WO2013120038A2 - Morpholino targeting dux4 for treating fshd - Google Patents

Description

MORPHOLINO TARGETING DUX4 FOR TREATING FSHD

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/597,480, filed on February 10, 2012, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under award number R01 HL081186 awarded by the National Heart, Lung, and Blood Institute (NHLBI) and R01 AR055685 awarded by the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/National Institute of Health (NIH). The government has certain rights in this invention.

BACKGROUND

Facioscapulohumeral muscular dystrophy (FSHD) is a dominantly inherited degenerative myopathy affecting approximately one in 20,000 individuals. FSHD affected patients can present with asymmetric weakness and wasting of facial muscles. FSHD affected patients can also present with shoulder muscle wasting and/or upper arm muscle wasting. FSHD disease pathology can progress to include distal anterior leg weakness, abdominal weakness, or hip-girdle muscle weakness (see, e.g., Tawil and Van Der Marrel, 2006. Muscle Nerve. 34(1): 1-15). A genetic alteration that can be associated with FSHD is a contraction within the D4Z4 array, a 3.3 kb macrosatellite repeat located at the distal tip of chromosome 4 (4q35.2). The D4Z4 array copy number can be polymorphic presumably due to intra-chromosomal conversion or crossover events. Individuals often carry chromosome 4 D4Z4 alleles bearing on the order of 100 repeats (see, e.g., Lemmers et al., 2007. Am. J. Hum. Genet. 81(5):884-94). FSHD patients presenting with the FSHD phenotype can carry 10 or fewer chromosome 4 D4Z4 repeats (see, e.g., Wijmenga et al., 1992. Nat. Genet. 2(l):26-30).

However, loss of all 4qter D4Z4 repeats does not result in FSHD (see, e.g., Tupler et al., 1996. J.

Med. Genet. 33:371-375), suggesting that haploinsufficiency of D4A4 is not the cause of the disease implying further that at least one copy of D4Z4 or even nearby sequences may be needed (Gabellini et al., 2002. Cell. 110(3):339-48).

The molecular mechanism by which a contraction within the D4Z4 array results in a dominant muscular dystrophy is not fully understood.

SUMMARY

The present inventors have recognized, among other things, that a problem to be solved is the development of a therapy for FSHD. In an embodiment, the present subject matter can provide a solution to this problem, such as by identifying a causative gene. In another embodiment, the present subject matter can provide a solution to this problem, such as by identifying a therapeutic molecular target. The present subject matter provides an antisense morpholino oligonucleotide, methods to prepare an antisense morpholino oligonucleotide, and uses of an antisense morpholino

oligonucleotide, for example, to bind an RNA sequence located within the 5' untranslated region (UTR) upstream of the DUX4 translational start site. In one model, either a contraction of the D4Z4 array or a second site mutation or epigenetic alteration allows for an increase in transcription at the 4q32.2 locus and this can result in FSHD. In accordance with the present subject matter, the increase in transcription can lead to an overexpression of a gene product that is deleterious to skeletal muscle development and function. In an embodiment of the present invention, the expression of a gene product can be lowered.

In accordance with one aspect of the invention, a target sequence within a gene can be identified. A morpholino oligonucleotide can be designed to interact with the particular target sequence. The interaction can be sequence specific, so as to ultimately result in a stable

morpholino:RNA duplex. Repression of gene product expression can be achieved by targeting a sequence within the UTR of an RNA message directly upstream of a translational start site.

For instance, an embodiment provides a morpholino oligonucleotide capable of inhibiting expression of a facioscapulohumeral muscular dystrophy candidate gene located within the polymorphic D4Z4 repeat region of chromosome 4q. In an embodiment, the morpholino

oligonucleotide can inhibit DUX4 expression. Another embodiment provides a dendrimer delivery moiety.

Further provided is a method of disrupting DUX4 expression using the morpholino oligonucleotide. In accordance with one aspect of the present subject matter, a designed morpholino oligonucleotide can bind to the DUX4 gene product. In one example, the morpholino oligonucleotide can bind within the DUX4 5' UTR of the RNA message. For example, the morpholino can disrupt translation of DUX4 by creating a stable, sequence specific morpholino:RNA duplex within DUX4. In one example, the morpholino can disrupt ribosome binding such that DUX4 translation initiation is inhibited.

An embodiment provides an antisense oligonucleotide comprised of morpholino subunits and phosphorous-containing intersubunit linkages comprising a sequence of at least 12 contiguous base- pairing moieties complementary to SEQ ID NO: 3 (gcacagtccggc tgaggtgcac gggagcccgc cggcctctct ctgcccgcgt ccgtccgtgaaattccggcc ggggctcacc gcgATGgccc tcccgacacc ctcggacagc (the ATG of DUX4 is capitalized; the sequence upstream of the ATG represents the UTR) wherein the antisense oligonucleotide is capable of binding to mRNA to form a heteroduplex structure. In an embodiment, the heteroduplex structure has a T_m of dissociation of at least 45° C. In one embodiment, the morpholino subunits are joined by phosphorodiamidate backbone linkages. In another embodiment, the antisense oligonucleotide contains between 14-24 base-pairing moieties. In one embodiment, the antisense oligonucleotide comprises a targeting sequence of 25 contiguous base-pairing moieties complementary to SEQ ID NO:3. In an embodiment, the antisense oligonucleotide comprises SEQ ID NO: l.

One embodiment provides a composition comprising the antisense oligonucleotides described herein and a pharmaceutically acceptable carrier.

An embodiment provides a method of inhibiting expression of DUX4 comprising administering to a subject (e.g., a human) in need thereof an effective amount of the antisense oligonucleotides described herein or compositions containing them. In one embodiment, the antisense oligonucleotide is administered in a manner effective to reduce or eliminate a detectable symptom of FSHD in the subject.

Another embodiment provides a method to treat facioscapulohumeral muscular dystrophy comprising administering to a subject in need thereof an effective amount of the antisense oligonucleotides described herein or compositions containing them.

An embodiment provides a method of treating facioscapulohumeral muscular dystrophy comprising: administering a morpholino oligonucleotide to a subject in need thereof; wherein the morpholino oligonucleotide is administered in a dose effective to inhibit expression of a

facioscapulohumeral muscular dystrophy candidate gene located within the polymorphic D4Z4 repeat region of chromosome 4q. In one embodiment, the administration of a morpholino oligonucleotide is in a dose effective to inhibit expression of DUX4. In another embodiment, the administration of a morpholino oligonucleotide is capable of inhibiting DUX4 expression by binding to a sequence within the 5' UTR of DUX4. In one embodiment, the administration is by intraperitoneal or intravenous injection. In another embodiment, the administration comprises a dendrimer delivery moiety, such as an octa-guanidine dendrimer delivery moiety. In an embodiment, the translation of DUX4 is inhibited. In another embodiment, the nuclear processing of DUX4 RNA is inhibited.

One embodiment provides a method of treating facioscapulohumeral muscular dystrophy in a subject, comprising administering to a subject an effective amount of an antisense oligonucleotide of 12-40 morpholino subunits linked by phosphorous-containing intersubunit linkages which join a morpholino nitrogen of one subunit to a 5' exocyclic carbon of an adjacent subunit,; and comprising a sequence which forms a heteroduplex with a AUG start-site region of DUX4 or a region 5' thereof and/or 3' thereof; wherein the antisense oligonucleotide inhibits production of DUX4 mRNA or protein. These and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments discussed in the present document. FIGURES 1 A-C. (A) provides a representation of chromosome 4 (not to scale), wherein the highly polymorphic D4Z4 repeat region, located on the q terminus at 4q35.2 is depicted. The red box indicates the region shown in Fig IB. (B) depicts a representation of several FSHD candidate genes located within 5Mb of the D4Z4 array (including, but not limited to, ANT1, FRG1, TUBB4Q, and FRG2). The representation further depicts a representation of one 3.3kb D4Z4 unit, wherein each of the 3.3kb D4Z4 units harbor two homeoboxes within a single open reading frame comprising the DUX4 gene. (C) depicts the MK1 morpholino (SEQ ID NOs: l and 2). The MK1 morpholino oligonucleotide was designed to inhibit DUX4 expression by binding to a sequence located within the 5'UTR of DUX4 directly upstream of the ATG (so as to prevent translation).

FIGURES 2A-B. (A) provides the western blot analysis of DUX4 expression in 5'UTR

DUX4 mES cells that were induced with doxycycline and treated with various amounts of MK1 morpholino oligonucleotide. (B) provides the DUX4 percent expression after protein levels were normalized to that of GAPDH.

DETAILED DESCRIPTION

I. Definitions

The terms below, as used herein, have the following meanings, unless otherwise indicated: A "morpholino oligonucleotide " is an oligonucleotide composed of morpholino subunit structures, where (i) the structures are linked together by phosphorus- containing linkages, one to three atoms long, joining the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, and (ii) purine or pyrimidine base-pairing moieties effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide. The purine or pyrimidine base-pairing moiety is typically adenine, cytosine, guanine, uracil, thymine or inosine. The synthesis, structures, and binding characteristics of morpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337, all of which are incorporated herein by reference.

As used herein, the term "target", relative to the RNA, refers a nucleotide region that surrounds the AUG start codon of a messenger RNA.

The term "target sequence" or "target" refers to a portion of the target RNA against which the oligonucleotide sequence is directed, that is, the sequence to which the oligonucleotide will hybridize by Watson-Crick base pairing of a complementary sequence. The target sequence may be a contiguous region of the mRNA.

The term "AUG start site region" includes a 125 nucleotide region in both the 5' and 3' direction relative to the AUG start codon of mRNAs. For example, the region includes about 25 nucleotides downstream (i.e., in a 3' direction) and 100 nucleotides upstream (i.e., in a 5' direction).

