US20120225034A1 - Agents useful in treating facioscapulohumeral muscular dystrophy - Google Patents

Agents useful in treating facioscapulohumeral muscular dystrophy Download PDF

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US20120225034A1
US20120225034A1 US13/225,384 US201113225384A US2012225034A1 US 20120225034 A1 US20120225034 A1 US 20120225034A1 US 201113225384 A US201113225384 A US 201113225384A US 2012225034 A1 US2012225034 A1 US 2012225034A1
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dux4
dux4c
nucleic acid
bases
rnai
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Alexandra Belayew
Frédérique Coppée
Céline Vanderplanck
Stephen Donald Wilton
Eugénie Ansseau
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Universite de Mons
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Universite de Mons
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Assigned to UNIVERSITE DE MONS reassignment UNIVERSITE DE MONS ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ANSSEAU, EUGENIE, BELAYEW, ALEXANDRA, COPPEE, FREDERIQUE, VANDERPLANCK, CELINE, WILTON, STEPHEN DONALD
Publication of US20120225034A1 publication Critical patent/US20120225034A1/en
Priority to US14/078,133 priority Critical patent/US20140105873A1/en
Priority to US15/047,258 priority patent/US9988628B2/en
Priority to US15/873,751 priority patent/US20180265870A1/en
Priority to US16/562,030 priority patent/US10907157B2/en
Priority to US17/153,215 priority patent/US11898143B2/en
Priority to US17/153,067 priority patent/US20210163941A1/en
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Definitions

  • the invention generally relates to diseases and conditions the treatment of which can benefit from reducing the expression of double homeobox 4 and/or double homeobox 4c.
  • diseases and conditions include inter alia those comprising increased levels and/or increased activity of double homeobox 4 and/or double homeobox 4c, and more particularly include facioscapulohumeral muscular dystrophy.
  • diseases and conditions also include those comprising expression of a fusion protein between DUX4 or DUX4c and another, unrelated protein, more particularly wherein the disease or condition is a tumour, even more particularly a sarcoma such as Ewing's family tumours, paediatric undifferentiated soft tissue sarcomas and rhabdomyosarcomas.
  • the invention concerns agents, more specifically antisense agents and RNA interference agents, capable of reducing or abolishing the expression of double homeobox 4 and/or double homeobox 4c, and elaborates methods, uses and further aspects employing such agents.
  • Facioscapulohumeral muscular dystrophy also known as Landouzy-Dejerine muscular dystrophy is an autosomal dominant muscle disorder affecting about 1/20,000 births. It is characterised by progressive weakness and atrophy of the muscles from the face, the upper-arms and shoulder girdle to the lower limbs.
  • FSHD is genetically linked to contractions of the D4Z4 repeat array on the 4q35 subtelomeric region.
  • Non-affected individuals typically have between 11-100 copies of the 3.3-kb D4Z4 element, while FSHD patients only have 1-10 copies.
  • a typical feature associated with the genetic defect is a decrease in DNA methylation of the contracted D4Z4 array as compared to non-affected individuals (van Overveld et al. 2005 (Ann Neurol. 58: 569-76.)).
  • a small group of patients with a typical FSHD phenotype does present more than 10 copies of the D4Z4 element, their DNA methylation level is low, similarly to that found in contracted D4Z4 arrays.
  • DNA hypomethylation is typically associated with an open chromatin structure suitable for transcription (de Greer et al. 2009 (Hum Mutat. 30: 1449-59)).
  • Gabri ⁇ ls et al. 1999 (Gene 236(1): 25-32) identified the double homeobox 4 (DUX4) gene within each D4Z4 element repeated in the array.
  • the DUX4 sequence was later corrected as published by Kowaljow et al. 2007 (Neuromuscul Disord 17: 611-23) and is available under the NCBI Genbank accession number: AF117653.2.
