SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed in electronic format via EFS-Web and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is 37921_0003U2_SL. The size of the text file is 17 KB, the text file was created on Mar. 19, 2020.

FIELD OF DISCLOSURE

The disclosure relates to compositions comprising nucleotide sequences comprising LNA gapmers and methods of using and administering such nucleotide sequences for, among other things, delivery of nucleic acid sequences to one or a plurality of cells.

BACKGROUND

Muscular Dystrophy (MD) comprises a group of 30 or more inherited disorders which are characterized by muscle weakness and replacement of muscle cells with connective tissue and fat (Emery, 1991). Although all MDs share common characteristics such as muscle weakness and muscle cell death, the disorders differ from one another in terms of their severities (i.e. progression variability), age of onset as well as muscles and other organ systems affected (Barakat-Haddad et al., 2016).

Facioscapulohumeral Muscular Dystrophy (FSHD) is an autosomal dominant disorder characterized by early involvement of muscle weakness and atrophy in facial muscles and shoulder girdle muscles (Padberg, 1982). With disease progression, muscle weakness and atrophy often spreads to the upper arms, pelvic girdle and lower limb muscles (Padberg, 1982). FSHD is the third most common muscular dystrophy, with a birth incidence of approximately 1 in 14,000 (Mostacciuolo et al., 2009). The frequency of occurrence for FSHD is often underestimated due to the high degree of clinical variability in addition to the large proportion of individuals who remain asymptomatic or experience mild symptoms (Deenen et al., 2014). In 1884, Landouzy and Dejerine were first to describe the classical FSHD phenotype with the disease being formerly known as Landouzy-Dejerine's disease (Landouzy & Dejerine, 1884 and Landouzy & Dejerine, 1886). However, it wasn't until 1980 when a rise in interest led to a greater understanding of FSHD, its clinical variability and its genetic complexities (Wijmenga, Padberg & Moerer et al., 1991). When the disorder was first described, FSHD and its association to chromosome 4 was unknown. However, since then, the fundamental cause of FSHD and the finding of D4Z4 reduced alleles at 4q35 has become clearer (Upadhyaya et al., 1997).

FSHD is associated with the deletion of a subset of D4Z4 macrosatellite repeat arrays in the subtelomeric region of chromosome 4, 4q35 (FIG.1). The genetic defect in FSHD was first identified by a reduction seen in an EcoRI fragment of genomic DNA using the p13E-11 probe compared to healthy individuals (Wijmenga et al., 1992). Non-affected control individuals typically contain between 11 and 100 D4Z4 repeats, with EcoRI fragments being 40-300 kb in size, while FSHD patients carry between 1 and 10 repeats on one allele, with EcoRI fragments being 10-38 kb in size (FIG.1) (Lunt, 1998). The deletion of a subset of D4Z4 macrosatellite repeat arrays occurs in early embryonic development through mitotic rearrangement (van der Maarel et al., 2000).

There are two forms of FSHD, FSHD1, which occurs in over 95% of cases, and the less common form, FSHD2, which occurs in the other 5% of individuals (Jones et al., 2012). FSHD1 is genetically linked to contractions of the macrosatellite D4Z4 repeat array (Wijmenga et al., 1990), whereas FSHD2 shows chromatin relaxation at D4Z4, but does not have a contraction of the D4Z4 locus (de Greef et al., 2010). In majority of patients with FSHD2, the disease is caused by digenic inheritance of a heterozygous mutation in the chromatin modifier gene on chromosome 18, Structural Maintenance of Chromosomes Flexible Hinge Domain Containing 1 (SMCHD1), as well as a distally located PAS-containing chromosome 4 (Lemmers et al., 2012). SMCHD1 is an important gene known for its role in regulating the repression of the D4Z4 array via DNA CpG methylation (Lemmers et al., 2012). Recent evidence however suggests that there is evidence for locus heterogeneity with FSHD2, due to the lack of SMCHD1 mutations seen in approximately 20% of FSHD2 affected patients. This evidence suggesting the presence of other modifier loci that too are potentially affecting the structure of D4Z4 (Lemmers et al., 2012).