Target and the oligonucleotide sequence analog are described as "complementary" to one another when hybridization occurs in an antiparallel configuration. A oligonucleotide analog sequence may have "near" or "substantial" complementarity to the target sequence and still function for the purpose of the present invention, that is, still be "complementary." For example, the oligonucleotide analog sequence compounds employed herein can have one mismatch with the target sequence out of 10 nucleotides, or one mismatch out of 20. Alternatively, the antisense oligonucleotides employed have at least 90% sequence homology, such as at least 95% sequence homology, with the target as designated herein.

An oligonucleotide "specifically hybridizes" to a target if the oligomer hybridizes to the target under physiological conditions, with a T_m greater than about 45°C, such as at least about 50°C, including about 60°C to about 80°C or higher. Such hybridization may also correspond to stringent hybridization conditions. At a given ionic strength and pH, the T_m is the temperature at which 50% of a target sequence hybridizes to a complementary polynucleotide. Such hybridization may occur with "near" or "substantial" complementary of the antisense oligomer to the target sequence, as well as with exact complementarity.

A "heteroduplex" refers to a duplex between an oligonucleotide analog sequence and the complementary portion of a target RNA.

"Inhibit" generally means to reduce expression of RNA or protein or its activity as compared to an untreated subject or sample (such as by inhibiting about 10%, about 20%, about 30%, about 40%, about 50%, about 60% about 70% about 80%, about 90% or about 100%).

An "effective amount" generally means an amount that provides the desired local or systemic effect and or performance. For example, an "effective amount" of an antisense oligonucleotide, targeted against DUX4, is an amount effective to treat or reduce at least one symptom of FSHD or DUX4 production.

The term "isolated" refers to protein(s)/polypeptide(s), nucleic acid(s)/oligonucleotide(s), factor(s), cell or cells which are not associated with one or more protein(s)/polypeptide(s), nucleic acid(s)/oligonucleotide(s), factors, cells or one or more cellular components that are associated with the protein(s)/polypeptide(s), nucleic acid(s)/oligonucleotide(s), factor(s), cell or cells in vivo.

Subject means a vertebrate, such as a mammal, including a human. Mammals include, but are not limited to, humans, farm animals, sport animals and companion animals. Included in the term "animal" is dog, cat, fish, gerbil, guinea pig, hamster, horse, rabbit, swine, mouse, monkey (e.g., ape, gorilla, chimpanzee, orangutan) rat, sheep, goat, cow and bird.

As used herein, "treat," "treating" or "treatment" includes treating, ameliorating, or inhibiting a disease related condition and/ or a symptom of a disease related condition.

II. Target

In accordance with the present subject matter, antisense oligonucleotides, including morpholino antisense oligonucleotides can knockdown gene expression. An antisense oligonucleotide in accordance with the present subject matter can target, for example, the 5' UTR region of any number of FSHD candidate genes, particularly those located within the D4Z4 repeat region, such as, but not limited to FRG1, FRG2, CRYM, ANT1, ALP, PITX1, LRP2BP, TUBB4Q or DUX4.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by a contraction of the D4Z4 macros atellite repeat array, located at the distal tip of chromosome 4(4q35.2). This modification alters chromatin and results in transcription from D4Z4. The D4Z4 transcript encodes DUX4 (NM_001127386 mRNA; XP_001720134 protein,) a protein that causes pathological changes in myoblasts, including impaired differentiation, stress sensitivity and cell death.

1 ccccccccca ccaccaccac caccaccacc ccgccggccg gccccaggcc tcgacgccct 61 gggtcccttc cggggtgggg cgggctgtcc caggggggct caccgccatt catgaagggg

121 tggagcctgc ctgcctgtgg gcctttacaa gggcggctgg ctggctggct ggctgtccgg

181 gcaggcctcc tggctgcacc tgccgcagtg cacagtccgg ctgaggtgca cgggagcccg

241 ccggcctctc tctgcccgcg tccgtccgtg aaattccggc cggggctcac cgcgatggcc

301 ctcccgacac cctcggacag caccctcccc gcggaagccc ggggacgagg acggcgacgg 361 agactcgttt ggaccccgag ccaaagcgag gccctgcgag cctgctttga gcggaacccg

421 tacccgggca tcgccaccag agaacggctg gcccaggcca tcggcattcc ggagcccagg

481 gtccagattt ggtttcagaa tgagaggtca cgccagctga ggcagcaccg gcgggaatct

541 cggccctggc ccgggagacg cggcccgcca gaaggccggc gaaagcggac cgccgtcacc 601 ggatcccaga ccgccctgct cctccgagcc tttgagaagg atcgctttcc aggcatcgcc

661 gcccgggagg agctggccag agagacgggc ctcccggagt ccaggattca gatctggttt

721 cagaatcgaa gggccaggca cccgggacag ggtggcaggg cgcccgcgca ggcaggcggc

781 ctgtgcagcg cggcccccgg cgggggtcac cctgctccct cgtgggtcgc cttcgcccac

841 accggcgcgt ggggaacggg gcttcccgca ccccacgtgc cctgcgcgcc tggggctctc

901 ccacaggggg ctttcgtgag ccaggcagcg agggccgccc ccgcgctgca gcccagccag 961 gccgcgccgg cagagggggt ctcccaacct gccccggcgc gcggggattt cgcctacgcc 1021 gccccggctc ctccggacgg ggcgctctcc caccctcagg ctcctcggtg gcctccgcac

1081 ccgggcaaaa gccgggagga ccgggacccg cagcgcgacg gcctgccggg cccctgcgcg 1141 gtggcacagc ctgggcccgc tcaagcgggg ccgcagggcc aaggggtgct tgcgccaccc 1201 acgtcccagg ggagtccgtg gtggggctgg ggccggggtc cccaggtcgc cggggcggcg 1261 tgggaacccc aagccggggc agctccacct ccccagcccg cgcccccgga cgcctccgcc 1321 tccgcgcggc aggggcagat gcaaggcatc ccggcgccct cccaggcgct ccaggagccg

1381 gcgccctggt ctgcactccc ctgcggcctg ctgctggatg agctcctggc gagcccggag

1441 tttctgcagc aggcgcaacc tctcctagaa acggaggccc cgggggagct ggaggcctcg

1501 gaagaggccg cctcgctgga agcacccctc agcgaggaag aataccgggc tctgctggag 1561 gagctttagg acgcggggtt gggacggggt cgggtggttc ggggcagggc g (SEQ ID NO:3)

MKGWSLPACGPLQGRLAGWLAVRAGLLAAPAAVHSPAEVHGSPP ASLCPRPSVKFRPGLTAMALPTPSDSTLPAEARGRGRRRRLVWTPSQSEALRACFERN PYPGIATRERLAQAIGIPEPRVQIWFQNERSRQLRQHRRESRPWPGRRGPPEGRRKRT

AVTGS QT ALLLR AFEKDRFPGIAAREEL ARETGLPES RIQrWFQNRR ARHPGQGGR AP AQAGGLCSAAPGGGHPAPSWVAFAHTGAWGTGLPAPHVPCAPGALPQGAFVSQAARAA PALQPSQAAPAEGVSQPAPARGDFAYAAPAPPDGALSHPQAPRWPPHPGKSREDRDPQ

RDGLPGPCAVAQPGPAQAGPQGQGVLAPPTSQGSPWWGWGRGPQVAGAAWEPQAGAAP PPQPAPPDASASARQGQMQGIPAPSQALQEPAPWSALPCGLLLDELLASPEFLQQAQPLLETE

APGELEASEEAASLEAPLSEEEYRALLEEL (SEQ ID NO:4)

These results position DUX4 as a molecular target for FSHD therapeutics, in that progression of the disease may be delayed or inhibited by altering expression of DUX4 (mRNA and/or protein). Double homeobox, 4 also known as DUX4 is a protein which in humans is encoded by the DUX4 gene. This gene is located within a D4Z4 repeat array in the subtelomeric region of chromosome 4q. The D4Z4 repeat is polymorphic in length; a similar D4Z4 repeat array has been identified on chromosome 10. Each D4Z4 repeat unit has an open reading frame (named DUX4) that contains two homeoboxes; the repeat-array and ORF is conserved in other mammals.

Despite ongoing advances in uncovering the molecular mechanisms governing FSHD, no therapy yet exits. To this end, the inventors have developed and describe herein an antisense morpholino-based oligonucleotide designed to disrupt DUX4 translation by specifically binding to, for example, the 5'UTR upstream of the DUX4 translational start site. Successful inhibition of DUX4 translation following treatment of cells with such morpholinos is described herein. This morpholino represents a novel FSHD therapeutic.

A. Target Sequences

The targets selected are sequences that span, are just downstream (within about 25 bases) or upstream (within 100 bases) of the AUG start codon of selected FSHD candidate genes. The sequence for the DUX4 AUG start-site region is identified herein as SEQ ID NO:3. In an example, an antisense oligonucleotide is complementary to at least 12 contiguous bases in SEQ IDNO:3. An antisense morpholino oligonucleotide was designed to bind to an RNA sequence located within the 5'UTR upstream of the DUX4 translational start site. An example of a DUX4 antisense morpholino oligonucleotide (MK1) has the following sequence: 5 ' - AATTTC ACGG ACGG ACGCGGGC AG A-3 ' (SEQ ID NO: 1). Each morpholino oligonucleotide is comprised of a short chain of subunits, each subunit being comprised of a nucleic acid base, a morpholine ring, and a non-ionic

phosphorodiamidate intersubunit linkage (GeneTools, Philomath, OR). In embodiments, a morpholino oligonucleotide is conjugated to fluorescine reporter (MKl-fluor or MK1 -inverse- fluor) or as a Vivo-Morpholino covalently linked to an octa-guanidne dendrimer delivery moiety (Vivo- MK1, Vivo-MKl -inverse).