  • DUX4 protein was expressed in primary myoblasts and biopsies of patients with FSHD but not in non-affected individuals, and that the DUX4 protein is a transcription factor targeting a large set of genes including inter cilia genes encoding further transcription factors, and that DUX4 gene activation at the FSHD locus initiates a transcription cascade leading to muscle atrophy, inflammation, decreased differentiation potential and oxidative stress, recapitulating the key features of FSHD (Bosnakowski et al. 2008 (EMBO J. 27(20): 2766-79); Kowabow et al. 2007 (Neuromuscul Disord 17: 611-23); Dixit et al. 2007 (Prot Natl Acad Sci USA 104: 18157-18162)). Double homeobox 4 is thus considered a major contributor to the pathology of FSHD muscles.
  • Dixit et al. 2007 also demonstrated in myoblast cultures that whereas transcription can initiate at any D4Z4 element within the repeat array, a prevalent stable DUX4 mRNA originates from the most distal D4Z4 unit and extends into the pLAM region which flanks the telomeric side of the D4Z4 array, whereby the pLAM region provides the DUX4 transcript with an intron and a polyadenylation signal ( FIGS. 1 and 2 ).
  • additional transcripts were identified that span several D4Z4 units, may have various parts spliced out, and may also comprise the pLAM region (Snider et al. 2009. Hum Mol Genet.
  • the homologous DUX4c gene was identified 42 kb centromeric of the D4Z4 array, within a truncated and inverted solitary D4Z4 unit.
  • the DUX4c gene encodes a 47-kDa protein with a double homeodomain identical to DUX4 but divergent in the carboxyl-terminal region.
  • the DUX4c protein is expressed at low levels in control muscles, it is induced in muscles of patients affected with Duchenne muscular dystrophy and is present at similar or yet higher levels in FSHD muscles. Additional experiments suggested that DUX4c could be involved in myoblast proliferation during muscle regeneration and that changes in its expression could contribute to the FSHD pathology (Ansseau et al. 2009 (PLoS One 4(10): e7482).
  • a fusion gene is seen that includes the 3′ region of the DUX4 gene as a result of chromosome rearrangements. Fusion between CIC, a human homolog of Drosophila capicua, and DUX4 was seen in Ewing's family tumours (EFTs) (Kawamura-Saito et al. 2006. Hum Mol Genet. 15: 2125-2137) and paediatric undifferentiated soft tissue sarcomas (USTS) (Yoshimoto et al. 2009. Cancer Genet Cytogenet 195: 1-11), and rhabdomyosarcomas (RMS) showed fusion between the EWSR1 gene and DUX4. (Sirvent et al. 2009. Cancer Genet Cytogenet 195: 12-08). As a consequence of fusion with the C-terminal fragment of DUX4 the resulting fusion proteins acquire an enhanced transcriptional activity, which leads to tumour formation.
  • EFTs Ewing's family tumours
  • antisense agents targeting sequence elements involved in splicing of DUX4 or DUX4c transcripts can reduce or abolish the production of the respective proteins.
  • This finding is unexpected, since antisense agents targeting sequence elements required for splicing were previously contemplated for therapeutic exon skipping to at least partly restore the functionality of defective proteins, such as for example to remove nonsense mutations or restore the reading frame disrupted by genomic deletions or duplications in the dystrophin gene in Duchenne muscular dystrophy (DMD) (Wilton et al, 2007 (Mol Ther 15(7): 1288-96); Adams et al. 2007 (BMC Mol Biol 8: 57)).
  • DMD Duchenne muscular dystrophy
  • the introns of the DUX4 transcript are located in its 3′ untranslated region (3′ UTR), which is unusual, and interference with splicing would therefore not be expected to alter the DUX4 coding sequence or the production of the DUX4 protein.
  • antisense agents targeting sequence elements involved in polyadenylation of DUX4 or DUX4c transcripts can reduce or abolish the production of the respective proteins.
  • the invention thus generally provides an antisense agent capable of reducing or abolishing the production of DUX4 or DUX4c proteins.
  • An antisense agent as intended herein may be capable of binding to (annealing with) DUX4 or DUX4c genes.
  • such antisense agent may be capable of binding to (annealing with) a sequence region in DUX4 or DUX4c (pre-mRNA) sequence.
