R ₂ is F. In some embodiments, R ₁ is fluoro and R ₂ is H; R ₁ is methoxy and R ₂ is H; and R ₁ is methoxyethoxy and R ₂ is H.

[0291]

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

[0292]

In some embodiments, the sugar substitute comprises a ring containing more than 5 atoms and more than one heteroatom. For example, a nucleoside containing a morpholino sugar moiety and its use in an oligomeric compound have been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Patent Nos. 5,698,685, 5,166,315, 5,185,444, and 5,034,506).

[0293]

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

.

[0294]

In some embodiments, the morpholino may be modified, for example, by adding or changing various substituents in the above-described morpholino structure. Such a sugar substitute is referred to herein as a "modified morpholino."

[0295]

In some embodiments, the oligonucleotide comprises one or more modified cyclohexenyl nucleosides, which are nucleosides containing a 6-membered cyclohexenyl in place of the pentofuranosyl residue of the natural nucleoside. Examples of modified cyclohexenyl nucleosides include, but are not limited to, those described in the art (see, e.g., according to published documents WO 2010/036696, published 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; WO 06/047842 and WO 01/049687; the contents of each of which are incorporated herein by reference in their entirety).

[0296]

Some modified cyclohexenyl nucleosides have the following formula:

,

Where

Bx is a heterocyclic base moiety; _T3 and _T4 each independently represent an internucleoside linking group linking a cyclohexenyl nucleoside analogue to the oligonucleotide compound; or one of _T3 and _T4 is an internucleoside linking group linking a tetrahydropyran nucleoside analogue to the 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; and q ₁ , q ₂ , q ₃ , q ₄ , q ₅ , q ₆ , q ₇ , q ₈ and q ₉ each independently represent H, C ₁ -C ₆ alkyl, substituted C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, substituted C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, substituted C ₂ -C ₆ alkynyl or another sugar substituent.

[0297]

Many other bicyclic and tricyclic ring systems of sugar substitutes are known in the art that can be used to modify the nucleoside for incorporation into an oligonucleotide (see, for example, the review: Leumann, Christian J., Bioorg. & Med. Chem., 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity.

[0298]

The method for producing modified sugar is well known to those skilled in the art. Some representative U.S. patents that describe the preparation of such modified sugars include, but are not limited to: U.S. Patent Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,567,811, 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,670,633, 5,700,920, 5,792,847, and 6,600,032, and WO 2005/121371, and each of which is incorporated herein by reference in its entirety.

[0299]

In a nucleotide containing a modified sugar moiety, the nucleobase moiety (natural, modified, or a combination of both) is retained during hybridization with a suitable target nucleic acid.

[0300]

Modified Nucleobases

A modification or substitution of a nucleobase (or bases) is structurally distinct from a natural or synthetic unmodified nucleobase and is further functionally interchangeable with such an unmodified nucleobase. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such a modification of nucleobases may impart nuclease stability, binding affinity, or some other useful biological properties to the modified oligonucleotide. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including substitution with 5-methylcytosine, are particularly useful for increasing the binding affinity of the modified oligonucleotide for the target nucleic acid. For example, substitution with 5-methylcytosine increases the stability of the nucleic acid duplex by 0.6-1.2ºC (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

[0301]

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

[0302]

Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of the modified oligonucleotide include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines (including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine).

[0303]

In some embodiments, a modified oligonucleotide targeting a DUX4 nucleic acid comprises one or more modified nucleobases. In some embodiments, a modified oligonucleotide targeting a DUX4 nucleic acid comprises one or more modified nucleobases. In some embodiments, a modified nucleobase is 5-methylcytosine. In some embodiments, each cytosine is 5-methylcytosine.

[0304]

Some motifs of modified oligonucleotides

In some embodiments, a modified oligonucleotide targeting a DUX4 nucleic acid has a chemically modified subunit arranged in a pattern or motif to impart properties to the modified oligonucleotide such as increased inhibitory activity, increased binding affinity for the target nucleic acid, or resistance to nuclease degradation in vivo.

[0305]

The chimeric modified oligonucleotide typically comprises at least one modified region to provide increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity to a target nucleic acid, and/or increased inhibitory activity. A second region of the chimeric modified oligonucleotide may optionally serve as a substrate for the intracellular endonuclease RNase H, which cleaves the RNA strand of the RNA:DNA duplex.

[0306]

A modified oligonucleotide having a gapmer motif is a chimeric modified oligonucleotide. In a gapmer, an internal region having several nucleotides that supports RNase H cleavage is located between external regions having several nucleotides that are chemically different from the nucleosides of the internal region. In the case of a modified oligonucleotide having a gapmer motif, the gap segment typically serves as a substrate for endonuclease cleavage, while the wing segment includes a modified nucleoside. In some embodiments of the invention, the gapmer regions differ in the types of sugar moieties that respectively contain different regions. In some embodiments, the types of sugar moieties that are used to differentiate the gapmer regions may include: β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides may include, among others, 2′-MOE and 2′-O- _CH3s ), and bicyclic sugar-modified nucleosides (such bicyclic sugar-modified nucleosides may include nucleosides containing LNA, GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Trz], and/or ALNA[Oxz]). The wing-gap-wing motif is often described as “XYZ,” where “X” denotes the length of the 5′-wing, “Y” denotes the length of the gap region, and “Z” denotes the length of the 3′-wing region. As used herein, a gapmer described as "XYZ" has a steric configuration such that a gap segment is located immediately adjacent to each 5"-wing segment and 3"-wing segment. Thus, there are no intervening nucleotides between a 5"-wing segment and a gap segment or between a gap segment and a 3"-wing segment. The modified oligonucleotides described herein may have a gapmer motif. In some embodiments, X and Z are the same, while in other embodiments, they are different. In a preferred embodiment, Y is from 8 to 16 nucleotides. X, Y or Z can be any number consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleotides. Therefore, gapmers include, but are not limited to, for example, 2-10-3, 2-14-2, 2-15-2, 2-16-2, 3-6-7, 3-7-5, 3-8-3, 3-8-4, 3-8-5, 3-9-2, 3-9-3, 3-9-4, 3-9-5, 3-9-8, 3-10-2, 3-10-3, 3-10-4, 3-11-3, 3-12-3, 3-14-3, 4-7-4, 4-7-5, 4-8-3, 4-8-4, 4-9-3, 4-10-3, 5-6-4, 5-6-5, 5-7-3, 5-7-4, 5-8-3, 5-8-4 or 7-6-3.

[0307]

In some embodiments, a modified oligonucleotide targeting a DUX4 nucleic acid has a 3-10-3 gapmer motif. In some embodiments, a modified oligonucleotide targeting a DUX4 nucleic acid has a 3-9-3 gapmer motif. In some embodiments, a modified oligonucleotide targeting a DUX4 nucleic acid has a 3-9-4 gapmer motif. In some embodiments, a modified oligonucleotide targeting a DUX4 nucleic acid has a 3-8-5 gapmer motif.

[0308]

In some embodiments, the 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 immediately contiguous nucleotides in the nucleobase sequence of any of the exemplified modified oligonucleotides described herein (e.g., at least 8 immediately contiguous nucleotides in the nucleobase sequence described in any of SEQ ID NOs: 2, 3, 4, 7-64, 69-97 or 102-109 in the sequence list).

[0309]

In some embodiments, the present invention relates to an oligomeric compound comprising an oligonucleotide. In some embodiments, the oligonucleotide comprises one or more chemical modifications. In some embodiments, the chemically modified oligonucleotide comprises one or more modified sugars. In some embodiments, the chemically modified oligonucleotide comprises one or more modified nucleobases. In some embodiments, the chemically modified oligonucleotide comprises one or more modified internucleoside linkages. In some embodiments, the chemical modifications (sugar modification, nucleobase modification, and/or linkage modification) define patterns or motifs. In some embodiments, the patterns of chemical modifications of sugar moieties, internucleoside linkages, and nucleobases are independent of one another. Thus, an oligonucleotide can be described by its sugar modification motif, internucleoside linkage motif, and/or nucleobase modification motif (as used herein, a nucleobase modification motif describes a chemical modification relative to the nucleobases regardless of the nucleobase sequence).

[0310]

Some sugar motifs

In some embodiments, the oligonucleotide comprises one or more types of modified sugar moieties and/or natural sugar moieties arranged along the oligonucleotide or a region thereof in a specific order or sugar modification motif. Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.

[0311]

In some embodiments, the oligonucleotide comprises or consists of a region comprising a gapmer sugar modification motif that comprises two outer regions, i.e., "wing segments," and one inner region, i.e., a "gap segment." The three regions of the gapmer motif (the 5'-wing segment, the gap segment, and the 3'-wing segment) form a continuous sequence of nucleosides in which at least some of the sugar moieties of the nucleosides of each of the wing segments differ from at least some of the sugar moieties of the nucleosides of the gap segment. In particular, at least the sugar moieties of the nucleosides of the wing segments that are closest to the gap segment (the 3'-most outer nucleoside of the 5'-wing segment and the 5'-most outer nucleoside of the 3'-wing segment) are different from the sugar moieties of the adjacent nucleosides of the gap segment, and thus the boundary sides between the wing segments and the gap segment are defined. In some embodiments, the sugar components in the gap segment are identical to each other. In some embodiments, the gap segment includes one or more nucleosides comprising a sugar moiety that is different from the sugar moiety of one or more other nucleosides of the gap segment. In some embodiments, the sugar modification motifs of the two wing segments are identical to each other (symmetrical gapmer). In some embodiments of the invention, the sugar modification motif of the 5'-wing segment is different from the sugar modification motif of the 3'-wing segment (asymmetric gapmer).

[0312]

Some segments of the 5'' wing

In some embodiments, the 5′ wing segment of the gapmer consists of 1 to 5 linked nucleosides. In some embodiments, the 5′ wing segment of the gapmer consists of 2 to 5 linked nucleosides. In some embodiments, the 5′ wing segment of the gapmer consists of 3 to 5 linked nucleosides. In some embodiments, the 5′ wing segment of the gapmer consists of 4 or 5 linked nucleosides. In some embodiments, the 5′ wing segment of the gapmer consists of 1 to 4 linked nucleosides. In some embodiments, the 5′ wing segment of the gapmer consists of 1 to 3 linked nucleosides. In some embodiments, the 5′ wing segment of the gapmer consists of 1 or 2 linked nucleosides. In some embodiments, the 5′ wing segment of the gapmer consists of 2 to 4 linked nucleosides. In some embodiments, the 5′-wing segment of the gapmer consists of 2 or 3 linked nucleosides. In some embodiments, the 5′-wing segment of the gapmer consists of 3 or 4 linked nucleosides. In some embodiments, the 5′-wing segment of the gapmer consists of 1 nucleoside. In some embodiments, the 5′-wing segment of the gapmer consists of 2 linked nucleosides. In some embodiments, the 5′-wing segment of the gapmer consists of 3 linked nucleosides. In some embodiments, the 5′-wing segment of the gapmer consists of 4 linked nucleosides. In some embodiments, the 5′-wing segment of the gapmer consists of 5 linked nucleosides.

[0313]

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

[0314]

In some embodiments, the 5′-wing segment of the gapmer comprises at least one non-bicyclic modified nucleoside. In some embodiments, the 5′-wing segment of the gapmer comprises at least one 2′-substituted nucleoside. In some embodiments, the 5′-wing segment of the gapmer comprises at least one, such as three, four, or five 2′-MOE nucleosides. In some embodiments, the 5′-wing segment of the gapmer comprises at least one 2′-OMe nucleoside. In some embodiments, each nucleoside of the 5′-wing segment of the gapmer is a non-bicyclic modified nucleoside. In some embodiments, each nucleoside of the 5′-wing segment of the gapmer is a 2′-substituted nucleoside. In some embodiments, each nucleoside of the 5'-wing segment of the gapmer is a 2'-MOE nucleoside. In some embodiments, each nucleoside of the 5'-wing segment of the gapmer is a 2'-OMe nucleoside.

[0315]

In some embodiments, the 5′-wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In some embodiments, the 5′-wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one 2′-substituted nucleoside. In some embodiments, the 5′-wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one 2′-MOE nucleoside. In some embodiments, the 5′-wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one 2′-OMe nucleoside. In some embodiments, the 5′-wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one 2′-deoxynucleoside.

[0316]

In some embodiments, the 5′-wing segment of the gapmer comprises at least one ethyl-hindered nucleoside and at least one non-bicyclic modified nucleoside. In some embodiments, the 5′-wing segment of the gapmer comprises at least one ethyl-hindered nucleoside and at least one 2′-substituted nucleoside. In some embodiments, the 5′-wing segment of the gapmer comprises at least one ethyl-hindered nucleoside and at least one 2′-MOE nucleoside. In some embodiments, the 5′-wing segment of the gapmer comprises at least one ethyl-hindered nucleoside and at least one 2′-OMe nucleoside. In some embodiments, the 5'-wing segment of the gapmer comprises at least one ethyl-hindered nucleoside and at least one 2'-deoxynucleoside.