Antisense oligonucleotides are designed to hybridize to a region of the target sequence. Selected targeting sequences can be made shorter, e.g., 12 bases, or longer, e.g., 40 bases, and include a number of mismatches, as long as the sequence is sufficiently complementary to allow hybridization with the target and forms a heteroduplex having a T_m of 45°C or greater.

More generally, the degree of complementarity between the antisense oligonucleotide and the target is sufficient to form a stable duplex. The region of complementarity of the antisense oligonucleotide with the target RNA sequence may be as short as 8-11 bases, but can be 12-15 bases or more, e.g. 12-20 bases, or 12-25 bases or greater. Oligomers as long as 40 bases may be suitable, where at least a minimum number of bases, e.g., 12 bases, are complementary to the target sequence.

The antisense oligonucleotide may be 100% complementary to the nucleic acid target sequence, or it may include mismatches, e.g., to accommodate variants. Mismatches, if present, are less destabilizing toward the end regions of the hybrid duplex than in the middle. The number of mismatches allowed will depend on the length of the oligomer, the percentage of G:C base pairs in the duplex, and the position of the mismatch(es) in the duplex, according to well understood principles of duplex stability. Although such an antisense oligonucleotide is not necessarily 100% complementary to the nucleic acid target sequence, it is effective to stably and specifically bind to the target sequence, such that a biological activity of the nucleic acid target, e.g., expression of DUX4 protein(s) and/ or mRNA, is modulated.

The stability of the duplex formed between the antisense oligonucleotide and the target sequence is a function of the binding T_m and the susceptibility of the duplex to cellular enzymatic cleavage. The T_m of an antisense compound with respect to complementary-sequence RNA may be measured by conventional methods, such as those described by Hames et al., Nucleic Acid

Hybridization, IRL Press, 1985, pp. 107-108 or as described in Miyada C. G. and Wallace R. B., 1987, Oligonucleotide hybridization techniques, Methods Enzymol. Vol. 154 pp. 94-107. Each antisense oligomer should have a binding T_m, with respect to a complementary-sequence RNA, of greater than body temperature and such as greater than 50°C, such as T_m's in the range 60-80°C or greater. According to well known principles, the T_m of an oligomer, with respect to a complementary- based RNA hybrid, can be increased by increasing the ratio of C:G paired bases in the duplex, and/or by increasing the length (in base pairs) of the heteroduplex. At the same time, for purposes of optimizing cellular uptake, it may be advantageous to limit the size of the oligomer.

In an example, an antisense oligonucleotide, including a morpholino, can be modified on the 3' end, the 5' end or both. In an example, the 3' modification can be, but is not limited to 3'- carboxyfluorescein, 3'-lissamine, 3'-dabcyl, 3'-primary amine, 3'-biotin, 3'-disulfide, 3' amine plus biotin, or any mixture thereof. In an example, the 5'-end modification can be a 5'-primary amine, or 5'-dabcyl.

The antisense compounds can be prepared by methods available to the art.

B. Uptake by Cells

The antisense oligonucleotide may be taken up by host cells by facilitated or active transport across the host cell membrane if administered in free (non-complexed) form, or by an endocytotic mechanism if administered in complexed form.

In one embodiment, the antisense oligonucleotide is a substrate for a membrane transporter system (i.e. a membrane protein or proteins) capable of facilitating transport or actively transporting the oligomer across the cell membrane.

The antisense oligonucleotide can be administered in complexed form, where the complexing agent is typically a polymer, e.g., a cationic lipid, polypeptide, or non-biological cationic polymer, having an opposite charge to any net charge on the antisense compound. Methods of forming complexes, including bilayer complexes, between anionic oligonucleotides and cationic lipid or other polymer components, are well known. For example, the liposomal composition Lipofectin™ (Feigner, Gadek et al. 1987), containing the cationic lipid DOTMA (N-[l -(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride) and the neutral phospholipid DOPE (dioleyl phosphatidyl

ethanolamine), is widely used. After administration, the complex is taken up by cells through an endocytotic mechanism, typically involving particle encapsulation in endosomal bodies.

The antisense compound may also be administered in conjugated form with an arginine-rich peptide linked covalently to the 5' or 3' end of the antisense oligomer. The peptide is typically 8-16 amino acids and consists of a mixture of arginine, and other amino acids including phenyalanine and cysteine. The use of arginine-rich peptide conjugates can be used to enhance cellular uptake of the antisense oligomer (See, e.g. Moulton, Nelson et al. 2004; Nelson, Stein et al. 2005).

In some instances, liposomes may be employed to facilitate uptake of the antisense oligonucleotides into cells. (See, e.g., Williams, S. A., Leukemia 10(12): 1980-1989, 1996;

Lappalainen et al., Antiviral Res. 23: 119, 1994; Uhlmann et al., "Antisense oligonucleotides: A new therapeutic principle," Chemical Reviews, Volume 90, No. 4, pages 544-584, 1990; Gregoriadis, G., Chapter 14, Liposomes, Drug Carriers in Biology and Medicine, pp. 287-341, Academic Press, 1979). Hydrogels may also be used as vehicles for antisense oligonucleotide administration, for example, as described in WO 93/01286. Alternatively, the oligonucleotides may be administered in microspheres or microparticles. (See, e.g., Wu, G. Y. and Wu, C. H., J. Biol. Chem. 262:4429-4432, 1987).

Alternatively, the use of gas-filled microbubbles complexed with the antisense oligonucleotides can enhance delivery to target tissues, as described in U.S. Pat. No. 6,245,747.

An in vivo delivery strategy can employ peptides based on a tat or a penetratin peptide to achieve delivery of an antisense oligonucleotide, such as a morpholino oligonucleotide. In an embodiment, an antisense oligonucleotide, such as a morpholino oligonucleotide, for in vivo use can be comprised of a morpholino oligonucleotide coupled with a dendrimer delivery moiety. In one embodiment, the antisense oligonucleotide, such as a morpholino oligonucleotide, can be covalently linked to an octa-guanidine dendrimer delivery moiety.

In an embodiment, a dendrimer can encapsulate a small molecule. In an embodiment, a surface of the dendrimer can be unconjugated (such as an amine terminated, cationic surface), or conjugated. Hydrogen bonding, van der Waals interaction, or electrostatic attraction between opposite charges on the dendrimer and cargo can be driving forces for the interaction leading to encapsulation. A surface of the dendrimer can be conjugated with, for example, succinic acid, benzene disulphonic acid, or polyethylene glycol 200 (PEGylated dendrimer). Cargo, such as a morpholino oligonucleotide, can be conjugated to the dendrimer periphery. In an embodiment, cargo can be conjugated to a dendrimer focal point.

A dendrimer based drug delivery system can include a dendrimer scaffold such as, but not limited to, triazine, polypropyleneimine, polyester 'bow tie,' polyamidoamine (PAMAM), or poly-L- lysine. In an embodiment, a triazine core can be a key element in assembling a dendritic molecular transporter. For example, two sites of a tri-functional triazine can be used for branching side arms. In an embodiment, a third functional site can be used linking covalently with a morpholino

oligonucleotide. The dendritic moiety can be conjugated with a morpholino oligomer while the oligomer remains on the synthesis resin. The protecting groups can be removed, and the oligomer can be detached from the synthesis resin. A subsequent perguanidinylation process can result in a conjugate of morpholino with a transporter moiety of a triazine core scaffold presenting eight guanidine head groups.

The properties of oligomers with any given backbone can be determined by a simple in vivo test, in which a labeled compound is administered to an animal, and a body fluid sample, taken from the animal several hours after the oligomer is administered, is assayed for the presence of heteroduplex with target RNA.

III. Treatment

A) The antisense compounds detailed above are useful in treating FSHD in a subject, including humans. Accordingly, one embodiment comprises contacting a cell with an antisense oligonucleotide effective to, for example, inhibit transcription, processing, and/or translation of a preselected mRNA. A method provided herein can include a morpholino oligonucleotide capable of knocking down gene expression. In an example, a morpholino oligonucleotide can block translation initiation in the cytosol by targeting the five prime untranslated region (5'UTR) of messenger RNA. For example, the morpholino oligonucleotide can interfere with translation of a target transcript by interfering with the progression of the ribosomal initiation complex. In an example, a morpholino oligonucleotide can modify pre-mRNA processing. A morpholino oligonucleotide can affect splicing in the nucleus by targeting splice junctions of splice regulatory sites. For example, a morpholino oligonucleotide can prevent binding of splice directing small nuclear ribonucleoprotein (such as snRNPs). In an example, the prevention of snRNP binding can inhibit the formation of complexes that could otherwise bind to targets at the border of an intron of pri-mRNA and would otherwise form the splice lariat structure. In an example, the morpholino oligonucleotide can modify splicing and can inhibit the formation of mature mRNA.

In an example, a morpholino oligonucleotide can inhibit miRNA maturation and activity by targeting mature miRNA or pri-miRNA. In an example, a morpholino oligonucleotide can inhibit a ribozyme. In an example, a morpholino oligonucleotide can result in a translational frameshift.

B) Administration of the Antisense Oligonucleotides

In an embodiment, an effective therapeutic antisense oligonucleotide, such as a morpholino oligonucleotide, can be designed to enter the cell of an adult animal in vivo. In an embodiment, systemic results can be achieved by intravenously injecting a morpholino. Routes of antisense oligonucleotide delivery include, but are not limited to, various systemic routes, including oral and parenteral routes, e.g., intravenous, subcutaneous, intraperitoneal, and intramuscular, as well as inhalation, transdermal and topical delivery. In an embodiment, localized delivery can be achieved by injecting an antisense oligonucleotide, such as a morpholino oligonucleotide, directly into the area of interest. The appropriate route may be determined by one of skill in the art, as appropriate to the condition of the subject under treatment.