  • Double homeobox 4 emerges as particularly implicated in the aetiology of facioscapulohumeral muscular dystrophy (FSHD).
  • FSHD facioscapulohumeral muscular dystrophy
  • an antisense agent capable of reducing or abolishing the production of DUX4 protein an antisense agent capable of binding to DUX4 gene; an antisense agent capable of binding to a sequence region in DUX4 (pre-mRNA) sequence.
  • an antisense agent capable of reducing or abolishing the production of DUX4 protein but not of DUX4c protein
  • an antisense agent capable of binding to DUX4 gene but not to DUX4c gene an antisense agent capable of binding to a sequence region in DUX4 (pre-mRNA) sequence but not to a sequence region in DUX4c (pre-mRNA) sequence.
  • an antisense agent capable of reducing or abolishing the production of DUX4c protein but not of DUX4 protein
  • an antisense agent capable of binding to a sequence region in DUX4c (pre-mRNA) sequence but not to a sequence region in DUX4 (pre-mRNA) sequence are: an antisense agent capable of reducing or abolishing the production of DUX4c protein but not of DUX4 protein; an antisense agent capable of binding to DUX4c gene but not to DUX4 gene; an antisense agent capable of binding to a sequence region in DUX4c (pre-mRNA) sequence but not to a sequence region in DUX4 (pre-mRNA) sequence.
  • the invention provides an antisense agent capable of binding to a sequence element required for splicing of the double homeobox 4 (DUX4) or double homeobox 4c (DUX4c) genes (as explained elsewhere in this specification, a mention of splicing or splicing of a gene generally refers to splicing of a gene's pre-mRNA to remove intervening sequence(s)).
  • the antisense agent can reduce or abolish the production of the respective DUX4 or DUX4c proteins.
  • such antisense agents might interfere with splicing of the DUX4 or DUX4c genes (pre-mRNA) or might act through another mechanism.
  • Double homeobox 4 emerges as particularly implicated in the aetiology of facioscapulohumeral muscular dystrophy (FSHD).
  • FSHD facioscapulohumeral muscular dystrophy
  • an antisense agent capable of binding to a sequence element required for splicing of the double homeobox 4 (DUX4) gene.
  • the antisense agent can reduce or abolish the production of DUX4 protein.
  • an antisense agent capable of binding to a sequence element required for splicing of the DUX4 gene but not of the DUX4c gene.
  • the antisense agent can reduce or abolish the production of DUX4 protein but does not reduce or abolish the production of DUX4c protein.
  • an antisense agent capable of binding to a sequence element required for splicing of the DUX4c gene but not of the DUX4 gene. The antisense agent can reduce or abolish the production of DUX4c protein but does not reduce or abolish the production of DUX4 protein.
  • Sequence elements required for splicing of the DUX4 or DUX4c genes as intended herein particularly denote cis sequence elements, i.e., those located within said DUX4 or DUX4c genes, respectively.
  • Sequence elements to be targeted by (i.e., selected to be bound by) antisense agents as disclosed herein may be preferably chosen from the group comprising or consisting of splice donor sites (i.e., 5′ splice sites), splice acceptor sites (i.e., 3′ splice sites), pyrimidine-rich or polypyrimidine tracts upstream of (i.e., 5′ relative to) splice acceptor sites, exon-intron boundaries, intron-exon boundaries, branch sites and exonic splicing enhancer elements of the DUX4 or DUX4c genes.
  • splice donor sites i.e., 5′ splice sites
  • splice acceptor sites i.e., 3′ splice sites
  • Splice donor sites and splice acceptor sites, exon-intron boundaries and intron-exon boundaries may be readily accessible for targeting and may thus constitute preferred sequence elements as intended herein.
  • particularly effective antisense agents as disclosed herein include those capable of binding to splice acceptor sites or intron-exon boundaries of the DUX4 or DUX4c genes.