[0317]

In some embodiments, the 5′-wing segment of the gapmer comprises at least one modified nucleoside selected from a 2′-MOE nucleoside, a 2′-OMe nucleoside, an LNA-containing nucleoside, a GuNA-containing nucleoside, an ALNA[Ms]-containing nucleoside, an ALNA[mU]-containing nucleoside, an ALNA[ipU]-containing nucleoside, an ALNA[Trz]-containing nucleoside, and/or an ALNA[Oxz]-containing nucleoside. In some embodiments, the 5′-wing segment of the gapmer comprises at least 2 modified nucleosides selected from a 2′-MOE nucleoside, a 2′-OMe nucleoside, an LNA-containing nucleoside, a GuNA-containing nucleoside, an ALNA[Ms]-containing nucleoside, an ALNA[mU]-containing nucleoside, an ALNA[ipU]-containing nucleoside, an ALNA[Trz]-containing nucleoside, and/or an ALNA[Oxz]-containing nucleoside. In some embodiments, the 5′-wing segment of the gapmer comprises at least 3 modified nucleosides selected from a 2′-MOE nucleoside, a 2′-OMe nucleoside, an LNA-containing nucleoside, a GuNA-containing nucleoside, an ALNA[Ms]-containing nucleoside, an ALNA[mU]-containing nucleoside, an ALNA[ipU]-containing nucleoside, an ALNA[Trz]-containing nucleoside, and/or an ALNA[Oxz]-containing nucleoside. In some embodiments, the 5′-wing segment of the gapmer comprises at least 4 modified nucleosides selected from a 2′-MOE nucleoside, a 2′-OMe nucleoside, an LNA-containing nucleoside, a GuNA-containing nucleoside, an ALNA[Ms]-containing nucleoside, an ALNA[mU]-containing nucleoside, an ALNA[ipU]-containing nucleoside, an ALNA[Trz]-containing nucleoside, and/or an ALNA[Oxz]-containing nucleoside. In some embodiments, the 5′-wing segment of the gapmer comprises at least 5 modified nucleosides selected from a 2′-MOE nucleoside, a 2′-OMe nucleoside, an LNA-containing nucleoside, a GuNA-containing nucleoside, an ALNA[Ms]-containing nucleoside, an ALNA[mU]-containing nucleoside, an ALNA[ipU]-containing nucleoside, an ALNA[Trz]-containing nucleoside, and/or an ALNA[Oxz]-containing nucleoside.

[0318]

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

[0319]

In some embodiments, a 5′-wing segment of a gapmer comprises three ethyl-hindered nucleosides. In some embodiments, a 5′-wing segment of a gapmer comprises two bicyclic nucleosides and two non-bicyclic modified nucleosides. In some embodiments, a 5′-wing segment of a gapmer comprises two ethyl-hindered nucleosides and two 2′-OMe nucleosides. In some embodiments, a 5′-wing segment of a gapmer comprises two bicyclic nucleosides and two non-bicyclic modified nucleosides. In some embodiments, a 5′-wing segment of a gapmer comprises two ethyl-hindered nucleosides and two 2′-OMe nucleosides. In some embodiments, the 5'-wing segment of the gapmer comprises two ethyl-hindered nucleosides and three 2'-OMe nucleosides.

[0320]

In some embodiments, the 5′-wing segment of the gapmer consists of one ALNA [Ms]-containing nucleoside. In some embodiments, the 5′-wing segment of the gapmer consists of two linked ALNA [Ms]-containing nucleosides. In some embodiments, the 5′-wing segment of the gapmer consists of three linked ALNA [Ms]-containing nucleosides. In some embodiments, the 5′-wing segment of the gapmer consists of four linked ALNA [Ms]-containing nucleosides. In some embodiments, the 5′-wing segment of the gapmer consists of five linked ALNA [Ms]-containing nucleosides. In some embodiments, the 5′-wing segment of the gapmer consists of one, two, or three linked ALNA [Ms]-containing nucleosides and one 2′-OMe nucleoside. In some embodiments, the 5′-wing segment of the gapmer consists of linked one, two, or three ALNA [Ms]-containing nucleosides and two 2′-OMe nucleosides. In some embodiments, the 5′-wing segment of the gapmer consists of linked one, two, or three ALNA [Ms]-containing nucleosides and three 2′-OMe nucleosides. In some embodiments, the 5′-wing segment of the gapmer consists of linked three ALNA [Ms]-containing nucleosides and one 2′-MOE nucleoside. In some embodiments, the 5′-wing segment of the gapmer consists of linked two ALNA [Ms]-containing nucleosides and two 2′-MOE nucleosides. In some embodiments, the 5′-wing segment of the gapmer consists of linked three LNA-containing nucleosides and two 2′-MOE nucleosides. In some embodiments, the 5'-wing segment of the gapmer consists of linked two LNA-containing nucleosides and two 2'-MOE nucleosides.

In some embodiments, the 5″-wing segment of the gapmer consists of linked one, two, or three ALNA [Ms]-containing nucleosides and one 5-methylcytosine. In some embodiments, the 5″-wing segment of the gapmer consists of linked one, two, or three ALNA [Ms]-containing nucleosides and two 5-methylcytosines. In some embodiments, the 5″-wing segment of the gapmer consists of linked one, two, or three ALNA [Ms]-containing nucleosides and three 5-methylcytosines.

[0321]

Some segments of the 3' wing

In some embodiments, the 3′ wing segment of a gapmer consists of 1 to 8 linked nucleosides. In some embodiments, the 3′ wing segment of a gapmer consists of 2 to 5 linked nucleosides. In some embodiments, the 3′ wing segment of a gapmer consists of 3 to 5 linked nucleosides. In some embodiments, the 3′ wing segment of a gapmer consists of 4 or 5 linked nucleosides. In some embodiments, the 3′ wing segment of a gapmer consists of 1 to 4 linked nucleosides. In some embodiments, the 3′ wing segment of a gapmer consists of 1 to 3 linked nucleosides. In some embodiments, the 3′ wing segment of a gapmer consists of 1 or 2 linked nucleosides. In some embodiments, the 3′ wing segment of a gapmer consists of 2 to 4 linked nucleosides. In some embodiments, the 3′ wing segment of a gapmer consists of 2 or 3 linked nucleosides. In some embodiments, the 3′ wing segment of a gapmer consists of 3 or 4 linked nucleosides. In some embodiments, the 3′ wing segment of a gapmer consists of 1 nucleoside. In some embodiments, the 3′ wing segment of a gapmer consists of 2 linked nucleosides. In some embodiments, the 3′ wing segment of a gapmer consists of 3 linked nucleosides. In some embodiments, the 3′ wing segment of a gapmer consists of 4 linked nucleosides. In some embodiments, the 3′ wing segment of a gapmer consists of 5 linked nucleosides. In some embodiments, the 3′ wing segment of a gapmer consists of 6 linked nucleosides. In some embodiments, the 3′ wing segment of a gapmer consists of 7 linked nucleosides. In some embodiments, the 3'-wing segment of the gapmer consists of 8 linked nucleosides.

[0322]

In some embodiments, the 3′-wing segment of the gapmer comprises at least one bicyclic nucleoside. In some embodiments, the 3′-wing segment of the gapmer comprises at least two bicyclic nucleosides. In some embodiments, the 3′-wing segment of the gapmer comprises at least three bicyclic nucleosides. In some embodiments, the 3′-wing segment of the gapmer comprises at least four bicyclic nucleosides. In some embodiments, the 3′-wing segment of the gapmer comprises at least one ethyl-hindered nucleoside. In some embodiments, the 3′-wing segment of the gapmer comprises at least one LNA-containing nucleoside, GuNA-containing nucleoside, ALNA[Ms]-containing nucleoside, ALNA[mU]-containing nucleoside, ALNA[ipU]-containing nucleoside, ALNA[Trz]-containing nucleoside, and/or ALNA[Oxz]-containing nucleoside. In some embodiments, each nucleoside of the 3′-wing segment of the gapmer is a bicyclic nucleoside. In some embodiments, each nucleoside of the 3′-wing segment of the gapmer is an ethyl-hindered nucleoside. In some embodiments, each nucleoside of the 3′-wing segment of the gapmer is an LNA-containing nucleoside, a GuNA-containing nucleoside, an ALNA [Ms]-containing nucleoside, an ALNA [mU]-containing nucleoside, an ALNA [ipU]-containing nucleoside, an ALNA [Trz]-containing nucleoside, and/or an ALNA [Oxz]-containing nucleoside.

[0323]

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

[0324]

In some embodiments, the 3′-wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one non-bicyclic modified nucleoside. In some embodiments, the 3′-wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one 2′-substituted nucleoside. In some embodiments, the 3′-wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one 2′-MOE nucleoside. In some embodiments, the 3′-wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one 2′-OMe nucleoside. In some embodiments, the 3′-wing segment of the gapmer comprises at least one bicyclic nucleoside and at least one 2′-deoxynucleoside.

[0325]

In some embodiments, a 3′-wing segment of a gapmer comprises at least one ethyl-hindered nucleoside and at least one non-bicyclic modified nucleoside. In some embodiments, a 3′-wing segment of a gapmer comprises at least one ethyl-hindered nucleoside and at least one 2′-substituted nucleoside. In some embodiments, a 3′-wing segment of a gapmer comprises at least one ethyl-hindered nucleoside and at least one 2′-MOE nucleoside. In some embodiments, a 3′-wing segment of a gapmer comprises at least one ethyl-hindered nucleoside and at least one 2′-OMe nucleoside. In some embodiments, the 3'-wing segment of the gapmer comprises at least one ethyl-hindered nucleoside and at least one 2'-deoxynucleoside.

[0326]

In some embodiments, the 3′-wing segment of the gapmer comprises at least one modified nucleoside selected from a 2′-MOE nucleoside, a 2′-OMe nucleoside, an LNA-containing nucleoside, a GuNA-containing nucleoside, an ALNA[Ms]-containing nucleoside, an ALNA[mU]-containing nucleoside, an ALNA[ipU]-containing nucleoside, an ALNA[Trz]-containing nucleoside, and/or an ALNA[Oxz]-containing nucleoside. In some embodiments, the 3′-wing segment of the gapmer comprises at least two modified nucleosides selected from a 2′-MOE nucleoside, a 2′-OMe nucleoside, an LNA-containing nucleoside, a GuNA-containing nucleoside, an ALNA[Ms]-containing nucleoside, an ALNA[mU]-containing nucleoside, an ALNA[ipU]-containing nucleoside, an ALNA[Trz]-containing nucleoside, and/or an ALNA[Oxz]-containing nucleoside. In some embodiments, the 3′-wing segment of the gapmer comprises at least three modified nucleosides selected from a 2′-MOE nucleoside, a 2′-OMe nucleoside, an LNA-containing nucleoside, a GuNA-containing nucleoside, an ALNA[Ms]-containing nucleoside, an ALNA[mU]-containing nucleoside, an ALNA[ipU]-containing nucleoside, an ALNA[Trz]-containing nucleoside, and/or an ALNA[Oxz]-containing nucleoside. In some embodiments, the 3′-wing segment of the gapmer comprises at least four modified nucleosides selected from a 2′-MOE nucleoside, a 2′-OMe nucleoside, an LNA-containing nucleoside, a GuNA-containing nucleoside, an ALNA[Ms]-containing nucleoside, an ALNA[mU]-containing nucleoside, an ALNA[ipU]-containing nucleoside, an ALNA[Trz]-containing nucleoside, and/or an ALNA[Oxz]-containing nucleoside. In some embodiments, the 3′-wing segment of the gapmer comprises at least five modified nucleosides selected from a 2′-MOE nucleoside, a 2′-OMe nucleoside, an LNA-containing nucleoside, a GuNA-containing nucleoside, an ALNA[Ms]-containing nucleoside, an ALNA[mU]-containing nucleoside, an ALNA[ipU]-containing nucleoside, an ALNA[Trz]-containing nucleoside, and/or an ALNA[Oxz]-containing nucleoside. In some embodiments, the 3′-wing segment of the gapmer comprises at least six modified nucleosides selected from a 2′-MOE nucleoside, a 2′-OMe nucleoside, an LNA-containing nucleoside, a GuNA-containing nucleoside, an ALNA[Ms]-containing nucleoside, an ALNA[mU]-containing nucleoside, an ALNA[ipU]-containing nucleoside, an ALNA[Trz]-containing nucleoside, and/or an ALNA[Oxz]-containing nucleoside. In some embodiments, the 3′-wing segment of the gapmer comprises at least seven modified nucleosides selected from a 2′-MOE nucleoside, a 2′-OMe nucleoside, an LNA-containing nucleoside, a GuNA-containing nucleoside, an ALNA[Ms]-containing nucleoside, an ALNA[mU]-containing nucleoside, an ALNA[ipU]-containing nucleoside, an ALNA[Trz]-containing nucleoside, and/or an ALNA[Oxz]-containing nucleoside. In some embodiments, the 3′-wing segment of the gapmer comprises at least eight modified nucleosides selected from a 2′-MOE nucleoside, a 2′-OMe nucleoside, an LNA-containing nucleoside, a GuNA-containing nucleoside, an ALNA[Ms]-containing nucleoside, an ALNA[mU]-containing nucleoside, an ALNA[ipU]-containing nucleoside, an ALNA[Trz]-containing nucleoside, and/or an ALNA[Oxz]-containing nucleoside.