The antisense oligonucleotide may be administered in any convenient vehicle which is physiologically acceptable. Such a composition may include any of a variety of standard

pharmaceutically accepted carriers employed by those of ordinary skill in the art. Examples include, but are not limited to, saline, phosphate buffered saline (PBS), water, aqueous ethanol, emulsions, such as oil/water emulsions or triglyceride emulsions, tablets and capsules. The choice of suitable physiologically acceptable carrier will vary dependent upon the chosen mode of administration.

The antisense oligonucleotide is generally administered in an amount and manner effective to result in a peak blood concentration of at least 200-400 nM antisense oligonucleotide. Typically, one or more doses of antisense oligonucleotide are administered, generally at regular intervals, for a period of about one to two weeks. Doses for oral administration can be from about 5-1000 mg oligomer per 70 kg individual. In some cases, doses of greater than 500 mg oligomer/patient may be administered. For i.v., i.p or s.q. administration, doses can include from about 100-1000 mg oligomer per 70 kg body weight. The antisense oligonucleotide may be administered at regular intervals for a short time period, e.g., daily for two weeks or less. However, in some cases the oligomer is administered intermittently over a longer period of time. The treatment regimen may be adjusted (dose, frequency, route, etc.) as indicated, based on the results of immunoassays, other biochemical tests and physiological examination of the subject under treatment.

Example I

Design of morpholino and morpholino- mediated inhibition of DUX4 expression

Materials and Methods

Generating a DUX4 expression construct with 5' UTR. A 2.7 kb construct encoding the DUX4 ORF and 3' genomic sequences to the Eco RI site was extended by subcloning additional 5' UTR sequence. This was accomplished by synthesizing primers, Fwd (5'- ATTCTACTCGAGGC AC AGTCCGGCTGAGGTGC A-3 ' ; SEQ ID NO:4) and Rev (5'- C ACGAGGGAGC AGGGTGAC-3 ' ; SEQ ID NO:5), amplifying a 5' sequence using a genomic

EcoRI DNA fragment bearing two D4Z4 repeats named λ42 (van Deutekom et al., 1993). The Xhol- BstEII fragment from this PCR product was used to replace the smaller XhoI-BstEII fragment present in p2Lox-DUX4(2.7) (Bosnakovski, Xu et al. 2008). The 5' extension was designed based on the 5' end of the DUX4 transcript as determined by 5' RACE (Dixit, Ansseau et al. 2007).

Cell culture. Murine myoblast cell line C2C12 was purchased from ATCC. Derivative cell lines were made by inserting into C2C12 myoblasts and murine ES cells using an inducible cassette exchange system, where the gene of interest is targeted into a doxycycline regulated locus as described in Bosnakovshi et al., 2008. EMBO J. 27:2766-2779 and Iacovino et al., 2011. Stem Cells. 29(10): 1580- 1588. Several cell lines were generated in which expression of the DUX4-encoding D4Z4 transcript could be regulated by doxycycline. Each derivative cell line contained the endogenous DUX4 5'UTR in some respect. Expression constructs were designed to express DUX4 (GenBack accession no. AF117653) sequence or from a larger construct containing sequence downstream of the ORF.

1 gaattctatc tggtacccag agggaagggg gttcccagtg agggcaggac caggcttcat

61 gcacctcttc aggaatgttc tcctcatagt ccagcctcaa ggtgtgcatc ctctgtgtgc

121 atggagtcca tggcaggctc tgcctgggga gccgtccagc tgcacacctg caatgtggtg

181 gtgaccctca tgaatgggtg gttctgggcc ccatggctgg cagcagagag ggagatgttc

241 agccaccaag cccagagccc tgccacaggc ttctgtgagg cctccatctg ctctgggttc

301 ttgccctgag aggctgccct gaagtcaaac agaagcaggt gggcctctct tccagggctg

361 ctctctcccc cactgacagc tccctagagg gagactcaga cagcggggac agattcctca

421 ggcataagca ctggagttta ggctggccag ttcattccat acgcccacat gacatgacac

481 aaggcagagg ctgtgggaca aaggtattgc cttttcttct ggcatgagga atggcttagg

541 aagcagggga tggtggggct ggggttgagt gatgggctgt gggccacaag gagtgggtgg

601 gcgctgagaa agtgtcctgg ttgtctgtcc atagacgcag aatgagtggc atcccaggag

661 cctgtgaggg gctggcagag acttactggt tccagtaaaa gccccatgtg gatgcagtaa

721 tgctgcctgc tggtccttgg ctgtaattac aaacaggtac atgaggtacc catgcatctt 781 gaagctctca gggagtgggt tccagctgct catggtaggc acttttagtc actgaacatg 841 cttcaggcat gtccaagctt gattaagcca ggcatcttgc tgtgaggccc tccacttcac 901 taagaacact cttccttgct tcccctggaa gttggacctt ccagttctgg ttctggagac 961 acgatggccc ctcctggacc cctgggagaa tgtgctcagg tgacacacag ttgatggggc 1021 ccatttccaa gccattcttc catttcccac tgtttgaggg acccgaggcc ggtgacaagc 1081 acagagccac ccaaggccag ctgtctgcac ctaaatgtga tgcttgtctg gatgtctcag 1141 ggccagaacc ctccaggtga gatggcctgg tcctcaccac ctggcgtccg tgctcccttt 1201 tcctctgttc aatcctggcg ccaatgcctc cctcaactct caggtcacca ttggagaaga 1261 tgctcaggaa gaacaagcag ctgcagttaa ccctgctgaa agtggcagat gggtccaggc 1321 tcttgagctc gtcttggaca tggaacatgt ggatacaggc tttgagcagt gtgtgtagct 1381 ctttcaggaa ggaagggaaa agggtgttac ccgggtccta caccctggaa cgacccttct 1441 cagacagtaa atagttggca gggtgcggtc atgtgtgatt ttagttttca actttaggct 1501 ttcattttca aattccacaa taaacacata aggtggagtt ctggtttcag cacacacaca 1561 cacacacaca aacacacaca cacacacaca cacagtctct ctctctgtat gtctctttct 1621 gtctctctct ctctccttcc tgcaaggatc cttgttaaca agaaaccttc tgccaaatgc 1681 ctctgaagca caggcaggtc ttggggagcc acaaggccac ttcctctttg tgcactagtg 1741 tcttgggtag gcatagcttt cagagctctg gggcctccac aaccttgccc tgctgtccag 1801 gggcagccct catgcagggg tgtcctaaga acttttcagg atgcacaagt tcagcactgt 1861 cttccaatgt gtgtttcacg atattttaat ggtggttctt ttgggaaaaa ggaaaggttc 1921 tgtgatcaat tatgggacac attgagctac agatcttttt cacaattgct cttaacaagc 1981 aggtagaccc tgagaacatg agtagcttcc ccgcaggtaa cttgagtgca tgagaacttt 2041 tgctttacaa ccatgccaat ctcacctcag cagttggcag tgctgcacgg ggcagacttc 2101 cctactcaaa ggctgtgaag cttttctttc ttttttttta aacattattt ttctttatag

2161 aattttgttg ggctgatatc aagcctggct tggtactgcc tcattttttt tggaatcaga 2221 acgctgttct ttaactcacg ggttgtgaag ttagaaggtg ctggtgtgac agcctgacaa 2281 gcagagcgca gctccaatcc caccttcatg ctctcatctg acgcagagcc ctcagagaag 2341 tggggaagtg cttcctggcc ctgcttctgg gggccgtccc caaggcagtc caccgaactt 2401 ccaaaacagc cttccctcac acacagccct gagccctcct gccgctcctc aatgttgcac 2461 atctctgaga agtggtccag catgttgctg tccaggggca gtgagaagca ggtgcggtga 2521 cacatgtctt cacggaccat gagcaccggg taaatctcct gcacaatctc cttgggggac 2581 accttgaggg agaaagcccc aacaactgat ggcatgccac atggcagaaa gcaaagactt 2641 accctttccc cagcccaaag tcctgagaat catgccaaaa atccttggtt tcccactttt 2701 taaaaatttt aaaattaaaa tcccaggttc cgcgtataca tgccatgccc acctgcacct 2761 gtgtgtgtgt gtgtgtttgt gcacgcagga cagagcctgg cccattgact attcctgcag 2821 accaagaaaa atccctatgc agagtaaggg gagatggaag aaacgaggga gagaaaatg: 2881 cagccttgcc tcctcccttg cccagtgcta aggtccccag ggcaaatggc ttttgccttc 2941 aacttcacct taacaacata caaaatatat tcatttttac ttccgtcact ttcttaacat