  • Antisense agents as disclosed herein may preferably bind to a whole sequence element required for splicing DUX4 or DUX4c (i.e., may wholly overlap with or wholly anneal to such sequence element). Alternatively, antisense agents as disclosed herein may bind to one or more portions of a sequence element required for splicing DUX4 or DUX4c (e.g., may partly overlap with or partly anneal to such sequence element).
  • binding to a sequence element required for splicing also encompasses antisense agents that bind at a position sufficiently close to said element.
  • the antisense agents may bind at a position sufficiently close to said element to disrupt the binding and function of splicing machinery that would normally mediate a particular splicing reaction occurring at that element (e.g., such agents may bind to pre-mRNA at a position within about 3, about 6, or about 9 bases of said element).
  • the invention provides an antisense agent capable of binding to a sequence element required for polyadenylation of DUX4 or DUX4c genes (as explained elsewhere in this specification, a mention of polyadenylation or polyadenylation of a gene generally refers to polyadenylation of a gene's pre-mRNA).
  • the antisense agent can reduce or abolish the production of the DUX4 or DUX4c proteins.
  • such antisense agents might interfere with polyadenylation of the DUX4 or DUX4c genes (pre-mRNA) or might act through another mechanism.
  • Double homeobox 4 emerges as particularly implicated in the aetiology of FSHD.
  • an antisense agent capable of binding to a sequence element required for polyadenylation of the DUX4 gene.
  • the antisense agent can reduce or abolish the production of DUX4 protein.
  • an antisense agent capable of binding to a sequence element required for polyadenylation of the DUX4 gene but not of the DUX4c gene.
  • the antisense agent can reduce or abolish the production of DUX4 protein but does not reduce or abolish the production of DUX4c protein.
  • an antisense agent capable of binding to a sequence element required for polyadenylation of the DUX4c gene but not of the DUX4 gene.
  • the antisense agent can reduce or abolish the production of DUX4c protein but does not reduce or abolish the production of DUX4 protein.
  • an antisense agent capable of binding to a sequence element required for polyadenylation of the DUX4 gene but not capable of binding to the DUX4c gene.
  • the antisense agent can reduce or abolish the production of DUX4 protein but does not reduce or abolish the production of DUX4c protein.
  • the antisense agent can reduce or abolish the production of DUX4c protein but does not reduce or abolish the production of DUX4 protein.
  • Sequence elements required for polyadenylation of the DUX4 or DUX4c genes as intended herein particularly denote cis sequence elements, i.e., those located within said DUX4 or DUX4c genes, respectively.
  • Sequence elements to be targeted by (i.e., selected to be bound by) antisense agents capable of binding to a sequence element required for polyadenylation of the DUX4 or DUX4c genes may be preferably polyadenylation signals (such as more preferably the polyadenylation signal ATTAAA) of the DUX4 or DUX4c genes.
  • Antisense agents capable of binding to a sequence element required for polyadenylation of the DUX4 or DUX4c genes may preferably bind to a whole sequence element required for polyadenylation of DUX4 or DUX4c (i.e., may wholly overlap with or wholly anneal to such sequence element).
  • antisense agents capable of binding to a sequence element required for polyadenylation of the DUX4 or DUX4c genes may bind to one or more portions of a sequence element required for polyadenylation of DUX4 or DUX4c (e.g., may partly overlap with or partly anneal to such sequence element).
  • binding to a sequence element required for polyadenylation also encompasses antisense agents that bind at a position sufficiently close to said element (e.g., such agents may bind to pre-mRNA at a position within about 3, about 6, or about 9 bases of said element).
  • Antisense agents as intended herein preferably comprise or denote antisense molecules such as more preferably antisense nucleic acid molecules or antisense nucleic acid analogue molecules.
  • antisense agents may refer to antisense oligonucleotides or antisense oligonucleotide analogues.
  • such antisense agents or molecules may be between about 10 and about 100 nucleotides or nucleotide analogues in length, preferably between about 12 and about 80 nucleotides or nucleotide analogues in length, also preferably between about 15 and about 50 nucleotides or nucleotide analogues in length, more preferably between about 20 and about 40 (such as, e.g., between about 20 and about 30) nucleotides or nucleotide analogues in length.