[0327]

In some embodiments, a gapmer 3′ wing segment comprises two LNA-containing nucleosides. In some embodiments, a gapmer 3′ wing segment comprises three LNA-containing nucleosides. In some embodiments, a gapmer 3′ wing segment comprises four LNA-containing nucleosides. In some embodiments, a gapmer 3′ wing segment comprises three ALNA [Ms]-containing nucleosides. In some embodiments, a gapmer 3′ wing segment comprises two GuNA-containing nucleosides. In some embodiments, a gapmer 3′ wing segment comprises three GuNA-containing nucleosides. In some embodiments, a gapmer 3′ wing segment comprises three ALNA [mU]-containing nucleosides. In some embodiments, a gapmer 3′ wing segment comprises three ALNA [ipU]-containing nucleosides. In some embodiments, the 3′-wing segment of the gapmer includes two LNA-containing nucleosides and one GuNA-containing nucleoside. In some embodiments, the 3′-wing segment of the gapmer includes three ALNA [Trz]-containing nucleosides. In some embodiments, the 3′-wing segment of the gapmer includes three ALNA [Ms]-containing nucleosides and one 2′-OMe nucleoside. In some embodiments, the 3′-wing segment of the gapmer includes three ALNA [Ms]-containing nucleosides and two 2′-OMe-containing nucleosides. In some embodiments, the 3′-wing segment of the gapmer includes three 2′-MOE nucleosides. In some embodiments, the 3′-wing segment of the gapmer includes four 2′-MOE nucleosides. In some embodiments, a 3′-wing segment of a gapmer comprises five 2′-MOE nucleosides. In some embodiments, a 3′-wing segment of a gapmer comprises three ALNA [Ms]-containing nucleosides and one 2′-MOE-containing nucleoside. In some embodiments, a 3′-wing segment of a gapmer comprises three ALNA [Ms]-containing nucleosides and two 2′-MOE nucleosides. In some embodiments, a 3′-wing segment of a gapmer comprises two ALNA [Ms]-containing nucleosides and two 2′-MOE nucleosides. In some embodiments, a 3′-wing segment of a gapmer comprises two ALNA [Ms]-containing nucleosides and three 2′-MOE nucleosides. In some embodiments, the 3′-wing segment of the gapmer includes three ALNA [Ms]-containing nucleosides and five 2′-MOE nucleosides. In some embodiments, the 3′-wing segment of the gapmer includes one ALNA [Ms]-containing nucleoside and three 2′-MOE nucleosides. In some embodiments, the 3′-wing segment of the gapmer includes two LNA-containing nucleosides and two 2′-MOE nucleosides.

[0328]

In some embodiments, a 3′-wing segment of a gapmer comprises three ethyl-hindered nucleosides. In some embodiments, a 3′-wing segment of a gapmer comprises two bicyclic nucleosides and two non-bicyclic modified nucleosides. In some embodiments, a 3′-wing segment of a gapmer comprises two ethyl-hindered nucleosides and two 2′-OMe nucleosides. In some embodiments, a 3′-wing segment of a gapmer comprises two bicyclic nucleosides and two non-bicyclic modified nucleosides. In some embodiments, a 3′-wing segment of a gapmer comprises two ethyl-hindered nucleosides and two 2′-OMe nucleosides. In some embodiments, the 3'-wing segment of the gapmer comprises two ethyl-hindered nucleosides and three 2'-OMe nucleosides.

[0329]

In some embodiments, the 3′-wing segment of the gapmer consists of one ALNA [Ms]-containing nucleoside. In some embodiments, the 3′-wing segment of the gapmer consists of two linked ALNA [Ms]-containing nucleosides. In some embodiments, the 3′-wing segment of the gapmer consists of three linked ALNA [Ms]-containing nucleosides. In some embodiments, the 3′-wing segment of the gapmer consists of four linked ALNA [Ms]-containing nucleosides. In some embodiments, the 3′-wing segment of the gapmer consists of five linked ALNA [Ms]-containing nucleosides. In some embodiments, the 3′-wing segment of the gapmer consists of three, four, or five linked 2′-MOE nucleosides. In some embodiments, the 3′-wing segment of a gapmer consists of linked one, two, or three ALNA [Ms]-containing nucleosides and one 2′-OMe nucleoside. In some embodiments, the 3′-wing segment of a gapmer consists of linked one, two, or three ALNA [Ms]-containing nucleosides and two 2′-OMe nucleosides. In some embodiments, the 3′-wing segment of a gapmer consists of linked one, two, or three ALNA [Ms]-containing nucleosides and three 2′-OMe nucleosides. In some embodiments, the 3′-wing segment of a gapmer consists of linked one, two, or three ALNA [Ms]-containing nucleosides and one 2′-MOE nucleoside. In some embodiments, the 3′-wing segment of a gapmer consists of linked one, two, or three ALNA [Ms]-containing nucleosides and two 2′-MOE nucleosides. In some embodiments, the 3′-wing segment of a gapmer consists of linked one, two, or three ALNA [Ms]-containing nucleosides and three 2′-MOE nucleosides. In some embodiments, the 3′-wing segment of a gapmer consists of linked one, two, or three ALNA [Ms]-containing nucleosides and five 2′-MOE nucleosides. In some embodiments, the 3′-wing segment of a gapmer consists of linked two LNA-containing nucleosides and two 2′-MOE nucleosides.

In some embodiments, the 3′-wing segment of a gapmer consists of linked one, two, or three ALNA [Ms]-containing nucleosides and one 5-methylcytosine. In some embodiments, the 3′-wing segment of a gapmer consists of linked one, two, or three ALNA [Ms]-containing nucleosides and two 5-methylcytosines. In some embodiments, the 3′-wing segment of a gapmer consists of linked one, two, or three ALNA [Ms]-containing nucleosides and three 5-methylcytosines. In some embodiments, the 3′-wing segment of a gapmer consists of linked one, two, three, four, or five 2′-MOE nucleosides and one 5-methylcytosine.

[0330]

In some embodiments, the 5′-wing segment of the gapmer consists of one ALNA [Ms]-containing nucleoside and the 3′-wing segment consists of one ALNA [Ms]-containing nucleoside. In some embodiments, the 5′-wing segment of the gapmer consists of two linked ALNA [Ms]-containing nucleosides and the 3′-wing segment consists of two linked ALNA [Ms]-containing nucleosides. In some embodiments, the 5′-wing segment of the gapmer consists of three linked ALNA [Ms]-containing nucleosides and the 3′-wing segment consists of three linked ALNA [Ms]-containing nucleosides. In some embodiments, the 5'-wing segment of the gapmer consists of four linked ALNA [Ms]-containing nucleosides and the 3'-wing segment consists of four linked ALNA [Ms]-containing nucleosides. In some embodiments, the 5'-wing segment of the gapmer consists of five linked ALNA [Ms]-containing nucleosides and the 3'-wing segment consists of five linked ALNA [Ms]-containing nucleosides.

In some embodiments, the 5′-wing segment of the gapmer consists of linked one, two, or three ALNA [Ms]-containing nucleosides and one 2′-OMe nucleoside; and the 3′-wing segment consists of linked one, two, or three ALNA [Ms]-containing nucleosides and one 2′-OMe nucleoside. In some embodiments, the 5′-wing segment of the gapmer consists of linked one, two, or three ALNA [Ms]-containing nucleosides and two 2′-OMe nucleosides; and the 3′-wing segment consists of linked one, two, or three ALNA [Ms]-containing nucleosides and two 2′-OMe nucleosides. In some embodiments, the 5'-wing segment of the gapmer consists of linked one, two, or three ALNA [Ms]-containing nucleosides and three 2'-OMe nucleosides; and the 3'-wing segment consists of linked one, two, or three ALNA [Ms]-containing nucleosides and three 2'-OMe nucleosides.

In some embodiments, the 5′-wing segment of a gapmer consists of linked one, two, or three ALNA [Ms]-containing nucleosides and one or two 2′-MOE nucleosides; and the 3′-wing segment consists of linked one, two, or three ALNA [Ms]-containing nucleosides and one or two 2′-MOE nucleosides. In some embodiments, the 5′-wing segment of a gapmer consists of one, two, or three linked ALNA [Ms]-containing nucleosides; and the 3′-wing segment consists of linked one, two, or three ALNA [Ms]-containing nucleosides and one, two, or three 2′-MOE nucleosides. In some embodiments, the 5′-wing segment of a gapmer consists of one, two, or three linked ALNA [Ms]-containing nucleosides; and the 3″-wing segment consists of one, two, three, four, or five linked 2″-MOE nucleosides. In some embodiments, the 5″-wing segment of the gapmer consists of one, two, three, four, or five linked 2″-MOE nucleosides; and the 3″-wing segment consists of one, two, or three linked ALNA[Ms]-containing nucleosides.

In some embodiments, the 5″-wing segment of the gapmer consists of one, two, or three linked ALNA [Ms]-containing nucleosides and includes one 5-methylcytosine; and the 3″-wing segment of the gapmer consists of one, two, or three linked ALNA [Ms]-containing nucleosides and includes one 5-methylcytosine. In some embodiments, the 5″-wing segment of the gapmer consists of one, two, or three linked ALNA [Ms]-containing nucleosides and includes two 5-methylcytosines; and the 3″-wing segment of the gapmer consists of one, two, or three linked ALNA [Ms]-containing nucleosides and includes one 5-methylcytosine. In some embodiments, the 5″-wing segment of the gapmer consists of one, two, or three linked ALNA [Ms]-containing nucleosides and includes two 5-methylcytosine; and the 3′-wing segment of the gapmer consists of one, two, or three linked ALNA [Ms]-containing nucleosides and includes two 5-methylcytosines. In some embodiments, the 5′-wing segment of the gapmer consists of one, two, or three linked ALNA [Ms]-containing nucleosides and includes three 5-methylcytosines; and the 3′-wing segment of the gapmer consists of one, two, or three linked ALNA [Ms]-containing nucleosides and includes three 5-methylcytosines. In some embodiments, the 5′-wing segment of the gapmer consists of one, two, or three linked ALNA [Ms]-containing nucleosides and includes one 5-methylcytosine; and the 3′-wing segment of the gapmer consists of one, two, three, four, or five linked 2′-MOE nucleosides and includes one 5-methylcytosine. In some embodiments, the 5'-wing segment of the gapmer consists of one, two, or three linked ALNA [Ms]-containing nucleosides; and the 3'-wing segment of the gapmer consists of one, two, or three linked ALNA [Ms]-containing nucleosides and one, two, or three 2'-MOE nucleosides and includes one 5-methylcytosine.

[0331]

In some embodiments, the gap segment of the gapmer comprises 10 immediately adjacent nucleosides and comprises a 2'-OMe nucleoside as the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, or 10th nucleoside, and the remaining nucleosides are deoxynucleosides.

[0332]

Compositions and methods for preparing pharmaceutical compositions

The modified oligonucleotide can be mixed with one or more pharmaceutically acceptable active or inactive substances to prepare a pharmaceutical composition or preparation. The composition and method of preparing the pharmaceutical composition depend on a number of criteria, which include, but are not limited to, the route of administration, the degree of the disease, or the dose to be administered.

[0333]

A modified oligonucleotide targeting a DUX4 nucleic acid can be used in a pharmaceutical composition by combining the modified oligonucleotide with a suitable pharmaceutically acceptable diluent or carrier. Examples of a pharmaceutically acceptable diluent include phosphate buffered saline (PBS). PBS is a diluent suitable for use in a composition intended for parenteral delivery. Therefore, in one embodiment, a pharmaceutical composition comprising a modified oligonucleotide targeting a DUX4 nucleic acid and a pharmaceutically acceptable diluent is used in the method described herein. In some embodiments, the pharmaceutically acceptable diluent is PBS.

[0334]

Pharmaceutical compositions containing modified oligonucleotides include any pharmaceutically acceptable salt, ester, or salt of such ester, or any other oligonucleotides that can provide (directly or indirectly) biologically active metabolites or moieties thereof when administered to animals, including humans. Therefore, for example, the present disclosure also relates to a pharmaceutically acceptable salt of a modified oligonucleotide, a prodrug, a pharmaceutically acceptable salt of such a prodrug, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium salts and potassium salts.

[0335]

A prodrug may include the inclusion of additional nucleosides at one or both ends of the modified oligonucleotide that are cleaved by endogenous nucleases in the body to form an active modified oligonucleotide.

[0336]

Conjugated modified oligonucleotide

The modified oligonucleotide may be covalently linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the resulting modified oligonucleotide. Typical conjugate moieties include cholesterol moieties and lipid moieties. Additional conjugate moieties include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.

[0337]

For example, the modified oligonucleotide may also be modified to have one or more stabilizing groups, which are typically attached to one or both ends of the modified oligonucleotide to improve properties such as nuclease stability. A cap structure is included in the stabilizing group. These terminal modifications may protect the modified oligonucleotide containing the terminal nucleic acid from degradation by exonucleases and may facilitate intracellular delivery and/or localization. The cap may be present at the 5' end (5' cap) or at the 3' end (3' cap), or may be present at both ends. Cap structures are well known in the art and include, for example, an inverted deoxy abasic cap. Additional 3' and 5' stabilizing groups that may be used to cap one or both ends of the modified oligonucleotide to impart nuclease stability include those described in WO 03/004602.