The open reading frame (ORF) for double homeobox 4 (DUX4) gene was mapped in each unit of the D4Z4 repeat arrays remaining after partial deletion associated with FSHD (Gabriels et al., 1999). 4q variants were discovered distal to D4Z4, 4qA and 4qB, both of which occur commonly in the Caucasian population (van Geel et al., 2002). Interestingly, for FSHD, the polyadenylation site is required for a pathogenic contraction of the D4Z4 array and is only intact on the permissive 4qA allele, whereas this signal is missing on the non-permissive 4qB allele (FIG.1) (Lemmers et al., 2010).

Two full-length isoforms of DUX4 exist with alternative splicing in the 3′ untranslated region (Snider et al., 2010). One full-length isoform of DUX4 is detected in FSHD skeletal muscle cells which contains the entire DUX4 ORF and in the 3′UTR has one or two spliced introns, ending in exon 3 (Snider et al., 2009). A second full-length DUX4 mRNA (fl-DUX4) isoform was characterized in induced pluripotent stem cells and in human testis cells by the addition of four exons and a more downstream polyadenylation signal in exon 7 (FIG.2) (Snider et al., 2010). In control muscles and in other somatic tissues, a shorter DUX4 mRNA variant (s-DUX4) was discovered because of its ability to remove the carboxyterminal end of DUX4 while maintaining the amino-terminal double-homeobox domains, ending in exon 3. s-DUX4 is unique from fl-DUX4 in that it encodes a non-pathogenic protein (Snider et al., 2010).

A high correlation between FSHD disease severity and fragment size has been identified with individuals with a large deletion of the D4Z4 array having earlier-onset disease in addition to rapid progression, compared to those patients with smaller contractions of the D4Z4 locus (Zatz et al., 1995, Tawil et al., 1996 & Bindoff et al., 2006). Currently the existence and precise mechanism for anticipation in FSHD remains uncertain.

It is currently estimated that 10%-30% of FSHD patients carry a new mutation (Padberg et al., 1995 & Zatz et al., 1995), and show an array of D4Z4 array lengths varying from 1 to more than 50 units (van der Maarel et al., 2000). Several studies have shown that approximately half of these reported D4Z4 rearrangements are mitotic in origin, and likely occur from postzygotic array contraction during early cell divisions in embryogenesis, resulting in somatic mosaicism. The other half of de novo cases likely occur before fertilization (Upadhyaya et al., 1995, Lemmers et al., 2004 & van der Maarel et al., 2000).

DUX4 is a double-homeodomain transcription factor encoded within the D4Z4 tandem repeat. DUX4 is a nuclear protein that is normally transcriptionally silenced in healthy individuals after early development; however in FSHD, truncations of the D4Z4 array leading to fewer than 11 units cause a failure to maintain complete suppression of full-length DUX4. Chromatin relaxation of the D4Z4 array (inability to suppress DUX4 fully), causes occasional bursts of DUX4 expression in a small number of skeletal muscle nuclei (Snider et al., 2010). In healthy individuals, DUX4 is normally expressed in early development and silenced during cellular differentiation but maintains expression levels within the seminiferous tubules in male testis (Snider et al., 2010). The primary role of DUX4 remains unclear, however some previously identified targets include cancer-testis antigens, as well as a broad set of genes involved in germ cell maintenance and development and stem cell biology (Geng et al., 2012).