3001 tacaaattgt atctttatat atgatttgta ttttcacaga gatttaagaa tttaatgcac

3061 cattatagta gaaaattgta tatctgtgta tatatttaca ttgaacagag agctttatat

3121 tttcatgtgg ttttatgatg ctgtccagca tcatttaatt tttcaacata attaactctc

3181 tttagcattt tttttcctag ggttattcta gtagttaaca acctcagctt ttttatttta

3241 atctttgaaa gtctttattt ttttctaatt tttgaaatac agtatttccc agatcaatta

3301 ttattggttg ctagtatttt ttctttcatc gctttgaaat ctggaaagtt cttagcatcc

3361 ccgctttttc tctgaaataa tgtttatgcc attttctccc tctattcttt ttaaaagact

3421 ctatctctga atgtattggt ctacttgatg gtgtccagta agtcttatat ttcacccgta

3481 attttcccat tctttaaaat attagtttcc aggactcaat atttgtgaat aatatatgtt

3541 caattttctt ttttctgctc cattgtttgc tgttgtgtct ctgtagtgaa tttataaaac

3601 tcagttatta tattcttcaa ctctatgatt tctgttgggt ttttaaaaat agtttttatc

3661 tctttgttga tattttgctc attcgttatt tttaaatttc actcagttgt ctctttctgt

3721 tatagttttg ctcactgaga atgcataaga tgattatttt aagttctcca tcagatatgc

3781 aaaaatcttt atttgttaaa attcagtttc tgaatattta tgtttttctt ccaatgggga

3841 atattttctg ccttctctgt gtgccttgtg attttttttt ttaaagagat ctggggatct

3901 atacagcact catcaaatct agcatttaaa gactggctca gtaaaggggg ataccgacag

3961 caatagtcca ggctatagat tctaggtgct tcacaaacac attcccaaat atattttctc

4021 tggacttcgc tgtgtttcca agttaaagag aattttttct caatgtattt tagattctat

4081 tgtatatttt cttccccagt tggctgtctg tggtattgca gtttcactag tgctgtagca

4141 aacactcatc tttcttctca gcagacacaa actgtcatct atatgactcc atcatgtcct

4201 tcagcactcc acatcaggag agaaagaatc tagtcattag acaatttatc aaaaagccaa

4261 acatttcaac acatattcta ctgttttaat tctctcctga aggagatact gggagttggg

4321 cattttctca ttagcccagt tactgttctg ggtgaaaaaa taaactgcag tggacaggct

4381 gtaagccaga cttcatcaaa tttctgcacc aatgaaaaaa aaatttacaa gagaaaaaca

4441 aaaaacccta ttaaacgtca cggacaaggc cagagtttga atatactgtg gtcatctctg

4501 ctccagtgca aactgtttcc agaaagccta cttctatttt ccttgctgta acagaggaac

4561 atttcctgtc ttatgtttat tctactctgc aatcccctaa ggctttttct ctccctccca

4621 gaatcttaaa gtgcattcga actcacaggc aaaatcctcc cagaatcttg tgagaacata

4681 aatgatctga ctagtttggc attgcttttg gggatctggg aaaatctgtg cacacttctg

4741 gagacccttg tcatgccatt ttttataaat ctattgtgcc tcaagtcaga agtgtgtgag

4801 gggagatggg gagacattgg gatgcgcgcg cctggggctc tcccacaggg ggctttcgtg

4861 agccaggcag cgagggccgc ccccgcgctg cagcccagcc aggccgcgac ggcagagggg

4921 gtctcccaac ctgccccggc gcgcggggat ttcgcctacg ccgccccggc tcctccggac

4981 ggggcgctct cccaccctca ggctcctcgg tggcctccgc acccgggcaa aagccgggag

5041 gaccgggacc cgcagcgcga cggcctgccg ggcccctgcg cggtggcaca gcctgggccc 5101 gctcaagcgg ggccgcaggg ccaaggggtg cttgcgccac ccacgtccca ggggagtccg 5161 tggtggggct ggggccgggt ccccaggtcg ccggggcggc gtgggaaccc caagccgggg 5221 caagcttcca cctccccagc ccgcgccccc ggacgcctcc gcctccgcgc ggcaggggca 5281 gatgcaaggc atcccggcgc cctcccaggc gctccaggag ccggcgccct ggtctgcact 5341 cccctgcggc ctgctgctgg atgagctcct ggcgagcccg gagtttctgc agcaggcgca 5401 acctctccta gaaacggagg ccccggggga gctggaggcc tcggaagagg ccgcctcgct 5461 ggaagcaccc ctcagcgagg aagaataccg ggctctgctg gaggagcttt aggacgcggg 5521 gttgggacgg ggtcgggtgg ttcggggcag ggcggtggcc tctctttcgc ggggaacacc 5581 tggctggcta cggaggggcg tgtctccgcc ccgccccctc caccgggctg accggcctgg 5641 gattcctgcc ttctaggtct aggcccggtg agagactcca caccgcggag aactgccatt 5701 ctttcctggg catcccgggg atcccagagc cggcccaggt accagcaggt gggccgccta 5761 ctgcgcacgc gcgggtttgc gggcagccgc ctgggctgtg ggagcagccc gggcagagct 5821 ctcctgcctc tccaccagcc caccccgccg cctgaccgcc ccctccccac cccccacccc 5881 ccacccccgg aaaacgcgtc gtcccctggg ctgggtggag acccccgtcc cgcgaaacac 5941 cgggccccgc gcagcgtccg ggcctgacac cgctccggcg gctcgcctcc tatgcgcccc 6001 cgcgccaccg tcgcccgccc gcccgggccc ctgcagccgc ccaggtgcca gcacggagcg 6061 cctggcggcg gaacgcagac cccaggcccg gcgcacaccg gggacgctga gcgttccagg 6121 cgggagggaa ggcgggcaga gatggagaga ggaacgggag acctagaggg gcggaaggac 6181 gggcggaggg acgttaggag ggagggaggg aggcagggag gcagggagga acggagggaa 6241 agacagagcg acgcagggac tgggggcggg cgggagggag ccggggaacg gggggaggaa 6301 ggcagggagg aaaagcggtc ctcggcctcc gggagtagcg ggacccccgc cctccgggaa 6361 aacggtcagc gtccggcgcg ggctgagggc tgggcccaca gccgccgcgc cggccggcgg 6421 ggcaccaccc attcgccccg gttccgtggc ccagggagtg ggcggtttcc tccgggacaa 6481 aagaccggga ctcgggttgc cgtcgggtct tcacccgcgc ggttcacaga ccgcacatcc 6541 ccaggctgag ccctgcaacg cggcgcgagg ccgacagccc cggccacgga ggagccacac 6601 gcaggacgac ggaggcgtga ttttggtttc cgcgtggctt tgccctccgc aaggcggcct 6661 gttgctcacg tctctccggc ccccgaaagg ctggccatgc cgactgtttg ctcccggagc 6721 tctgcgggca cccggaaaca tgcagggaag ggtgcaagcc cggcacggtg ccttcgctct 6781 ccttgccagg ttccaaaccg gccacactgc agactcccca cgttgccgca cgcgggaatc 6841 catcgtcagg ccatcacgcc ggggaggcat ctcctctctg gggtctcgct ctggtcttct 6901 acgtggaaat gaacgagagc cacacgcctg cgtgtgcgag accgtcccgg caacggcgac 6961 gcccacaggc attgcctcct tcacggagag agggcctggc acactcaaga ctcccacgga 7021 ggttcagttc cacactcccc tccaccctcc caggctggtt tctccctgct gccgacgcgt 7081 gggagcccag agagcggctt cccgttcccg cgggatccct ggagaggtcc ggagagccgg 7141 cccccgaaac gcgcccccct cccccctccc ccctctcccc cttcctcttc gtctctccgg 7201 ccccaccacc accaccgcca ccacgccctc cccccccacc cccccccccc accaccacca 7261 ccaccacccc gccggccggc cccaggcctc gacgccctgg ggtcccttcc ggggtggggc 7321 gggctgtccc aggggggctc accgccattc atgaaggggt ggagcctgcc tgcctgtggg 7381 cctttacaag ggcggctggc tggctggccg gctgtccggg caggcctcct ggctgcacct 7441 gccgcagtgc acagtccggc tgaggtgcac gggagcccgc cggcctctct ctgcccgcgt 7501 ccgtccgtga aattccggcc ggggctcacc gcgatggccc tcccgacacc ctcggacagc 7561 accctccccg cggaagcccg gggacgagga cggcgacgga gactcgtttg gaccccgagc 7621 caaagcgagg ccctgcgagc ctgctttgag cggaacccgt acccgggcat cgccaccaga 7681 gaacggctgg cccaggccat cggcattccg gagcccaggg tccagatttg gtttcagaat 7741 gagaggtcac gccagctgag gcagcaccgg cgggaatctc ggccctggcc cgggagacgc 7801 ggcccgccag aaggccggcg aaagcggacc gccgtcaccg gatcccagac cgccctgctc 7861 ctccgagcct ttgagaagga tcgctttcca ggcatcgccg cccgggagga gctggccaga 7921 gagacgggcc tcccggagtc caggattcag atctggtttc agaatcgaag ggccaggcac 7981 ccgggacagg gtggcagggc gcccgcgcag gcaggcggcc tgtgcagcgc ggcccccggc 8041 gggggtcacc ctgctccctc gtgggtcgcc ttcgcccaca ccggcgcgtg gggaacgggg 8101 cttcccgcac cccacgtgcc ctgcgcgcct ggggctctcc cacagggggc tttcgtgagc 8161 caggcagcga gggccgcccc cgcgctgcag cccagccagg ccgcgccggc agaggggatc 8221 tcccaacctg ccccggcgcg cggggatttc gcctacgccg ccccggctcc tccggacggg 8281 gcgctctccc accctcaggc tcctcggtgg cctccgcacc cgggcaaaag ccgggaggac 8341 cgggacccgc agcgcgacgg cctgccgggc ccctgcgcgg tggcacagcc tgggcccgct 8401 caagcggggc cgcagggcca aggggtgctt gcgccaccca cgtcccaggg gagtccgtgg 8461 tggggctggg gccggggtcc ccaggtcgcc ggggcggcgt gggaacccca agccggggca 8521 gctccacctc cccagcccgc gcccccggac gcctccgcct ccgcgcggca ggggcagatg 8581 caaggcatcc cggcgccctc ccaggcgctc caggagccgg cgccctggtc tgcactcccc 8641 tgcggcctgc tgctggatga gctcctggcg agcccggagt ttctgcagca ggcgcaacct 8701 ctcctagaaa cggaggcccc gggggagctg gaggcctcgg aagaggccgc ctcgctggaa 8761 gcacccctca gcgaggaaga ataccgggct ctgctggagg agctttagga cgcggggttg 8821 ggacggggtc gggtggttcg gggcagggcg gtggcctctc tttcgcgggg aacacctggc 8881 tggctacgga ggggcgtgtc tccgccccgc cccctccacc gggctgaccg gcctgggatt 8941 cctgccttct aggtctaggc ccggtgagag actccacacc gcggagaact gccattcttt 9001 cctgggcatc ccggggatcc cagagccggc ccaggtacca gcaggtgggc cgcctactgc 9061 gcacgcgcgg gtttgcgggc agccgcctgg gctgtgggag cagcccgggc agagctctcc 9121 tgcctctcca ccagcccacc ccgccgcctg accgccccct ccccaccccc caccccccac 9181 ccccggaaaa cgcgtcgtcc cctgggctgg gtggagaccc ccgtcccgcg aaacaccggg 9241 ccccgcgcag cgtccgggcc tgacaccgct ccggcggctc gcctcctatg cgcccccgcg 9301 ccaccgtcgc ccgcccgccc gggcccctgc agccgcccag gtgccagcac ggagcgcctg 9361 gcggcggaac gcagacccca ggcccggcgc acaccgggga cgctgagcgt tccaggcggg 9421 agggaaggcg ggcagagatg gagagaggaa cgggagacct agaggggcgg aaggacgggc 9481 ggagggacgt taggagggag ggagggaggc agggaggcag ggaggaacgg agggaaagac 9541 agagcgacgc agggactggg ggcgggcggg agggagccgg ggaacggggg gaggaaggca 9601 gggaggaaaa gcggtcctcg gcctccggga gtagcgggac ccccgccctc cgggaaaacg 9661 gtcagcgtcc ggcgcgggct gagggctggg cccacagccg ccgcgccggc cggcggggca 9721 ccacccattc gccccggttc cgtggcccag ggagtgggcg gtttcctccg ggacaaaaga 9781 ccgggactcg ggttgccgtc gggtcttcac ccgcgcggtt cacagaccgc acatccccag 9841 gctgagccct gcaacgcggc gcgaggccga cagccccggc cacggaggag ccacacgcag 9901 gacgacggag gcgtgatttt ggtttccgcg tggctttgcc ctccgcaagg cggcctgttg 9961 ctcacgtctc tccggccccc gaaaggctgg ccatgccgac tgtttgctcc cggagctctg 10021 cgggcacccg gaaacatgca gggaagggtg caagcccggc acggtgcctt cgctctcctt 10081 gccaggttcc aaaccggcca cactgcagac tccccacgtt gccgcacgcg ggaatccatc 10141 gtcaggccat cacgccgggg aggcatctcc tctctggggt ctcgctctgg tcttctacgt 10201 ggaaatgaac gagagccaca cgcctgcgtg tgcgagaccg tcccggcaac ggcgacgccc 10261 acaggcattg cctccttcac ggagagaggg cctggcacac tcaagactcc cacggaggtt 10321 cagttccaca ctcccctcca ccctcccagg ctggtttctc cctgctgccg acgcgtggga 10381 gcccagagag cggcttcccg ttcccgcggg atccctggag aggtccggag agccggcccc 10441 cgaaacgcgc ccccctcccc cctcccccct ctcccccttc ctcttcgtct ctccggcccc 10501 accaccacca ccgccaccac gccctccccc cccccccccc ccccccacca ccaccaccac 10561 caccccgccg gccggcccca ggcctcgacg ccctgggtcc cttccggggt ggggcgggct 10621 gtcccagggg ggctcaccgc cattcatgaa ggggtggagc ctgcctgcct gtgggccttt 10681 acaagggcgg ctggctggct ggctggctgt ccgggcaggc ctcctggctg cacctgccgc 10741 agtgcacagt ccggctgagg tgcacgggag cccgccggcc tctctctgcc cgcgtccgtc 10801 cgtgaaattc cggccggggc tcaccgcgat ggccctcccg acaccctcgg acagcaccct 10861 ccccgcggaa gcccggggac gaggacggcg acggagactc gtttggaccc cgagccaaag 10921 cgaggccctg cgagcctgct ttgagcggaa cccgtacccg ggcatcgcca ccagagaacg 10981 gctggcccag gccatcggca ttccggagcc cagggtccag atttggtttc agaatgagag 11041 gtcacgccag ctgaggcagc accggcggga atctcggccc tggcccggga gacgcggccc 11101 gccagaaggc cggcgaaagc ggaccgccgt caccggatcc cagaccgccc tgctcctccg 11161 agcctttgag aaggatcgct ttccaggcat cgccgcccgg gaggagctgg ccagagagac 11221 gggcctcccg gagtccagga ttcagatctg gtttcagaat cgaagggcca ggcacccggg 11281 acagggtggc agggcgcccg cgcaggcagg cggcctgtgc agcgcggccc ccggcggggg 11341 tcaccctgct ccctcgtggg tcgccttcgc ccacaccggc gcgtggggaa cggggcttcc 11401 cgcaccccac gtgccctgcg cgcctggggc tctcccacag ggggctttcg tgagccaggc 11461 agcgagggcc gcccccgcgc tgcagcccag ccaggccgcg ccggcagagg ggatctccca 11521 acctgccccg gcgcgcgggg atttcgccta cgccgccccg gctcctccgg acggggcgct 11581 ctcccaccct caggctcctc gctggcctcc gcacccgggc aaaagccggg aggaccggga 11641 cccgcagcgc gacggcctgc cgggcccctg cgcggtggca cagcctgggc ccgctcaagc