[0338]

Cell culture and treatment with modified oligonucleotides

The effect of the modified oligonucleotide on the level, activity, or expression of DUX4 nucleic acid can be assayed in vitro in a variety of cell types. Cell types used for such assays are available from commercial suppliers (e.g., American Type Culture Collection, Manassus, VA; Zen-Bio, Inc., Research Triangle Park, NC; Clonetics Corporation, Walkersville, MD), and cells are cultured according to the supplier's instructions using commercially available reagents (e.g., Invitrogen Life Technologies, Carlsbad, CA). Examples of cell types include, but are not limited to, C2C12 cells, HepG2 cells, Hep3B cells, primary hepatocytes, A549 cells, GM04281 fibroblasts, and LLC-MK2 cells. These cells can be used to transfect a vector expressing human DUX4 mRNA. The vector is preferably expressed as a fusion protein with a reporter gene such as luciferase or GFP, and an example is the psiCHECK-2 vector (Promega).

[0339]

In vitro study of modified oligonucleotide

The present document describes a method for treating cells with a modified oligonucleotide, which can be appropriately modified according to the type of the modified oligonucleotide.

[0340]

Typically, cells are treated with the modified oligonucleotide when the cells reach approximately 60-80% confluence in culture.

[0341]

The modified oligonucleotide can be introduced into cells, for example, using the lipofection method.

One of the reagents commonly used to introduce the modified oligonucleotide into cultured cells is the cationic lipid transfection reagent, LIPOFECTIN (registered trademark) (Invitrogen, Carlsbad, CA). The modified oligonucleotide is mixed with LIPOFECTIN (registered trademark) in OPTI-MEM (registered trademark) (Invitrogen, Carlsbad, CA) to achieve the desired final concentration of the modified oligonucleotide and the concentration of LIPOFECTIN (registered trademark), which is typically in the range of 2-12 μg/mL per 100 nM modified oligonucleotide.

[0342]

Another reagent used to introduce the modified oligonucleotide into cultured cells is LIPOFECTAMINE 2000 (registered trademark) (Invitrogen, Carlsbad, CA). The modified oligonucleotide is mixed with LIPOFECTAMINE 2000 (registered trademark) in OPTI-MEM (registered trademark) 1 serum reconstitution medium (Invitrogen, Carlsbad, CA) to achieve the desired concentration of the modified oligonucleotide and the concentration of LIPOFECTAMINE (registered trademark), which is typically in the range of 2-12 μg/mL per 100 nM modified oligonucleotide.

[0343]

Another reagent used to introduce the modified oligonucleotide into cultured cells is Cytofectin (registered trademark) (Invitrogen, Carlsbad, CA). The modified oligonucleotide is mixed with Cytofectin (registered trademark) in OPTI-MEM (registered trademark) 1 serum reconstitution medium (Invitrogen, Carlsbad, CA) to achieve the desired concentration of modified oligonucleotide and Cytofectin (registered trademark) concentration, which is typically in the range of 2-12 μg/mL per 100 nM modified oligonucleotide.

[0344]

Another method used to introduce a modified oligonucleotide into cultured cells is electroporation.

[0345]

The modified oligonucleotide can be introduced into cells without using LIPOFECTION or the like. The method of suppressing the expression of the target gene of the modified oligonucleotide in this case is called the gymnosis method.

[0346]

The cells are treated with the modified oligonucleotide using a conventional method. Typically, the cells are harvested 16-48 hours after treatment with the modified oligonucleotide, at which time the level of RNA or protein of the target nucleic acid is measured using a method known in the art and described herein. Typically, when treatment is performed in multiple replicates, the data are presented as the average of the replicate treatments.

[0347]

The concentration of the modified oligonucleotide used varies from cell line to cell line. The method for determining the optimal concentration of the modified oligonucleotide with respect to a particular cell line is well known in the art. The modified oligonucleotide is typically used at a concentration of 1 to 300 nM in transfection using LIPOFECTAMINE2000 (registered trademark), LIPOFECTIN or Cytofectin. The modified oligonucleotide is used at a higher concentration in the range of 625 to 20,000 nM in transfection using electroporation. Based on the degree of inhibition of gene expression at each concentration, the IC ₅₀ concentration of the modified oligonucleotide can be calculated, which inhibits 50% of the gene expression.

[0348]

RNA isolation

RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. The method for isolating RNA is well known in the art. RNA is prepared using a method well known in the art, such as using TRIZOL reagent (registered trademark) (Invitrogen, Carlsbad, CA) according to the manufacturer's recommended protocol.

[0349]

Analysis of inhibition of target level or expression

Inhibition of the level or expression of DUX4 nucleic acid can be analyzed using various methods known in the art. For example, the target level of nucleic acid can be quantified, for example, by Northern blotting, competitive polymerase chain reaction (PCR), or quantitative real-time PCR. RNA analysis can be performed on total cellular RNA or poly (A) + mRNA. The method for isolating RNA is well known in the art. Northern blotting is also routinely performed in the art. Quantitative real-time PCR can be readily performed using a commercially available ABI PRISM (registered trademark) 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, CA, and used according to the manufacturer's instructions.

[0350]

Quantitative analysis of target RNA levels using real-time PCR

Quantitative determination of target RNA levels can be accomplished by quantitative real-time PCR using the ABI PRISM (registered trademark) 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. The quantitative real-time PCR method is well known in the art.

[0351]

Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction to generate complementary DNA (cDNA), which is then used as a substrate for real-time PCR amplification. The RT and real-time PCR reactions are performed sequentially in a single sample well. RT and real-time PCR reagents were obtained from Invitrogen (Carlsbad, CA). The RT and real-time PCR reactions are performed using methods well known to those skilled in the art.

[0352]

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

[0353]

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

[0354]

Protein level analysis

Antisense inhibition of DUX4 nucleic acid can be assessed by measuring the level of DUX4 protein. The level of DUX4 protein can be assayed or quantified using a variety of methods well known in the art, such as immunoprecipitation, Western blotting (immunoblotting), enzyme-linked immunosorbent assay (ELISA), protein assays, protein activity assays (e.g., caspase activity assays), immunohistochemistry, immunocytochemistry, or fluorescence-activated cell sorting (FACS). An antibody directed to the target can be identified and obtained from a variety of sources, such as the MSRS antibody catalog (Aerie Corporation, Birmingham, MI), or can be prepared using conventional methods for producing monoclonal or polyclonal antibodies well known in the art.

[0355]

Gene expression analysis

The expression level of the DUX4 gene can also be measured using a reporter gene such as luciferase. For example, using the psiCHECK-2 vector (Promega), the expression of the DUX4 gene can be measured by the luminescence value of Renilla luciferase, which is a fusion protein with DUX4, and adjusted for the luminescence value of Firefly luciferase present in the same vector, non-specific effects such as cell death can be excluded.

[0356]

In vivo study of modified oligonucleotide

The modified oligonucleotide is tested in an animal to assess its ability to inhibit DUX4 expression and induce a phenotypic change. The study can be performed in a normal animal or in an experimental disease model such as a DUX4 transgenic mouse model (Jones, T. et al., PLoS One 2018; 13(2), article number e0192657) or a mouse expressing the DUX4 gene using a gene-expressing AAV virus (Wallace, L M et al., Mol Ther 2012; 20(7):1417, Wallace, L M et al., Ann Neurol 2011; 69(3):540).

[0357]

For administration to an animal, the modified oligonucleotide is prepared in a pharmaceutically acceptable diluent, such as phosphate-buffered saline. Administration includes the parenteral route. Following a 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 DUX4 protein levels is also measured.

[0358]

Some antisense mechanisms

FSHD is caused by abnormal expression of the DUX4 gene (particularly, DUX4-FL splice variants) in muscles. On the other hand, DUX4 is also expressed in the testes and the like in healthy individuals. In some DUX4 splice variants expressed in the testes and the like, in addition to DUX4-FL, splice variants of exon 1, exon 2, exon 6, exon 7 and/or exon 1, exon 2, exon 4, exon 5, exon 6, exon 7 splice variants are expressed (the above-mentioned Non-Patent Document 1).

[0359]

Some biomarkers

At least in part, for example, the expression of genes MBD3L2, ZSCAN4, TRIM43, DEFB103, ZNF217 or the like is modulated by the accumulation level of DUX4 protein (the above-mentioned non-patent document 2). In addition, the level of creatinine kinase in the blood can be measured as a marker of myopathy.

[0360]

Some indications

In some embodiments, provided herein is a method of treating an individual comprising administering one or more pharmaceutical compositions described herein. In some embodiments, the individual suffers from FSHD.

[0361]

Thus, the present document provides a method for alleviating a symptom associated with FSHD in a subject in need thereof. In some embodiments, a method is provided for reducing the frequency of one or more symptoms associated with FSHD. In some embodiments, the invention provides a method for reducing the severity of a symptom associated with FSHD. In some embodiments, the symptoms associated with FSHD include muscle stiffness, myotonia, facial weakness, ptosis of the eyelids, inability to whistle, decreased ability to change facial expression, melancholic or angry facial expression, difficulty pronouncing words, scapular muscle weakness (deformities such as winged scapulae and sloping shoulders), lower limb weakness, hearing loss, and heart disease.

[0362]

In some embodiments, the method of the invention comprises administering to an individual in need thereof a therapeutically effective amount of a compound targeting a DUX4 nucleic acid.

[0363]

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

[0364]

In some embodiments of the invention, a pharmaceutical composition comprising a modified oligonucleotide targeting DUX4 is used in the preparation of a medicament for the treatment of a patient suffering from or predisposed to a DUX4-associated disease, such as FSHD.

[0365]

In some embodiments, the method described herein comprises administering a compound comprising a modified oligonucleotide having immediately adjacent portions of the nucleobases described herein, as set forth in SEQ ID NO: 2, 3, 4, 7-64, 69-97, or 102-109 in the sequence listing.

[0366]

Introduction

In some embodiments, the compound and pharmaceutical composition described herein are administered parenterally.

[0367]

In some embodiments, parenteral administration is by infusion. The infusion may be continuous or prolonged, short-term or intermittent. In some embodiments, the administered pharmaceutical agent is delivered by pump. In some embodiments, parenteral administration is by injection (e.g., by bolus injection). The injection preparation may be administered by syringe.

[0368]

Examples of parenteral administration include subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intracavitary administration, or intracranial administration such as intrathecal or intraventricular administration. Administration may be continuous or prolonged, short-term or intermittent.

[0369]

In some embodiments, delivery of a compound or pharmaceutical composition described herein results in a reduction in target mRNA and/or protein levels by at least 70%. In some embodiments, delivery of a compound or composition described herein results in a 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% reduction in target mRNA and/or target protein levels 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.

[0370]

In some embodiments of the invention, the modified oligonucleotide is delivered by injection or infusion once daily, once every three days, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, once every six months, twice a year, or once a year.

[0371]

Some combination treatments

In some embodiments, a first agent comprising a modified oligonucleotide of the present invention is co-administered with one or more second agents. In some embodiments, such second agents are for treating the same FSHD as the first agent described herein. In some embodiments, such second agents are for treating a disease, disorder, or condition other than the first agent described herein. In some embodiments, such second agents are for treating an undesirable side effect of one or more pharmaceutical compositions described herein. In some embodiments, the second agent is co-administered with the first agent to treat an undesirable effect of the first agent. In some embodiments, the second agent is co-administered with the first agent to produce a combination effect. In some embodiments, the second agent is co-administered with the first agent to produce a synergistic effect.

[0372]

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

[0373]

Some connections

In some embodiments, the compound described herein can produce an oligomer using a phosphoramidite method using commercially available amidites (including LNA) to synthesize DNA and RNA. The artificial nucleic acid GuNA can produce an oligomer using the method described in WO 2014/046212 and WO 2017/047816. The artificial nucleic acids ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Trz], and ALNA [Oxz] can produce an oligomer using the method described in Japanese Patent Application No. 2018-212424.

[0374]

In some embodiments, a compound described herein has one or more in vitro and/or in vivo properties that are improved over a suitable comparative compound.

[0375]

In some embodiments, a compound designated as Compound No. 1, having the sequence (5″ to 3″) ngagattcccgccggt (n is 5-methylcytosine, incorporated herein as SEQ ID NO: 2), i.e., a gapmer in which the 5″ wing and the 3″ wing, each consisting of three LNA-containing nucleosides, is a compound in which each internucleoside linkage is a phosphorothioate linkage.

[0376]

In some embodiments, a compound designated as Compound No. 2, having the sequence (5″ to 3″) gnagttctccgcggt (n is 5-methylcytosine, incorporated herein as SEQ ID NO: 3 in the sequence listing), i.e., a gapmer in which each 5″-wing and 3″-wing consists of three ALNA[Ms]-containing nucleosides, is a compound in which each internucleoside linkage is a phosphorothioate linkage.

[0377]

In some embodiments, the compound designated as Compound No. 3, having the sequence (5″ to 3″) gnntagacagcgtngg (n is 5-methylcytosine, incorporated herein as SEQ ID NO: 4 in the sequence listing), i.e., a gapmer in which each 5″-wing and 3″-wing consists of three LNA-containing nucleosides, is a compound in which each internucleoside linkage is a phosphorothioate linkage.