Unlike other disorders which are caused by structural mutations within the disease gene, FSHD involves an intricate cascade of epigenetic events after the deletion of a subset of D4Z4 macrosatellite repeat arrays (van der Maarel, Frants & Padberg, 2007). Several studies have shown that DUX4 expression induces the expression of genes involved in muscle atrophy, such as muscle RING finger 1 (MuRF1) and atrogin-1/MAFbx, sensitizes cells to oxidative stress, thereby inducing the expression of mu-crystallin (CRYM), inhibits the MYOD1 gene resulting in differentiation abnormalities, and causes a p53-dependent myopathy in mouse muscles in vivo (de Palma et al., 2008, Vie et al., 1997 and Wallace et al., 2011). Recent evidence has also shown that paired-like homeodomain 1 (PITX1), tripartite motif containing 43 (TRIM43), and methyl-CpG binding domain protein 3-like 2 (MBD3L2) are also upregulated in FSHD (Dixit et al., 2007 and Ferreboeuf et al., 2014) (FIG.3).

Despite the progress made in understanding the underlying genetic and pathophysiological complexities of FSHD, no curative treatment options have been established. Currently, standard disease management options include physical therapy, exercise, management of respiratory dysfunction and orthopedic intervention (Tawil et al., 2014).

SUMMARY OF EMBODIMENTS

The present disclosure relates to compositions comprising a nucleotide sequence. In some embodiments, the nucleotide sequence comprises: (i) an RNA-binding domain from about 5 to about 30 nucleotides complementary to a region of a mammalian double homeobox 4 (DUX4); and (ii) at least one locked nucleic acid (LNA) domain comprising from about 1 to about 5 nucleotides, or a salt thereof.

The present disclosure also relates to a composition comprising a nucleotide sequence from about 8 to about 120 nucleotides. In some embodiments, the nucleotide sequence is complementary to at least about 8 contiguous nucleotides from position 675 to position 688 of exon 1 of a human DUX4, or a salt thereof. In some embodiments, the nucleotide sequence is complementary to at least about 8 contiguous nucleotides from position 182 to position 197 of exon 3 of a human DUX4, or a salt thereof. In some embodiments, the nucleotide sequence is complementary to at least about 8 contiguous nucleotides from position 98 to position 116 of exon 3 of a human DUX4, or a salt thereof. In some embodiments, the nucleotide sequence comprises a central DNA gap domain flanked by a first LNA domain at a 5′ end of the nucleotide sequence and a second LNA domain on a 3′ end of the nucleotide sequence. In some embodiments, the nucleotide sequence comprises one or a plurality of nucleotide sequences chosen from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 or combination thereof. In some embodiments, the composition comprises at least 2 nucleic acid molecules, each nucleic acid molecule comprising one or a plurality of nucleotide sequences chosen from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 or combination thereof.

The present disclosure also relates to a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises: (i) a therapeutically effective amount of any composition described herein; and (ii) a pharmaceutically acceptable carrier.

The present disclosure also relates to a method of treating a dystrophin-related disorder. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of one or a combination of any composition described herein, and/or any pharmaceutical composition described herein.

The present disclosure also relates to a method of modulating or inhibiting expression of DUX4 in a subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of one or a combination of any composition described herein, and/or any pharmaceutical composition described herein.

The present disclosure also relates to a method of targeting a DUX4 mRNA in a cell. In some embodiments, the cell is a muscle cell. In some embodiments, the method comprises contacting one or a combination of any composition described herein, and/or any pharmaceutical composition described herein, with the cell. In some embodiments, the cell is a muscle cell. In some embodiments, the method of targeting a DUX4 mRNA comprises contacting any one or plurality of the nucleotide sequences disclosed herein with a cell expressing DUX4 mRNA at a concentration sufficient and for a time sufficient to hybridize the one or plurality of nuelcotide sequences to the DUX4 mRNA. In some embodiments, the step of contecting is preceded by administering the one or plurality of nucleotide sequences In some embodiments, the one or plurality of the nucleotide sequences disclosed herein comprises a marker or tag that is capable of detection upon exposure to a stimulus, such as a particular wavelength of light, magnetic field or an ezyme with a known reaction product. In some embodiments, the method of targeting a DUX4 mRNA in a cell further comprises exposing the one or plurality of nucleotide sequences comprising a marker or tag to a stimulus such that the marker or tag is visible after administration of an effective amount of the one or plurality of nucleotide sequences.