11701 ggggccgcag ggccaagggg tgcttgcgcc acccacgtcc caggggagtc cgtggtgggg

11761 ctggggccgg ggtccccagg tcgccggggc ggcgtgggaa ccccaagccg gggcagctcc

11821 acctccccag cccgcgcccc cggacgcctc cgcctccgcg cggcaggggc agatgcaagg

11881 catcccggcg ccctcccagg cgctccagga gccggcgccc tggtctgcac tcccctgcgg

11941 cctgctgctg gatgagctcc tggcgagccc ggagtttctg cagcaggcgc aacctctcct

12001 agaaacggag gccccggggg agctggaggc ctcggaagag gccgcctcgc tggaagcacc

12061 cctcagcgag gaagaatacc gggctctgct ggaggagctt taggacgcgg ggttgggacg

12121 gggtcgggtg gttcggggca gggccgtggc ctctctttcg cggggaacac ctggctggct

12181 acggaggggc gtgtctccgc cccgccccct ccaccgggct gaccggcctg ggattcctgc

12241 cttctaggtc taggcccggt gagagactcc acaccgcgga gaactgccat tctttcctgg

12301 gcatcccggg gatcccagag ccggcccagg tacctgcgca cgcgcgggtt tgcgggcagc

12361 cgcctgggct gtgggagcag cccgggcaga gctctcctgc ctctccacca gcccaccccg

12421 ccgcctgacc gccccctccc caccccccac cccccacccc cggaaaacgc gtcgtcccct

12481 gggctgggtg gagacccccg tcccgcgaaa caccgggccc cgcgcagcgt ccgggcctga

12541 ctccgctccg gcggctcgcc tcctgtgtgc ccccgcgcca ccgtcgcccg cccgcccggg

12601 cccctgcagc ctcccagctg ccagcgcgga gctcctggcg gtcaaaagca tacctctgtc

12661 tgtctttgcc cgcttcctgg ctagacctgc gcgcagtgcg caccccggct gacgtgcaag

12721 ggagctcgct ggcctctctg tgcccttgtt cttccgtgaa attctggctg aatgtctccc

12781 cccaccttcc gacgctgtct aggcaaacct ggattagagt tacatctcct ggatgattag

12841 ttcagagata tattaaaatg ccccctccct gtggatccta tagaagattt gcatcttttg

12901 tgtgatgagt gcagagatat gtcacaatat cccctgtaga aaaagcctga aattggttta

12961 cataacttcg gtgatcagtg cagatgtgtt tcagaactcc atagtagact gaacctagag

13021 aatggttaca tcacttaggt gatcagtgta gagatatgtt aaaattctcg tgtagacaga

13081 gcctagacaa ttgttacatc acctagtgat cagtgcaggg ataagtcata aagcctcctg

13141 taggcagagt gtaggcaagt gttccctccc tgggctgatc agtgcagaga tatctcacaa

13201 agcccctata agccaaacct tgacaagggt tacatcacct gtttgagcag tggaaatata

13261 tatcacaaag ccccctgtag acaaagccca gacaattttt acatctcctg agtgagcatt

13321 ggagagatct gtcacaatgc ccctgtaggc agagcttaga caagtgttac atcacctggg

13381 tgatcagtgc agagatatgt caaaacgctc ctgtagtctg aacctagaca ggagttacat

13441 caccttgggg atcagtgcag agatacgtga gaattcc (SEQ ID NO: 8)

MALPTPSDSTLPAEARGRGRRRRLVWTPSQSEALRACFERNPYP GIATRERLAQAIGIPEPRVQIWFQNERSRQLRQHRRESRPWPGRRGPPEGRRKRTAVT GSQTALLLRAFEKDRFPGIAAREELARETGLPESRIQIWFQNRRARHPGQGGRAPAQA GGLCSAAPGGGHPAPSWVAFAHTGAWGTGLPAPHVPCAPGALPQGAFVSQAARAAPAL QPS Q AAP AEGISQP AP ARGDFAY AAP APPDGALS HPQ APR WPPHPGKS REDRDPQRDG LPGPCAVAQPGPAQAGPQGQGVLAPPTSQGSPWWGWGRGPQVAGAAWEPQAGAAPPPQ PAPPDASASARQGQMQGIPAPSQALQEPAPWSALPCGLLLDELLASPEFLQQAQPLLE TEAPGELEASEEAASLEAPLSEEEYRALLEEL (SEQ ID NO:9)

For example, the iC2C12 cell line was targeted with DUX4 (iC2C12-DUX4), the 5'UTR of DUX4 (iC2C12-5'UTR DUX4 ORF), or a 2.7 kb DNA sequence from the terminal D4Z4 repeat containing DUX4 (iC2C12-5'UTR DUX4(2.7)). The iC2C12 lines were seeded onto 75 cm² flasks at a cell concentration between 1.5 x 10⁵ and 1.0 x 10⁶. The cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% glutamax, and 1% antibiotic antimycotic at 37° C and 5% CO₂. Medium was replaced or added every 2 to 3 days. Cultures were not permitted to become confluent.