[0378]

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

[0379]

Non-limiting description and incorporation by reference

Although some of the compounds, compositions, and methods described herein have been described specifically in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to be limiting. Each reference, GENBANK accession number, and the like described in this application is incorporated herein by reference in its entirety.

[0380]

In the sequence listing accompanying this application, each sequence is designated as "RNA" or "DNA", as appropriate. However, in practice, these sequences may be modified by any combination of chemical modifications. One skilled in the art will readily appreciate that a designation such as "RNA" or "DNA" to describe a modified oligonucleotide is arbitrary in some instances. For example, an oligonucleotide comprising a nucleoside comprising a 2'-OH sugar moiety and a thymine base may be described as DNA having a modified sugar (2'-OH relative to the natural 2'-H of DNA) or as RNA having a modified base (thymine (methylated uracil) relative to the natural uracil of RNA).

[0381]

Therefore, the nucleic acid sequences provided herein, including but not limited to the sequences in the sequence listing, are intended to include, but are not limited to, nucleic acids comprising any combination of natural or modified RNA and/or DNA, including such nucleic acids that have modified nucleobases. As a non-limiting further example, an oligomeric compound having the nucleobase sequence "ATCGATCG" includes any oligomeric compound, modified or unmodified, having such a nucleobase sequence, which includes, but is not limited to, those compounds that contain RNA bases, such as those compounds having the sequence "AUCGAUCG", and those that have some DNA bases and some RNA bases, such as "AUCGATCG", and oligomeric compounds with other modified or natural bases, such as "ATmeCGAUCG" (here meC means a cytosine base containing a methyl group at position 5).

[Examples]

[0382]

Non-limiting description and incorporation by reference

Although some of the compounds, compositions, and methods described herein have been described specifically in accordance with certain embodiments, the following examples are intended to illustrate the compounds described herein only and are not intended to limit them. Each of the references cited in this application is incorporated herein by reference in its entirety.

[0383]

The structures of the artificial nucleic acids used in the present description are shown by the following structural formulas together with their corresponding abbreviations.

Structures and abbreviations of artificial nucleic acids

.

[0384]

Example 1

Synthesis and purification of modified oligonucleotide compound for in vitro evaluation

Using different amidites (LNA amidite was purchased from Chem Genes and Hongene Biotechnology Limited; 2'-OMe amidite was purchased from Sigma-Aldrich; GuNA was synthesized using the methods described in WO 2014/046212 and WO 2017/047816; and ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Trz], and ALNA [Oxz] were synthesized using the methods described in Japanese Patent Application No. 2018-212424), the modified oligonucleotide compound was synthesized at 0.2 or 1.0 μmol scale using CPG or a polystyrene carrier using an nS-8II automatic DNA/RNA oligonucleotide synthesizer (manufactured by Gene Design Inc.). All amidites were adjusted to 0.1 M acetonitrile; the coupling time for unnatural nucleosides was 10 min; and other steps were performed under standard nS-8II conditions. Activator 42 (Sigma-Aldrich) was used as an activator, and sulfurizing reagent II (Gren Research Corporation) was used for thiolation. 28% aqueous ammonia was added to the synthesized oligonucleotide and the reaction was carried out at 60-65ºC for 8 hours to cut out the support and deprotect the main part. After concentration and distillation of ammonia, purification was carried out by reverse-phase HPLC.

[0385]

Example 2

Synthesis and purification of modified oligonucleotide compound for in vivo evaluation

The modified oligonucleotide compound was synthesized using different amidites using a polystyrene support in a scale of 20-50 μm by using an automatic DNA/RNA oligonucleotide synthesizer AKTA oligopilot plus 10 (manufactured by GE Healthcare Japan). DNA amidite was adjusted to 0.1 M, unnatural amidite was adjusted to 0.05-0.1 M acetonitrile solution; the coupling recycling time for unnatural nucleosides was 20 minutes; and when the first base was introduced into the universal support, each of the coupling, thiolation and capping steps were performed twice in succession. The remaining steps were carried out under the standard conditions of AKTA oligopilot plus10. Activator 42 (Sigma-Aldrich) was used as the activator, and sulfurizing reagent II (Gren Research Corporation) was used for thiolation. The synthesized oligonucleotide was subjected to decyanethyl treatment on the solid phase using 20% diethylamine acetonitrile or 50% triethylamine/acetonitrile, 28% aqueous ammonia solution was added and the reaction was carried out at 60-65ºC for 8-24 hours to cut out from the carrier and deprotect the base part. After concentration and distillation of ammonia, purification was carried out using an anion exchange column. Excess salt contained after anion exchange was removed using a desalting column.

[0386]

Example 3

Confirmation of the purity of the modified oligonucleotide compound

Purification and confirmation of the purity of the synthesized modified oligonucleotide compounds were performed using reversed-phase HPLC under the following conditions. All compounds had a purity of 85% or greater.

Reversed-phase HPLC (purification)

Mobile phase:

Solution A: 400 mM hexafluoroisopropanol, 15 mM triethylamine

Solution B: methanol

Gradient: A:B=85:15→70:30 (10 min)

Columns used:

Column Preparative Waters XBridge @ Oligonucleotide BEH C18 OBDTM Prep, 130 Å, 2.5 µm, 10 mM*50 mM

Flow rate:

Preparative 5 ml/min

Column temperature: 60ºC

Detection: UV (260 nm)

Reversed Phase HPLC (Purity Verification)

Mobile phase:

Solution A: 400 mM hexafluoroisopropanol, 15 mM aqueous triethylamine solution

Solution B: methanol

Gradient: A:B=80:20→70:30 (6.5 min)

Columns used:

Analysis Waters ACQUITY UPLC column @ Oligonucleotide BEH C18, 130 Å 1.7 µm, 2.1 mM*50 mM

Flow rate: 0.2 ml/min

Column temperature: 60ºC

Detection: UV (260 nm)

Anion exchange purification

Mobile phase:

Solution A: 1 mM NaOH 20% acetonitrile aqueous solution

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

Column used: TSKgel SuperQ-5PW (13) φ21.1*15 mM

Flow rate: 7 ml/min

Column temperature: room temperature

Detection: UV (260 nm)

Desalination column

Mobile phase:

Solution A: 20% aqueous acetonitrile solution

Solution B: 20% aqueous acetonitrile solution

Columns used:

GE HiPrep 26/10 Desalting* 4 in order

Flow rate: 12 ml/min

Column temperature: room temperature

[0387]

Example 4

Determination of the molecular weight of a modified oligonucleotide compound

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

Mobile phase:

Solution A: 400 mM hexafluoroisopropanol, 15 mM aqueous triethylamine solution

Solution B: methanol

Gradient: A:B=80:20→70:30 (6.5 min)

Columns used:

Column Waters ACQUITY UPLC @ Oligonucleotide BEH C18, 130 Å 1.7 µm, 2.1 mM*50 mM

Flow rate: 0.2 ml/min

Column temperature: 60ºC

Detection: UV (260 nm)

[0388]

Example 5

Molecular weight of the synthesized modified oligonucleotide compound

The synthesized modified oligonucleotide compounds are shown in Table 1 below. In the compound designations, each nucleotide is represented by three letters. However, the 3'-terminal nucleotide is represented by two letters because there is no internucleoside linkage.

1) The first letter is capitalized and indicates the following nitrogen bases:

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

2) the second letter indicates the following sugar fragments:

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

3) the third letter indicates the following internucleoside bonds:

s=phosphorothioate, p=phosphodiester.

The target position indicates the 5'-target site of the mature DUX4 mRNA of the modified oligonucleotide (position of SEQ ID NO: 1 in the sequence listing, corresponding to the 3'-end of the modified oligonucleotide).

[Table 1-1]

Table 1 Compound
No. SEQ NO Position-
target Subsequence m/z Types of ions 1 2 233 MlsGlsAlsGdsAdsTdsTdsCdsCdsCdsGdsCdsCdsGlsGlsTl 1318.8 [M-4H]4- 2 3 1309 GmsMmsAmsGdsTdsTdsCdsTdsCdsCdsGdsCdsGmsGmsTm 1807.9 [M-3H]3- 3 4 1480 GlsMlsMlsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMlsGlsGl 1784.3 [M-3H]3- 4 7 232 GlsAlsTlsTdsCdsCdsCdsGdsCdsCdsGdsGlsTlsGl 4630,44 [M-H]- 5 8 233 AgsGgsAgsTdsTdsCdsCdsCdsGdsCdsCdsGgsGgsTg 1214.8 [M-4H]4- 6 9 233 GgsAgsGgsAdsTdsTdsCdsCdsCdsGdsCdsCdsGgsGgsTg 1300.8 [M-4H]4- 7 9 233 GlsAlsGlsAdsTdsTdsCdsCdsCdsGdsCdsCdsGlsGlsTl 1239.3 [M-4H]4- 8 10 233 MmsGmsAdsGmsAdsTdsTdsMdsMdsMdsGdsMdsMmsGdsGmsTm 1161.8 [M-5H]5- 9 10 233 MmsGdsAmsGmsAdsTdsTdsMdsMdsMdsGdsMdsMmsGmsGdsTm 1161.9 [M-5H]5- 10 2 233 MgsGgsAgsGdsAdsTdsTdsCdsCdsCdsGdsCdsCdsGgsGgsTg 1381,0 [M-4H]4- 11 10 233 MusGusAusGdsAdsTdsTdsMdsMdsMdsGdsMdsMdsGusGusTu 1136.5 [M-5H]5- 12 10 233 MpsGpsApsGdsAdsTdsTdsMdsMdsMdsGdsMdsMdsGpsGpsTp 1170.2 [M-5H]5- 13 11 234 GlsAlsGlsAdsTdsTdsCdsCdsCdsGdsCdsMlsGlsGl 4653,17 [M-H]- 14 11 234 GgsAgsGgsAdsTdsTdsCdsCdsCdsGdsCdsMgsGgsGg 1224.5 [M-4H]4- 15 12 234 GtsAtsGtsAdsTdsTdsMdsMdsMdsGdsMdsMtsGtsGt 1317.7 [M-4H]4- 16 12 234 GusAusGusAdsTdsTdsMdsMdsMdsGdsMdsMusGusGu 1261,0 [M-4H]4- 17 12 234 GmsAmsGmsAdsTdsTdsMdsMdsMdsGdsMdsMmsGmsGm 1292,5 [M-4H]4- 18 13 234 MmsGdsAmsGmsAdsTdsTdsMdsMdsMdsGdsMmsMmsGdsGm 1097,5 [M-5H]5- 19 14 234 MgsGgsAgsGdsAdsTdsTdsCdsCdsCdsGdsCdsMgsGgsGg 1304.5 [M-4H]4- 20 14 234 MlsGlsAlsGdsAdsTdsTdsCdsCdsCdsGdsCdsMlsGlsGl 1242.6 [M-4H]4- 21 15 1306 AlsGlsTlsTdsCdsTdsCdsCdsGdsCdsGdsGdsTdsGlsTlsGl 1769.7 [M-3H]3- 22 16 1307 GlsMlsAlsGdsTdsTdsCdsTdsCdsCdsGdsCdsGdsGdsTlsGlsTl 1876,0 [M-3H]3-

[Table 1-2]

(Table 1, continued) 23 17 1307 GlsGlsMlsAdsGdsTdsTdsCdsTdsCdsCdsGdsCdsGdsGdsTlsGlsTl 1990,8 [M-3H]3- 24 18 1308 MgsAgsGgsTdsTdsCdsTdsCdsCdsGdsCdsGdsGgsTgsGg 1736.7 [M-3H]3- 25 18 1308 MgsAgsGgsTdsTdsCdsTdsCdsCdsGdsCdsGdsGdsTgsGg 1284.9 [M-4H]4- 26 19 1308 MmsAmsGdsTmsTdsMdsTdsMdsMdsGdsMdsGmsGdsTmsGm 1096.2 [M-5H]5- 27 19 1308 MmsAdsGmsTmsTdsMdsTdsMdsMdsGdsMdsGmsGmsTdsGm 1370.6 [M-4H]4- 28 20 1308 GlsCdsAlsGlsTdsTdsCdsTdsCdsCdsGdsCdsGlsGlsTdsGl 1765.4 [M-3H]3- 29 20 1308 GlsCdsAlsGdsTlsTdsCdsTdsCdsCdsGdsCdsGlsGdsTdsGl 1755.3 [M-3H]3- 30 20 1308 GgsCdsAgsGgsTdsTdsCdsTdsCdsCdsGdsCdsGgsGgsTdsGg 1385.1 [M-4H]4- 31 20 1308 GlsCdsAlsGlsTdsTdsCdsTdsCdsCdsGdsCdsGlsGdsTlsGl 1323.4 [M-4H]4- 32 97 1308 GgsCdsAgsGdsTdsTdsCdsTdsCdsCdsGdsMgsGdsGgsTdsGg 1828.3 [M-3H]3- 33 21 1308 GlsMlsAlsGdsTdsTdsCdsTdsCdsCdsGdsCdsGdsGlsTlsGl 1769.4 [M-3H]3- 34 21 1308 GlsMlsAlsGlsTdsTdsCdsTdsCdsCdsGdsCdsGlsGlsTlsGl 1788.3 [M-3H]3- 35 21 1308 GlsMlsAdsGlsTdsTdsCdsTdsCdsCdsGdsCdsGlsGdsTlsGl 1769.2 [M-3H]3- 36 21 1308 GmsMmsAmsGdsTdsTdsCdsTdsCdsCdsGdsCdsGdsGmsTmsGm 1923,5 [M-3H]3- 37 21 1308 GgsMgsAgsGdsTdsTdsCdsTdsCdsCdsGdsCdsGdsGgsTgsGg 1851.7 [M-3H]3- 38 21 1308 GgsMgsAgsGdsTdsTdsCdsTdsCdsCdsGdsCdsGgsGdsTgsGg 1388.3 [M-4H]4- 39 21 1308 GgsMgsAdsGgsTdsTdsCdsTdsCdsCdsGdsCdsGgsGdsTgsGg 1389.1 [M-4H]4- 40 21 1308 GlsMlsAdsGlsTdsTdsCdsTdsCdsCdsGdsCdsGlsGlsTdsGl 1769,5 [M-3H]3- 41 22 1308 GmsMmsAdsGmsTdsTdsMdsTdsMdsMdsGdsMdsGmsGdsTmsGm 1165,5 [M-5H]5- 42 22 1308 GmsMdsAmsGmsTdsTdsMdsTdsMdsMdsGdsMdsGmsGmsTdsGm 1165.4 [M-5H]5- 43 22 1308 GmsMmsAdsGmsTdsTdsMdsTdsMdsMdsGdsMdsGmsGmsTdsGm 1165.2 [M-5H]5- 44 22 1308 GmsMmsAdsGmsTdsTdsMdsTdsMdsMdsGdsMdsGmsGdsTdsGm 1144.2 [M-5H]5- 45 22 1308 GmsMmsAdsGmsTdsTdsMdsTdsMdsMdsGdsMmsGdsGmsTdsGm 1165.2 [M-5H]5-

[Table 1-3]

(Table 1, continued) 46 22 1308 GlsMlsAdsGlsTdsTdsMdsTdsMdsMdsGdsMdsGlsGlsTdsGl 1341.2 [M-4H]4- 47 22 1308 GlsMlsAdsGlsTdsTdsMdsTdsMdsMdsGdsMdsGlsGdsTdsGl 1334.2 [M-4H]4- 48 23 1308 GlsGlsMlsAdsGdsTdsTdsCdsTdsCdsCdsGdsCdsGdsGlsTlsGl 1884,5 [M-3H]3- 49 24 1308 GmsGdsMmsAdsGmsTdsTdsMdsTdsMdsMdsGdsMdsGmsGdsTdsGm 1213.3 [M-5H]5- 50 24 1308 GmsGdsMmsAdsGmsTdsTdsMdsTdsMdsMdsGdsMdsGmsGmsTdsGm 1028,5 [M-6H]6- 51 24 1308 GlsGdsMlsAdsGlsTdsTdsMdsTdsMdsMdsGdsMdsGlsGdsTdsGl 1136.2 [M-5H]5- 52 24 1308 GlsGdsMlsAdsGlsTdsTdsMdsTdsMdsMdsGdsMdsGlsGlsTdsGl 1141.8 [M-5H]5- 53 25 1309 MgsAgsGgsTdsTdsCdsTdsCdsCdsGdsCdsGgsGgsTg 1621,0 [M-3H]3- 54 26 1309 GmsCdsAmsGmsTdsTdsCdsTdsCdsCdsGdsMmsGmsGdsTm 1356.7 [M-4H]4- 55 3 1309 GlsMlsAlsGdsTdsTdsCdsTdsCdsCdsGdsCdsGlsGlsTl 1654.3 [M-3H]3- 56 3 1309 GlsMlsAdsGdsTdsTdsCdsTdsCdsCdsGdsCdsGlsGlsTl 4936.4 [M-H]- 57 3 1309 GlsMlsAlsGdsTdsTdsCdsTdsCdsCdsGdsCdsGdsGlsTl 1645.4 [M-3H]3- 58 3 1309 GgsMgsAgsGdsTdsTdsCdsTdsCdsCdsGdsCdsGgsGgsTg 1736.8 [M-3H]3- 59 3 1309 GgsMgsAdsGdsTdsTdsCdsTdsCdsCdsGdsCdsGgsGgsTg 1285.1 [M-4H]4- 60 3 1309 GlsMlsAgsGdsTdsTdsCdsTdsCdsCdsGdsCdsGgsGlsTl 1261.1 [M-4H]4- 61 3 1309 GlsMgsAlsGdsTdsTdsCdsTdsCdsCdsGdsCdsGlsGgsTl 1261.1 [M-4H]4- 62 3 1309 GgsMlsAlsGdsTdsTdsCdsTdsCdsCdsGdsCdsGlsGlsTg 1261.1 [M-4H]4- 63 3 1309 GlsMlsAdsGlsTdsTdsCdsTdsCdsCdsGdsCdsGlsGlsTl 1240.6 [M-4H]4- 64 27 1309 GmsMmsAdsGmsTdsTdsCdsTdsCdsCdsGdsMmsGdsGmsTm 1360,1 [M-4H]4- 65 28 1309 GgsMgsAgsGdsTdsTdsMdsTdsMdsMdsGdsMdsGgsGgsTg 1316.1 [M-4H]4- 66 29 1309 GmsMmsAmsGdsTdsTdsMdsTdsMdsMdsGdsCosGmsGmsTm 1374.8 [M-4H]4- 67 29 1309 GmsMmsAmsGosTdsTdsMdsTdsMdsMdsGdsCosGmsGmsTm 1381.9 [M-4H]4- 68 29 1309 GmsMmsAmsGdsTdsTdsMdsTdsMdsMdsGosCosGmsGmsTm 1381.9 [M-4H]4-

[Table 1-4]

(Table 1, continued) 69 30 1309 GlsMlsAlsGdsTdsTdsMdsTdsMdsMdsGdsMdsGlsGlsTl 1254.6 [M-4H]4- 70 30 1309 GmsMmsAmsGdsTdsTdsMdsTdsMdsMdsGdsMdsGmsGmsTm 1370.2 [M-4H]4- 71 30 1309 GtsMtsAtsGdsTdsTdsMdsTdsMdsMdsGdsMdsGtsGtsTt 1395.9 [M-4H]4- 72 30 1309 GusMusAusGdsTdsTdsMdsTdsMdsMdsGdsMdsGusGusTu 1338.8 [M-4H]4- 73 30 1309 GpsMpsApsGdsTdsTdsMdsTdsMdsMdsGdsMdsGpsGpsTp 1381.3 [M-4H]4- 74 30 1309 GmsMmsAmsGosTdsTdsMdsTdsMdsMdsGdsMdsGmsGmsTm 1377.9 [M-4H]4- 75 31 1309 GmsMmsAmsGosUosTdsMdsTdsMdsMdsGdsCosGmsGmsTm 1385.9 [M-4H]4- 76 32 1309 GmsMmsAmsGosUosTdsMdsTdsMdsMdsGdsMdsGmsGmsTm 1381.9 [M-4H]4- 77 33 1309 GgsGgsCdsAgsGdsTdsTdsCdsTdsCdsCdsGdsCdsGgsGgsTg 1384.9 [M-4H]4- 78 33 1309 GlsGlsCdsAdsGlsTdsTdsCdsTdsCdsCdsGlsCdsGdsGlsTl 1323.6 [M-4H]4- 79 34 1309 GlsGlsMlsAdsGdsTdsTdsCdsTdsCdsCdsGdsCdsGlsGlsTl 1769.2 [M-3H]3- 80 34 1309 GlsGdsMlsAdsGdsTdsTdsCdsTdsCdsCdsGdsCdsGlsGdsTl 1750.7 [M-3H]3- 81 34 1309 GgsGgsMgsAdsGdsTdsTdsCdsTdsCdsCdsGdsCdsGgsGgsTg 1388.6 [M-4H]4- 82 34 1309 GmsGmsMmsAdsGdsTdsTdsCdsTdsCdsCdsGdsCdsGmsGmsTm 1442.3 [M-4H]4- 83 35 1309 GgsGgsMgsAdsGdsTdsTdsMdsTdsMdsMdsGdsMdsGgsGgsTg 1402.4 [M-4H]4- 84 35 1309 GlsGlsMlsAdsGdsTdsTdsMdsTdsMdsMdsGdsMdsGlsGlsTl 1340.9 [M-4H]4- 85 36 1309 TmsGmsGmsCdsAdsGdsTdsTdsCdsTdsCdsCdsGdsCdsGmsGmsTm 2025.9 [M-3H]3- 86 36 1309 TgsGgsGgsCdsAdsGdsTdsTdsCdsTdsCdsCdsGdsCdsGgsGgsTg 1953,3 [M-3H]3- 87 37 1310 GlsMlsAlsGdsTdsTdsCdsTdsCdsCdsGdsCdsGlsGl 1538.2 [M-3H]3- 88 37 1310 GgsMgsAgsGdsTdsTdsCdsTdsCdsCdsGdsCdsGgsGg 1204.8 [M-4H]4- 89 38 1310 GlsGlsCdsAdsGdsTdsTdsCdsTdsCdsCdsGdsMlsGlsGl 1653.1 [M-3H]3- 90 39 1310 GlsGlsMlsAdsGdsTdsTdsCdsTdsCdsCdsGdsCdsGlsGl 1653.4 [M-3H]3- 91 39 1310 GgsGgsMgsAdsGdsTdsTdsCdsTdsCdsCdsGdsCdsGgsGg 1291.1 [M-4H]4-

[Table 1-5]

(Table 1, continued) 92 40 1310 GgsGgsMgsAdsGdsTdsTdsCdsTdsCdsCdsGdsMgsGgsGg 1312,0 [M-4H]4- 93 41 1310 GlsGlsMlsAdsGdsTdsTdsMdsTdsMdsMdsGdsMdsGlsGl 1253.9 [M-4H]4- 94 42 1472 MlsGlsTlsCdsGdsGdsAdsAdsGdsGdsTdsGlsGlsGl 4774,58 [M-H]- 95 43 1472 GlsMlsGlsTdsCdsGdsGdsAdsAdsGdsGdsTdsGlsGlsGl 1705.7 [M-3H]3- 96 44 1472 AlsGlsMlsGdsTdsCdsGdsGdsAdsAdsGdsGdsTdsGlsGlsGl 1815.1 [M-3H]3- 97 45 1472 MgsAgsGgsCdsGdsTdsCdsGdsGdsAdsAdsGdsGdsTgsGgsGdsGg 1499.9 [M-4H]4- 98 46 1473 AlsGlsMlsGdsTdsCdsGdsGdsAdsAdsGdsGdsTlsGlsGl 1700,3 [M-3H]3- 99 46 1473 AgsGgsMgsGdsTdsCdsGdsGdsAdsAdsGdsGdsTgsGgsGg 1336.7 [M-4H]4- 100 47 1473 AlsGlsMlsGdsTdsMdsGdsGdsAdsAdsGdsGdsTlsGlsGl 1278.6 [M-4H]4- 101 48 1473 MlsAlsGlsCdsGdsTdsCdsGdsGdsAdsAdsGdsGdsTlsGlsGl 1801.8 [M-3H]3- 102 48 1473 MmsAmsGmsCdsGdsTdsCdsGdsGdsAdsAdsGdsGdsTmsGmsGm 1956,1 [M-3H]3- 103 48 1473 MgsAgsGgsCdsGdsTdsCdsGdsGdsAdsAdsGdsGdsTgsGgsGg 1883.9 [M-3H]3- 104 48 1473 MgsAgsGgsCdsGdsTdsCdsGdsGdsAdsAdsGdsGgsTgsGdsGg 1413,0 [M-4H]4- 105 49 1473 MgsAgsGgsMdsGdsTdsMdsGdsGdsAdsAdsGdsGdsTgsGgsGg 1419.9 [M-4H]4- 106 50 1473 AgsMgsAdsGgsCdsGdsTdsCdsGdsGdsAdsAdsGdsGdsTgsGgsGg 1495,5 [M-4H]4- 107 50 1473 AgsMgsAgsGdsCdsGdsTdsCdsGdsGdsAdsAdsGdsGdsTgsGgsGg 1495.1 [M-4H]4- 108 51 1474 MlsAlsGlsCdsGdsTdsCdsGdsGdsAdsAdsGdsGdsTlsGl 1678,1 [M-3H]3- 109 52 1474 AlsMlsAlsGdsCdsGdsTdsCdsGdsGdsAdsAdsGdsGlsTlsGl 1797,1 [M-3H]3- 110 52 1474 AlsMlsAlsGdsCdsGdsTdsCdsGdsGdsAdsAdsGlsGlsTlsGl 1354.3 [M-4H]4- 111 53 1475 GlsAlsMlsAlsGdsCdsGdsTdsCdsGdsGdsAdsAdsGlsGlsTl 1354.4 [M-4H]4- 112 54 1476 GlsAlsMlsAdsGdsCdsGdsTdsCdsGdsGdsAdsAlsGlsGl 5073.96 [M-H]- 113 55 1476 AlsGlsAlsCdsAdsGdsCdsGdsTdsCdsGdsGdsAdsAlsGlsGl 1795.1 [M-3H]3- 114 56 1480 MgsMgsTgsAdsGdsAdsCdsAdsGdsCdsGdsTdsMgsGgsGg 1313.8 [M-4H]4-