Inducible murine embryonic stem cells containing the 5'UTR DUX4 sequence (5'UTR DUX4 mES) were cultured in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12

(DMEM/F12) supplemented with 20% ES qualified FBS, 1% Glutamax, 1% Non-essential amino acids, Leukemia Inhibitory Factor (LIF), β-mercaptoethanol, and 1% penicillin/streptomycin at 37° C and 5% C0₂. Cells were passaged every 2 to 3 days, and medium was replaced or added every day. Differentiation of the ES cells was prevented.

Design of the DUX4 Morpholino. An antisense morpholino oligonucleotide was designed to bind to an RNA sequence located within the 5'UTR upstream of the DUX4 translational start site. The DUX4 antisense morpholino oligonucleotide (MK1) has the following sequence: 5'- AATTTC ACGGACGGACGCGGGC AGA-3 ' (SEQ ID NO: 1). A negative control was designed to contain an identical molecular composition, but with the inverse sequence of the DUX4 antisense morpholino oligonucleotide. The negative control inverse morpholino oligonucleotide (MKl-inverse) had the following sequence: 5'-AGACGGGCGCAGGCAGGCACTTTAA-3' (SEQ ID NO: 2). Each morpholino oligonucleotide was comprised of a short chain of subunits, each subunit being comprised of a nucleic acid base, a morpholine ring, and a non-ionic phosphorodiamidate intersubunit linkage (GeneTools, Philomath, OR). In some experiments, a morpholino oligonucleotide was conjugated to fluorescine reporter (MKl-fluor or MKl-inverse-fluor) or as a Vivo-Morpholino covalently linked to an octa-guanidne dendrimer delivery moiety (Vivo-MKl, Vivo-MKl-inverse).

Transfection of the Morpholino. Cells were transfected with morpholinos by way of the GeneTools Endo-Porter peptide. In brief, fresh culture medium was added to the cells and the desired concentration of morpholino was added and gently swirled to mix. Next 6 μΜ of the Endo-Poreter peptide reagent was added to each well and gently swirled to mix.

Western blot analysis. Cells were treated with doxycycline to induce expression of DUX4.

Induced cells were then treated with morpholino oligonucleotide at varying concentrations in the presence of 6μΜ Endo-porter (GeneTools, Philomath, OR). Forty-eight hours post-treatment, protein extracts were generated by lysing cells in IX lysis buffer/SDS loading dye (300mM Tris pH 6.8, 25% glycerol, 20% β-mercaptoethanol, 10% SDS, 0.02% bromophenol blue) at room temperature then boiled for 10 minutes at 99° C. Equal volumes of protein extract were electrophoresed through a 10% Tris-HCl polyacrylamide-SDS separation gel having a 4 % polyacrylamide stacking gel for one hour at 150 volts. The gel was transferred onto PVDF membrane using a Bio-Rad wet transfer system for one hour at 200 mAmps. The membrane was washed three times in IX tris buffered saline (TBS; 1M Tris, pH7, 5M sodium chloride). The membrane was then washed three times in TBST (TBS plus 0.05% Tween 20). The membrane was then blocked for one hour with Western Blocking Reagent (Roche Applied Science, Indianapolis, IN). The membrane was then washed again three times with IX TBS, then three times with IX TBST. The membrane was incubated overnight at 4° C in a suitable antibody. The membrane was then washed three times with IX TBS and a further three times with IX TBST. The membrane was next incubated for one hour at 4° C with a suitable secondary antibody. The signal was visualized using the Chemiluminescent Substrate Detection Kit (Thermo Fisher Scientific Inc., Rockford, Illinois). Protein expression levels were quantified using ImageJ software and normalized to GAPDH expression levels.

In all experiments, primary antibodies were diluted in Solution 1 of the SignalBoost

Immunoreaction Enhancer Kit (Calbiochem EMD4Biosciences). Secondary antibodies were diluted in Solution 2 of the SignalBoost™ Immunoreaction Enhancer Kit. Suitable antibodies can be obtained from polyclonal sera, monospecific sera or from monoclonal antibody culture. Techniques for producing and processing monoclonal and polyclonal sera are abundantly known in the art (e.g. Reinherz et al. (1979) J. Immunol. 123, 1312 , Ritz et al. Nature (1980) 283, 583, and Mayer and Walter, eds. Immunochemical Methods in Cell and Molecular Biology, Academic Press, London, 1987). Animals suitable for raising the antibodies are e.g. cows, rabbits, mice, goats, donkeys, or chickens.

More specifically, in the present invention a rabbit monoclonal antibody recognizing DUX4 (E5-5, diluted 1 in 100, Biomol) was utilized and produced a 45 kDa band (+/- 5 kDa). A murine monoclonal antibody recognizing DUX4 (9A12, diluted 1 in 100, Dixit et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 18157-18162) produced a 50 kDa band (+/- 5 kDa). A mouse monoclonal antibody recognizing Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) conjugated to peroxidase (G9295, diluted 1 in 1000, Sigma- Aldrich) produced a 37 kDa band (+/- 5 kDa). A peroxidase conjugated anti-rabbit IgG or a peroxidase conjugated anti-mouse IgG (whole molecule, SantaCruz, Gibbstwon, NJ) was also used.

Cell Survival/ ATP Assays. Cells were cultured as described above. DUX 4 expression was induced with doxycycline, prior to morpholino treatment, as described above. Cells were treated with morpholino. After 48 hours, cell viability was determined by quantifying the presence of ATP in each culture via CellTiter-Glo (Promega), as per the manufacturer's protocol.

Results Molecular design of a morpholino effective in disrupting DUX4 translation. The polymorphic D4Z4 repeat region is located on chromosome 4q (Figure 1 A). As discussed previously, contraction and/or hypo-methylation of the D4Z4 repeat regions can be associated with the FSHD disease phenotype. To study the role of FSHD candidate gene D4Z4, and to investigate the effects of a morpholino oligonucleotide on DUX4 expression in a model of FSHD, a morpholino-based oligonucleotide was designed to target a sequence within the DUX4 5'UTR of the RNA message directly upstream of the translational start site (Figures 1 A-C). One morpholino effective in knocking down gene expression was constructed of 25 morpholine ring subunits coupled via non-ionic phosphorodiamidate linkage (Figure 1C). Interaction with a sequence-specific RNA target can occur via a RNA-Induced Silencing Complex (RISC) and RNAse H-independent steric blocking mechanism. Rather than causing RNA degradation, a stable morpholino: RNA duplex is formed. An example of an effective DUX4 targeting morpholino, is a morpholino oligonucleotide that is targeted directly upstream of the DUX4 translational start site, thereby disrupting ribosome binding and reducing DUX4 translation initiation.

Successful morpholino-mediated inhibition of DUX4 expression. Cell lines in which expression of the DUX4-encoding D4Z4 transcript could be regulated by doxycycline were created as previously discussed. The ability of the MK1 morpholino to effectively block DUX4 translation was tested in the various cell lines. Inducible murine embryonic stem cells containing the 5'UTR DUX4 sequence (mES 5'UTR DUX4) were induced with 100 ng/ml or 50 ng/ml doxycycline, or were not subjected to induction at all. Cells were then transduced with the MK1 morpholino oligonucleotide or the inverse morpholino oligonucleotide at a concentration of 5 μΜ or 10 μΜ, or were left untreated. As previously described, the transduction was performed with the Endo-porter transfection system. Forty-eight hours following morpholino oligonucleotide treatment, cells were lysed and DUX4 expression levels were determined by western blot (Figure 2). Expression levels were normalized to GAPDH and quantified as a percentage of total expression determined by mES 5'UTR DUX4 cells treated with 100 ng/ml doxycycline alone.

Effective inhibition of DUX4 expression by MK1 was observed in a dose dependent manner in all cell populations induced with doxycycline and subsequently transfected with the MK1 morpholino oligonucleotide. After normalization, cells induced with 100 ng/ml doxycycline alone (hereinafter "100 dox +/MK1 -") represented 100% DUX4 expression (see Figure 2). Cells induced with 100 ng/ml doxycycline and treated with 5 μΜ MK1 morpholino oligonucleotide (hereinafter "MK1") exhibited a reduction of DUX4 expression by almost half, since DUX4 expression was approximately 57% the DUX4 expression in comparison to 100 dox +/MK1 - treated cells. When cells were induced with 100 ng/ml doxycycline and treated with 10 μΜ MK1 morpholino oligonucleotide, DUX4 expression was dramatically reduced to about 17% DUX4 expression in comparison to 100 dox +/MK1 - treated cells. The effect of MK1 in cells induced with lower levels of doxycycline was also investigated. Cells were induced with 50 ng/ml of doxycycline without exposure to MK1 and DUX4 expression was 31% of the DUX4 expression in 100 dox +/MK1 - treated cells. Cells that were induced with 50 ng/ml of doxycycline and treated with 5 μΜ MK1 produced 13% the amount of DUX4 produced in 100 dox +/MK1 - treated cells. Cells that were induced with 50 ng/ml of doxycycline and treated with 10 μΜ MK1 produced only 6% the amount of DUX4 produced in 100 dox +/MK1 - treated cells. These results demonstrate the effectiveness of MK1 to successfully disrupt DUX4 translation and expression.