[Table 1-6]

(Table 1, continued) 115 57 1480 GlsCdsMlsTlsAdsGdsAdsCdsAdsGdsCdsGdsTlsMlsGdsGl 1334.9 [M-4H]4- 116 58 1480 GlsMlsCdsTlsAdsGdsAdsCdsAdsGdsCdsGdsTlsCdsGlsGl 1331.3 [M-4H]4- 117 59 1480 GlsMlsCdsTlsAdsGdsAdsCdsAdsGdsCdsGdsTdsMlsGlsGl 1335.1 [M-4H]4- 118 59 1480 GlsMlsCdsTdsAdsGdsAlsCdsAdsGdsCdsGdsTdsMlsGlsGl 1335.2 [M-4H]4- 119 60 1480 GlsMlsMlsTdsAdsGdsAdsCdsAdsGdsCdsGlsTdsCdsGlsGl 1334.9 [M-4H]4- 120 60 1480 GlsMlsMlsTdsAdsGdsAdsCdsAdsGlsCdsGdsTdsCdsGlsGl 1335.2 [M-4H]4- 121 4 1480 GgsMgsMgsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMgsGgsGg 1399.7 [M-4H]4- 122 4 1480 GlsMlsMlsTlsAdsGdsAdsCdsAdsGdsCdsGdsTdsMlsGlsGl 1345.4 [M-4H]4- 123 4 1480 GmsMmsMmsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMmsGmsGm 1454,0 [M-4H]4- 124 61 1480 GlsMlsMlsTdsAdsGdsAdsMdsAdsGdsMdsGdsTdsMlsGlsGl 1345.3 [M-4H]4- 125 61 1480 GtsMtsMtsTdsAdsGdsAdsMdsAdsGdsMdsGdsTdsMtsGtsGt 1189.2 [M-5H]5- 126 61 1480 GusMusMusTdsAdsGdsAdsMdsAdsGdsMdsGdsTdsMusGusGu 1143.5 [M-5H]5- 127 61 1480 GmsMmsMmsTdsAdsGdsAdsMdsAdsGdsMdsGdsTdsMmsGmsGm 1168.8 [M-5H]5- 128 62 1480 GmsMmsMmsTdsAdsGdsAdsMdsAdsGdsMdsGdsUosMmsGmsGm 1172,0 [M-5H]5- 129 62 1480 GmsMmsMmsTdsAdsGdsAdsMdsAdsGdsMdsGosUosMmsGmsGm 1178,0 [M-5H]5- 130 63 1480 GmsMmsMmsUosAdsGdsAdsMdsAdsGdsMdsGdsTdsMmsGmsGm 1172,0 [M-5H]5- 131 63 1480 GmsMmsMmsUosAosGdsAdsMdsAdsGdsMdsGdsTdsMmsGmsGm 1178,0 [M-5H]5- 132 64 1480 GmsMmsMmsUosAdsGdsAdsMdsAdsGdsMdsGdsUosMmsGmsGm 1175.2 [M-5H]5- 133 65 214 MlsTlsMlsAdsGdsCdsTdsGdsGdsCdsGdsTlsGlsAl 4683,56 [M-H]- 134 66 1323 MlsMlsAlsGdsGdsAdsAdsAdsGdsAdsAdsTlsGlsGl 4748,41 [M-H]- 135 67 1458 GlsGlsGlsAdsGdsAdsCdsAdsTdsTdsCdsAlsGlsMl 4701,26 [M-H]- 136 68 1495 MlsTlsAlsAdsTdsCdsCdsAdsGdsGdsTdsTlsTlsGl 4641.35 [M-H]-

[0389]

Example 6

In vitro DUX4 knockdown activity assay (lipofection method)

C2C12 cells were seeded at a concentration of 1.25 x 10 ⁴ cells/cm ² per transfection reagent in which the DUX4-modified oligonucleotide and Lipofectamine RNAi reagent were mixed and cultured overnight in a CO ₂ incubator. The next day, the cells were transfected with a reporter plasmid in which the DUX4 sequence was cloned into multiple cloning sites of the psiCHECK-2 vector (Promega) using Lipofectamine 2000 reagent and cultured in a CO ₂ incubator for approximately 24 h. Thereafter, using the Dual-Glo Luciferase Assay System, intracellular Firefly luciferase and Renilla luminescence were determined using a plate reader. To correct for the effect of transfection efficiency and cell number on the luminescence due to Renilla luciferase activity, the ratio of Firefly luciferase activity to luminescence was calculated. The inhibition rate was calculated as a percentage of the rate of Renilla/Firefly decrease upon addition of the modified oligonucleotide, and the IC ₅₀ value was calculated from the concentrations at the two 50% points and the inhibition rate at these times (Table 2). Compared with compounds (Compound Nos. 1-132) that are complementary to positions 232-248, 1306-1325 or 1472-1495 of mature DUX4 mRNA, Compound Nos. 133 (complementary to positions 214-227 of SEQ ID NO: 1 in the sequence listing), 134 (complementary to positions 1323-1336 of SEQ ID NO: 1 in the sequence listing), 135 (complementary to positions 1458-1471 of SEQ ID NO: 1 in the sequence listing) and 136 (complementary to positions 1495-1508 of SEQ ID NO: 1 in the sequence listing) were found to have significantly lower inhibition levels.

[0390]

Example 7

In vitro DUX4 knockdown activity test (Gymnosis method)

C2C12 cells were seeded at 6× ¹⁰³ cells/ ^cm2 in DUX4-modified oligonucleotide solution and cultured in a _CO2 incubator for 2 nights. Two days later, the culture medium containing the DUX4-modified oligonucleotide solution was removed from the cells and the cells were washed with fresh culture medium. Thereafter, the cells were transfected with a reporter plasmid in which the DUX4 sequence was cloned into multiple cloning sites of the psiCHECK-2 vector (Promega) using Lipofectamine 2000 reagent and cultured in a _CO2 incubator for approximately 24 hours. Thereafter, using the Dual-Glo Luciferase Assay System, intracellular Firefly luciferase and Renilla luminescence were determined using a plate reader. To correct for the effect of transfection efficiency and cell number on the amount of luminescence due to Renilla luciferase activity, the ratio of Firefly luciferase activity to luminescence was calculated. The degree of inhibition was calculated as a percentage of the rate of decline of Renilla/Firefly when the modified oligonucleotide was added, and the IC ₅₀ value was calculated from the concentrations at the two 50% points and the degree of inhibition at these times. The results are shown in Table 2 below. Compared with compounds (Compound Nos. 1-132) containing a nucleobase sequence complementary to an equal length portion in the region of positions 232-248, 1306-1325 or 1472-1495 of mature DUX4 mRNA, Compound Nos. 133 (complementary to positions 214-227 of SEQ ID NO: 1 in the sequence listing), 134 (complementary to positions 1323-1336 of SEQ ID NO: 1 in the sequence listing), 135 (complementary to positions 1458-1471 of SEQ ID NO: 1 in the sequence listing) and 136 (complementary to positions 1495-1508 of SEQ ID NO: 1 in the sequence listing) were found to have significantly lower inhibition rates.

[Table 2-1]

Table 2 Connection No. IC ₅₀ [μM] Lipofection method The gymnosis method 1 0.004 0.108 2 0,008 0.084 3 0.003 0.142 4 0.098 0.829 5 0.027 0.341 6 0,007 0.128 7 0,009 0.378 8 0.022 0.495 9 0,018 0.340 10 0.006 0.182 11 0,008 0.114 12 0,020 0.423 13 0,015 0.242 14 0.002 0,079 15 0,010 0.128 16 0,008 0.091 17 0.006 0,079 18 0,016 0.394 19 0.006 0.158 20 0,018 0.724 21 0.006 0.964 22 0.004 0.280 23 0,007 0.365 24 0,018 0.188 25 0,009 0.447 26 0.043 0.259 27 0.037 0.201 28 0.003 0,080 29 0,010 0.556 30 0.006 0.559 31 0.006 0.117 32 0,007 0.404 33 0,007 0.272 34 0,008 0.073

[Table 2-2]

(Table 2, continued) 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

[Table 2-3]

(Table 2, continued) 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

[Table 2-4]

(Table 2, continued) 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

[0391]

Example 8

Synthesis of a modified oligonucleotide compound and in vitro DUX4 knockdown activity test (Gymnosis method)

Table 3 shows the newly synthesized modified oligonucleotide compounds and the results of in vitro DUX4 knockdown activity assays performed with the compounds in the same manner as in Example 7.

[0392]

In the compound notations, each nucleotide is represented by three letters. However, the 3'-terminal nucleotide is represented by two letters because there is no internucleoside linkage.

1) The first letter is capitalized and indicates the following nitrogen bases:

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

2) the second letter indicates the following sugar fragments:

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

3) the third letter indicates the following internucleoside bonds:

s=phosphorothioate, p=phosphodiester.

The target position indicates the 5'-target site of the mature DUX4 mRNA of the modified oligonucleotide (position of SEQ ID NO: 1 in the sequence listing, corresponding to the 3'-end of the modified oligonucleotide).

[0393]

Compared with compounds (Compound Nos. 137-247) containing a nucleobase sequence complementary to an equal-length portion in the region of positions 126-147, 232-248, 1306-1325, or 1472-1495 of mature DUX4 mRNA, 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) were found to have significantly lower levels inhibition.

[0394]

[Table 3-1]

[0395]

[Table 3-2]

[0396]

Example 9

In vivo DUX4 knockdown activity assay

An adeno-associated viral vector AAV-DUX4 (SignaGen Laboratories, Cat. No. SL100862) containing mature DUX4 mRNA with SEQ ID NO: 1 in the sequence listing was prepared. Under isoflurane (Pfizer Inc.) anesthesia, AAV-DUX4 was intramuscularly injected into the tibialis anterior muscle of 8-week-old C57BL/6J mice (male, Charles River, Japan) at a dose of 1E+10 VG/50 μl. Three days later, modified oligonucleotides targeting DUX4 were prepared in saline at concentrations of 1, 3, 10, and 50 mg/(5 ml)/kg and injected into 8-week-old C57BL/6J mice (male, Charles River, Japan) via the tail vein. After 72 h, whole blood was collected from the abdominal vena cava under cervical dislocation or isoflurane anesthesia (Pfizer Inc.), and the mice were sacrificed. The tibialis anterior muscle was then collected, immersed in RNAlater Soln (invitrogen), and frozen at -80ºC. The tissue was added with homogenization buffer of the Maxwell RSC JustRNA Tissue Kit (Promega), minced using a multi-bead shaker, and RNA was purified according to the protocol provided with the kit. 400 ng of RNA was reverse transcribed, and quantitative PCR was performed using the resulting cDNA. The knockdown activity of the modified oligonucleotide was expressed as the ratio of DUX4 to 18S rRNA relative to the vehicle group. The results for 1, 3, 10, and 50 mg/(5 ml)/kg are shown in Figs. 1–4. Compound #1 (a sequence complementary to positions 233-248 of mature mRNA DUX4), Compound #2 (a sequence complementary to positions 1309-1323 of mature mRNA DUX4), Compound #3 (a sequence complementary to positions 1480-1495 of mature mRNA DUX4), Compound #13 (a sequence complementary to positions 234-247 of mature mRNA DUX4), Compound #41 (a sequence complementary to positions 1308-1323 of mature mRNA DUX4), Compound #54 (a sequence complementary to positions 1309-1323 of mature mRNA DUX4), Compound #57 (a sequence Complementary to positions 1309-1323 of DUX4 mature mRNA), Compound #68 (a sequence complementary to positions 1309-1323 of DUX4 mature mRNA), Compound #78 (a sequence complementary to positions 1309-1324 of DUX4 mature mRNA), Compound #88 (a sequence complementary to positions 1310-1323 of DUX4 mature mRNA), Compound #104 (a sequence complementary to positions 1473-1488 of DUX4 mature mRNA), Compound #122 (a sequence complementary to positions 1480-1495 of DUX4 mature mRNA) were able to suppress the expression of the DUX4 gene in muscles even when introduced into a living organism.

[0397]

Example 10

Safety of modified oligonucleotide

When compounds No. 3, 42 and 123 were administered intravenously to 6-week-old ICR mice (male, Charles River, Japan) at a maximum dose of 100 mg/kg, liver toxicity (increase in blood ALT and AST and histopathological abnormalities), renal toxicity (increase in blood UN and creatinine and histopathological abnormalities), changes in general symptoms, mortality, etc. were not observed.

[0398]

Example 11

In vivo DUX4 knockdown activity assay in Tg mice

Nine-week-old male FLExDUX4-heteto/HSA-MCM-hetero:TG (DUX4-Tg) and FLExDUX4-wild/HSA-MCM-hetero:TG (MCM, control) mice (male transported to Charles River Japan from The Jackson Laboratory) were used. The modified oligonucleotide targeting DUX4 was prepared in saline so that the injected fluid of each dose was 5 ml/kg and administered weekly via the tail vein. One week after the 4-week administration, whole blood was collected from the abdominal vena cava under isoflurane anesthesia and the mouse was euthanized. EDTA plasma was separated and creatine kinase (CK) was determined. The lower limb muscle was collected, wet weight was measured and the muscle was subjected to gene expression analysis.

As shown in Fig. 5 and Fig. 6, Compound No. 3 and Compound No. 123 suppressed the expression of DUX4 mRNA. In addition, the blood CK level as a marker of myopathy was decreased. On the other hand, in the case of Compound No. 113 and Compound No. 247, there was no obvious effect on the expression of DUX4 mRNA and the blood CK level.

[0399]

Example 12

Continuous Dose Toxicity Test in Mice

The modified oligonucleotide targeting DUX4 was prepared in saline at a concentration of 100 mg/(5 ml)/kg and administered via the tail vein to 6-week-old ICR mice (male, Charles River Japan) for four consecutive days. Seventy-two hours after the last administration, blood was collected from the posterior vena cava under isoflurane anesthesia and clinical biochemical examination was performed. In addition, after the mouse was sacrificed by exsanguination, necropsy was performed and the liver and kidney were subjected to histopathological examination. In the case of compound #123, there was no mortality after administration and no change in general condition, food intake and body weight, as well as hepatotoxicity (increase in serum ALT and AST and histopathological abnormalities) and nephrotoxicity (increase in serum UN and creatinine and histopathological abnormalities) were observed. On the other hand, after administration of compound No. 113 and compound No. 247, although there was no mortality and no change in general condition, food intake and body weight, obvious hepatotoxicity (increased serum ALT, AST, GLDH, ALP, bilirubin and bile acid levels and histopathological abnormalities: degenerative necrosis of hepatocytes and hypertrophy of hepatocytes) and nephrotoxicity (increased serum creatinine) were observed in the case of both compounds. In addition, the concentrations of compound No. 123 in the liver and kidney were 323 and 251 μg/g, respectively, and there was no hepatotoxicity or nephrotoxicity, despite the fact that the tissue concentrations of compound No. 123 were comparable to or greater than the concentrations of 111 and 185 μg/g in the liver and kidney for compound No. 247 and the concentrations of 39.3 and 235 μg/g of compound No. 113 in the liver and kidney for compound No. 113.

[0400]

[Table 4]

Blood biochemistry in continuous administration test in mice

Subject of research Control group Connection No. 123 Control group Connection #247 Connection 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

[0401]

[Table 5]

Pathological results in continuous administration test in mice

Pathological examination results Control group Connection No. 123 Control group Connection #247 Connection #113 Degenerative 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

[0402]

Reference example

Below are the schemes of the methods for synthesizing ALNA [Ms]-containing nucleotides, ALNA [mU]-containing nucleotides, ALNA [ipU]-containing nucleotides, ALNA [Trz]-containing nucleotides, ALNA [Oxz]-containing nucleotides. The starting compounds (1a, 1d and 1g) can be synthesized using the method described in WO 2017/047816.

[0403]

Synthesis of ALNA [Ms]-T

[0404]

Synthesis of ALNA [Ms]-mC

[0405]

Synthesis of ALNA[Ms]-G

[0406]

Synthesis of ALNA [Ms]-A

[0407]

Synthesis of ALNA [mU]-T

[0408]

Synthesis of ALNA [mU]-mC

[0409]

Synthesis of ALNA [mU]-G

[0410]

Synthesis of ALNA [mU]-A

[0411]

Synthesis of ALNA [ipU]-T

[0412]

Synthesis of ALNA [ipU]-mC

[0413]

Synthesis of ALNA [ipU]-G

[0414]

Synthesis of ALNA [ipU]-A

[0415]

Synthesis of ALNA[Trz]-T

[0416]

Synthesis of ALNA [Trz]-mC

[0417]

Synthesis of ALNA[Trz]-G

[0418]

Synthesis of ALNA[Trz]-A

[0419]

Synthesis of ALNA[Oxz]-T

[0420]

Synthesis of ALNA [Oxz]-mC

[Industrial applicability]

[0421]

The modified oligonucleotide of the present invention can be used as a compound used for therapeutic treatment, prevention or slowing down the progression of a disease associated with DUX4.

[Explanatory translation of the sequence list]

[0422]

SEQ ID NO: 1 in the sequence listing shows the base sequences of DUX4 mature mRNA.

SEQ ID NOs. 2-4 and 7-109 in the sequence listing show the base sequences of the modified oligonucleotides.

SEQ ID NO: 5-6 in the sequence listing respectively show the base sequences of DUX4-FL2 and DUX4 as splice variants of SEQ ID NO: 1.

Claims (79)

1. A modified oligonucleotide for inhibiting the expression of dual homeobox 4, consisting of 12-30 residues, including: a nucleobase sequence that comprises at least 8 immediately adjacent nucleobase sequences and is complementary to an equal length portion at positions 126-147, 232-248, 1306-1325 or 1480-1495 from the 5'-end of the nucleobase sequence of the mature double homeobox 4 mRNA in SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide has at least 90% complementarity with a portion of equal length in the nucleobase sequence of the mature double homeobox 4 mRNA in SEQ ID NO: 1, and when at least 8 immediately adjacent nucleobase sequences comprise a nucleobase sequence that is complementary to a portion of equal length at positions 1480-1495 from the 5'-end of the nucleobase sequence of SEQ ID NO: 1, the modified oligonucleotide consists of a nucleobase sequence having at the 3'-end a base complementary to the base at position 1480 from the 5'-end of the nucleobase of SEQ ID NO: 1, wherein one or more modified nucleotides of the modified oligonucleotide include a modified sugar. 2. The modified oligonucleotide of claim 1, wherein the modified sugar is selected from the group consisting of a bicyclic sugar, a modified 2'-O-methoxyethyl-modified sugar, and a 2'-O-methyl-modified sugar. 3. The modified oligonucleotide of claim 2, wherein the bicyclic sugar is selected from the group consisting of LNA, GuNA, ALNA [Ms], ALNA [mU], ALNA [ipU], ALNA [Oxz] and ALNA [Trz]. 4. A modified oligonucleotide for inhibiting the expression of dual homeobox 4, consisting of 12-30 residues, containing: a nucleobase sequence that comprises at least 8 immediately adjacent nucleobase sequences and is complementary to an equal length portion at positions 1472-1495 from the 5'-end of the nucleobase sequence of the mature double homeobox 4 mRNA in SEQ ID NO: 1, wherein the nucleobase sequence of the modified oligonucleotide has at least 90% complementarity with an equal length portion of the nucleobase sequence of the mature dual homeobox 4 mRNA in SEQ ID NO: 1, and the modified oligonucleotide comprises at least one nucleoside that comprises a modified sugar selected from the group consisting of GuNA, ALNA[Ms], ALNA[mU], ALNA[ipU], ALNA[Oxz] and ALNA[Trz]. 5. A modified oligonucleotide according to claim 4, additionally containing: 2ʼ-O-methoxyethyl modified sugar and/or 2ʼ-O-methyl modified sugar. 6. A modified oligonucleotide according to any one of claims 1 to 5, wherein at least one modified nucleotide of the modified oligonucleotide comprises a modified nucleic base. 7. The modified oligonucleotide of claim 6, wherein the modified nucleic acid base is 5-methylcytosine. 8. A modified oligonucleotide according to any one of claims 1 to 7, wherein at least one internucleoside linkage is a modified internucleoside linkage. 9. The modified oligonucleotide of claim 8, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage. 10. A modified oligonucleotide according to any one of claims 1 to 9, comprising: 1) gap segment, 2) 5'-wing segment and 3) 3'-wing segment, wherein the gap segment is located between the 5'-wing segment and the 3'-wing segment, all nucleosides of the 5'-wing segment and the 3'-wing segment each comprise at least one modified sugar, and the gap segment comprises only nucleosides that do not contain a modified sugar, or comprises one or two nucleosides each of which contains a modified sugar, and includes other nucleosides that do not contain a modified sugar. 11. A modified oligonucleotide according to any one of claims 1 to 10, consisting of: a nucleobase sequence that is complementary to the nucleobase sequence at positions 128-143 from the 5'-end of the nucleobase sequence, the nucleobase sequence at positions 232-247 from the 5'-end, the nucleobase sequence at positions 233-248 from the 5'-end, the nucleobase sequence at positions 1309-1323 from the 5'-end, or the nucleobase sequence at positions 1480-1495 from the 5'-end of the mature double homeobox 4 mRNA of SEQ ID NO: 1. 12. A modified oligonucleotide according to any one of claims 1 to 11, consisting of: the base sequences gtggcgatgc ccgggt (SEQ ID NO: 75), gagattcccg cnggtg (SEQ ID NO: 78: n is 5-methylcytosine), ngagattcccgccggt (SEQ ID NO: 2: n is 5-methylcytosine), gnagttctccgcggt (SEQ ID NO: 3: n is 5-methylcytosine) or gnntagacagcgtngg (SEQ ID NO: 4: n is 5-methylcytosine). 13. A modified oligonucleotide according to claim 12, represented by the following formula, GlsMlsMlsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMlsGlsGl, where nucleic bases are represented by the following symbols: A=adenine, T=thymine, G=guanine, C=cytosine and M=5-methylcytosine; Sugar components are represented by the following symbols: l=LNA and d=2ʼ-deoxyribose; and internucleoside linkages are represented by the following symbol: s=phosphorothioate. 14. A modified oligonucleotide according to claim 12, represented by the following formula, GmsMmsMmsTdsAdsGdsAdsCdsAdsGdsCdsGdsTdsMmsGmsGm, where nucleic bases are represented by the following symbols: A=adenine, T=thymine, G=guanine, C=cytosine and M=5-methylcytosine; Sugar components are represented by the following symbols: m=ALNA [Ms] and d=2ʼ-deoxyribose; and internucleoside linkages are represented by the following symbol: s=phosphorothioate. 15. A modified oligonucleotide according to claim 12, represented by the following formula, GmsMmsAmsGdsTdsTdsCdsTdsCdsCdsGdsCdsGmsGmsTm, where nucleic bases are represented by the following symbols: A=adenine, T=thymine, G=guanine, C=cytosine and M=5-methylcytosine; Sugar components are represented by the following symbols: m=ALNA [Ms], and d=2ʼ-deoxyribose; and internucleoside bonds are represented by the following symbol: s=phosphorothioate. 16. A modified oligonucleotide according to claim 12, represented by the following formula, MlsGlsAlsGdsAdsTdsTdsCdsCdsCdsGdsCdsCdsGlsGlsTl, where nucleic bases are represented by the following symbols: A=adenine, T=thymine, G=guanine, C=cytosine, and M=5-methylcytosine; the sugar components are represented by the following symbols: l=LNA and d=2ʼ-deoxyribose; and internucleoside linkages are represented by the following symbol: s=phosphorothioate. 17. A modified oligonucleotide according to claim 13, represented by the following formula, or a salt thereof: 18. A modified oligonucleotide according to claim 14, represented by the following formula, or a salt thereof: 19. A modified oligonucleotide according to claim 15, represented by the following formula, or a salt thereof: 20. A modified oligonucleotide according to claim 16, represented by the following formula, or a salt thereof: 21. A modified oligonucleotide for inhibiting the expression of dual homeobox 4, represented by the following formula, or a salt thereof: 22. A modified oligonucleotide for inhibiting the expression of dual homeobox 4, represented by the following formula, or a salt thereof: 23. A modified oligonucleotide for inhibiting the expression of dual homeobox 4, represented by the following formula, or a salt thereof: 24. A modified oligonucleotide for inhibiting the expression of dual homeobox 4, represented by the following formula, or a salt thereof: 25. A pharmaceutical composition for the treatment, prevention or delay of the development of a disease associated with dual homeobox 4, comprising: an effective amount of a modified oligonucleotide according to any one of claims 1 to 24 or a pharmaceutically acceptable salt thereof, wherein the dual homeobox 4-associated disease is a disease selected from the group consisting of facioscapulohumeral muscular dystrophy, B-cell acute lymphocytic leukemia, differentiated round cell sarcoma, and fetal rhabdomyosarcoma. 26. The pharmaceutical composition according to claim 25, wherein the disease associated with double homeobox 4 is fascioscapulohumeral muscular dystrophy. 27. A method for the therapeutic treatment, prevention or slowing of the progression of a disease associated with dual homeobox 4 in a subject, wherein the method comprises administering to a subject in need thereof an effective amount of a modified oligonucleotide according to any one of claims 1-24, wherein the dual homeobox 4-associated disease is a disease selected from the group consisting of facioscapulohumeral muscular dystrophy, B-cell acute lymphocytic leukemia, differentiated round cell sarcoma, and fetal rhabdomyosarcoma. 28. Use of a modified oligonucleotide according to any one of claims 1 to 24 in the manufacture of a medicinal product for the therapeutic treatment, prevention or slowing of the progression of a disease associated with dual homeobox 4, wherein the disease associated with dual homeobox 4 is a disease selected from the group consisting of facioscapulohumeral muscular dystrophy, B-cell acute lymphocytic leukemia, differentiated round cell sarcoma and fetal rhabdomyosarcoma. 29. Use of a modified oligonucleotide according to any one of claims 1 to 24 for the therapeutic treatment, prevention or slowing down of the progression of a disease associated with dual homeobox 4, wherein the disease associated with dual homeobox 4 is a disease selected from the group consisting of facioscapulohumeral muscular dystrophy, B-cell acute lymphocytic leukemia, differentiated round cell sarcoma and fetal rhabdomyosarcoma.