Various Notes & Embodiments

Embodiment 1 includes subject matter (such as a morpholino oligonucleotide) capable of inhibiting expression of a facioscapulohumeral muscular dystrophy candidate gene located within the highly polymorphic D4Z4 repeat region of chromosome 4q.

In Embodiment 2, the subject matter of Embodiment 1 can optionally include a morpholino oligonucleotide capable of inhibiting DUX4 expression.

In Embodiment 3, the subject matter of one or any combination of Embodiments 1 - 2 can optionally include a dendrimer delivery moiety.

In Embodiment 4, the subject matter of one or any combination of Embodiments 1 - 3 can optionally include an octa-guanidine dendrimer delivery moiety.

In Embodiment 5, the subject matter of one or any combination of Embodiments 1 - 4 can optionally include a covalently linked morpholino oligonucleotide and octa-guanidine dendrimer delivery moiety.

In Embodiment 6, the subject matter of one or any combination of Embodiments 1 - 5 can optionally include a morpholino oligonucleotide capable of binding to a sequence within the 5' UTR of DUX4.

In Embodiment 7, the subject matter of one or any combination of Embodiments 1 - 6 can optionally include a morpholino oligonucleotide capable of interfering with pre-mRNA processing.

In Embodiment 8, the subject matter of one or any combination of Embodiments 1 - 7 can optionally include a morpholino oligonucleotide that is capable of inducing a translational frameshift.

In Embodiment 9, the subject matter of one or any combination of Embodiments 1 - 8 can optionally include a phosphorodiamidate backbone.

In Embodiment 10, the subject matter of one or any combination of Embodiments 1 - 9 can optionally include a morpholino oligonucleotide that is effective in lowering the toxicity of the facioscapulohumeral muscular dystrophy candidate gene DUX4.

Embodiment 11 can include, or can optionally be combined with the subject matter of one or any combination of Embodiments 1-10 to include, subject matter (such as of treating

facioscapulohumeral muscular dystrophy) comprising administering a morpholino oligonucleotide to a human in need thereof; wherein the morpholino oligonucleotide is administered in a dose effective to inhibit expression of a facioscapulohumeral muscular dystrophy candidate gene located within the highly polymorphic D4Z4 repeat region of chromosome 4q.

In Embodiment 12, the method of Embodiment 11 can optionally further comprise the administration of a morpholino oligonucleotide in a dose effective to inhibit expression of DUX4.

In Embodiment 13, the method of one or any combination of Embodiments 11 - 12 can optionally further comprise the administration of a morpholino oligonucleotide capable of inhibiting DUX4 expression by binding to a sequence within the 5' UTR of DUX4.

In Embodiment 14, the method of one or any combination of Embodiments 11 - 13 can optionally further comprise the step of intraperitoneal injection.

In Embodiment 15, the method of one or any combination of Embodiments 11 - 14 can optionally further comprise the step of intravenously injecting the morpholino oligonucleotide.

In Embodiment 16, the method of one or any combination of Embodiments 11 - 15 can optionally further comprise the administration of a dendrimer delivery moiety.

In Embodiment 17, the method of one or any combination of Embodiments 11 - 16 can optionally further comprise the administration of an octa-guanidine dendrimer delivery moiety.

In Embodiment 18, the method of one or any combination of Embodiments 11 - 17 can optionally further comprise the administration of a morpholino oligonucleotide capable of inhibiting translation of DUX4.

In Embodiment 19, the method of one or any combination of Embodiments 11 - 18 can optionally further comprise the administration of a morpholino oligonucleotide capable of inhibiting nuclear processing of DUX4 mRNA.

In Embodiment 20, the method of one or any combination of Embodiments 11 - 19 can optionally further comprise the administration of a morpholino oligonucleotide effective in lowering the toxicity of the facioscapulohumeral muscular dystrophy candidate gene DUX4.

These non-limiting embodiments can be combined in any permutation or combination.

The above detailed description includes references to the accompanying drawings, which form a part of the detailed description. The drawings show, by way of illustration, specific embodiments in which the invention can be practiced. Such embodiments can include elements in addition to those shown or described. However, the present inventors also contemplate embodiments in which only those elements shown or described are provided. Moreover, the present inventors also contemplate embodiments using any combination or permutation of those elements shown or described (or one or more aspects thereof), either with respect to a particular embodiment (or one or more aspects thereof), or with respect to other embodiments (or one or more aspects thereof) shown or described herein. In the event of inconsistent usages between this document or any documents so incorporated by reference, the usage in this document controls.

In this document, the terms "a" or "an" are used, as is common in patent documents, to include one or more than one, independent of any other instances or usages of "at least one" or "one or more." In this document, the term "or" is used to refer to a nonexclusive or, such that "A or B" includes "A but not B," "B but not A," and "A and B," unless otherwise indicated. In this document, the terms "including" and "in which" are used as the plain-English equivalents of the respective terms "comprising" and "wherein." Also, in the following claims, the terms "including" and "comprising" are open-ended, that is, a system, device, article, or process that includes elements in addition to those listed after such a term in a claim are still deemed to fall within the scope of that claim. Moreover, in the following claims, the terms "first," "second," and "third," etc. are used merely as labels, and are not intended to impose numerical requirements on their objects.

The above description is intended to be illustrative, and not restrictive. For example, the above-described embodiments (or one or more aspects thereof) may be used in combination with each other. Other embodiments can be used, such as by one of ordinary skill in the art upon reviewing the above description. The Abstract is provided to comply with 37 C.F.R. § 1.72(b), to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. Also, in the above Detailed Description, various features may be grouped together to streamline the disclosure. This should not be interpreted as intending that an unclaimed disclosed feature is essential to any claim. Rather, inventive subject matter may lie in less than all features of a particular disclosed embodiment. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separate embodiment, and it is contemplated that such embodiments can be combined with each other in various combinations or permutations. The scope of the invention should be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled.

All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.

Claims

WHAT IS CLAIMED IS:

1. An antisense oligonucleotide of 8-40 morpholino subunits linked by phosphorous-containing intersubunit linkages which join a morpholino nitrogen of one subunit to a 5' exocyclic carbon of an adjacent subunit, and comprising a sequence which forms a heteroduplex with an AUG start-site region of DUX4; wherein the antisense oligonucleotide inhibits production of DUX4 mRNA or protein.

2. An antisense oligonucleotide comprising morpholino subunits and phosphorous-containing intersubunit linkages comprising a sequence of at least 8 contiguous base-pairing moieties complementary to SEQ ID NO:3, wherein the antisense oligonucleotide is capable of binding to mRNA to form a heteroduplex structure.

3. The antisense oligonucleotide of claim 1 or 2,wherein the heteroduplex structure has a T_m of dissociation of at least 45° C.

4. The antisense oligonucleotide of claim 3, wherein the morpholino subunits are joined by phosphorodiamidate backbone linkages.

5. The antisense oligonucleotide of claim 1 or 2, wherein the antisense oligonucleotide contains between 14-24 base-pairing moieties.

6. The antisense oligonucleotide of claim 1 or 2, wherein the antisense oligonucleotide comprises a targeting sequence of 25 contiguous base-pairing moieties complementary to SEQ ID NO:3.

7. The antisense oligonucleotide of claim 1 or 2, wherein the antisense oligonucleotide comprises SEQ ID NO: 1.

8. A composition comprising the antisense oligonucleotide of claim 1 and a pharmaceutically acceptable carrier.

9. A method of inhibiting expression of DUX4 comprising administering to a subject in need thereof an effective amount of the antisense oligonucleotide of claim 1.

10. The method according to claim 9, where the antisense oligonucleotide is administered in a manner effective to reduce or eliminate a detectable symptom of facioscapulohumeral muscular dystrophy (FSHD) in the subject.

11. A method to treat facioscapulohumeral muscular dystrophy comprising administering to a subject in need thereof an effective amount of the antisense oligonucleotide of claim 1.

12. A method of treating facioscapulohumeral muscular dystrophy comprising:

administering a morpholino oligonucleotide to a subject in need thereof;

wherein the morpholino oligonucleotide is administered in a dose effective to inhibit expression of a facioscapulohumeral muscular dystrophy candidate gene located within the polymorphic D4Z4 repeat region of chromosome 4q.

13. The method of claim 12, further comprising the administration of a morpholino

oligonucleotide in a dose effective to inhibit expression of DUX4.

14. The method of claim 13, further comprising the administration of a morpholino

oligonucleotide capable of inhibiting DUX4 expression by binding to a sequence within the 5' UTR of DUX4.

15. The method of any one of claims 9-14, wherein the administration is by intraperitoneal or intravenous injection.

16. The method of any one of claims 9-14, further comprising the administration of a dendrimer delivery moiety.

18. The method of claim 16, wherein the dendrimer delivery moiety is an octa-guanidine dendrimer delivery moiety.

19. The method of claim 13, wherein the translation of DUX4 is inhibited.

20. The method of claim 13, wherein the nuclear processing of DUX4 RNA is inhibited.

21. A method of treating facioscapulohumeral muscular dystrophy in a subject, comprising administering to a subject an effective amount of an antisense oligonucleotide of 8-40 morpholino subunits linked by phosphorous-containing intersubunit linkages which join a morpholino nitrogen of one subunit to a 5' exocyclic carbon of an adjacent subunit, and comprising a sequence which forms a heteroduplex with a AUG start-site region of DUX4; wherein the antisense oligonucleotide inhibits production of DUX4 mRNA or protein.