  • antisense agents including antisense nucleic acid analogue molecules, such as, e.g., antisense oligonucleotide analogues, more preferably antisense oligonucleotide analogues comprising a 2′-O-methylated phosphorothioate backbone or more preferably antisense oligonucleotide analogues comprising a phosphorodiamidate morpholino backbone as schematically illustrated in FIGS. 24 and 25 , respectively.
  • antisense nucleic acid analogue molecules such as, e.g., antisense oligonucleotide analogues, more preferably antisense oligonucleotide analogues comprising a 2′-O-methylated phosphorothioate backbone or more preferably antisense oligonucleotide analogues comprising a phosphorodiamidate morpholino backbone as schematically illustrated in FIGS. 24 and 25 ,
  • an antisense agent as disclosed herein may be conjugated to a cell penetrating peptide (CPP) to enhance the cellular uptake of said antisense agents.
  • CPP cell penetrating peptide
  • such antisense agents or molecules may be configured to bind to (anneal with) a sequence region, more particularly a region in DUX4 or DUX4c (pre-mRNA) sequence, wherein said region is at least about 10 nucleotides in length, preferably at least about 12 nucleotides in length, also preferably at least about 15 nucleotides in length, more preferably at least about 20 nucleotides in length, even more preferably at least about 25 or at least about 30 nucleotides in length, such as for example between about 10 and about 100 nucleotides in length, preferably between about 12 and about 80 nucleotides in length, also preferably between about 15 and about 50 nucleotides in length, and more preferably between about 20 and about 40 (such as, e.g., between about 20 and about 30) nucleotides in length, wherein the reference to nucleotides may preferably denote consecutive nucleotides.
  • a DUX4 gene preferably intended for targeting by the antisense agents as disclosed herein resides in the distal-most D4Z4 unit which extends into the pLAM region flanking the telomeric side of the D4Z4 array.
  • Such DUX4 gene leads to production of comparably stable mRNA(s) (Dixit et al. 2007, supra).
  • DUX4 gene comprises two introns which are located in its 3′ UTR, namely intron 1 (or intron A) within the D4Z4 unit and intron 2 (or intron B) provided by the pLAM region.
  • Such DUX4 gene further comprises a polyadenylation signal ATTAAA located within the pLAM region.
  • a DUX4 gene as intended herein may also denote a DUX4 transcription units that span several D4Z4 units, may display alternative splicing, and may comprise the pLAM region, in particular as disclosed by Snider et al. 2009, supra.
  • such DUX4 transcripts may comprise intron 1, intron 1bis or intron 2a within the D4Z4 unit, intron 2 provided by the pLAM region or intron 2a bis provided by the D4Z4 unit and the pLAM region.
  • Such DUX4 transcript further comprises a polyadenylation signal ATTAAA located within the pLAM region.
  • sequence elements to be targeted by anti-DUX4 antisense agents as disclosed herein particularly sequence elements required for splicing of the DUX4 gene to be targeted by anti-DUX4 antisense agents capable of binding to such elements, preferably include those sequence elements (such as, e.g., splice donor sites, splice acceptor sites, exon-intron boundaries, intron-exon boundaries, pyrimidine-rich or polypyrimidine tracts, branch sites and/or exonic splicing enhancer elements) required for removal of said DUX4 intron 1, intron 1bis, intron 2, intron 2a or intron 2a his (preferably of intron 1 or 2) upon splicing of a DUX4 gene.
  • sequence elements such as, e.g., splice donor sites, splice acceptor sites, exon-intron boundaries, intron-exon boundaries, pyrimidine-rich or polypyrimidine tracts, branch sites and/or exonic splicing enhancer elements
  • the targeted DUX4 sequence elements may be chosen from the group comprising or consisting of splice donor sites of said DUX4 intron 1, intron 1 bis, intron 2, intron 2a or intron 2a bis(preferably of intron 1 or 2) and splice acceptor sites of said DUX4 intron 1, intron 1 bis, intron 2, intron 2a or intron 2a bis (preferably of intron 1 or 2); more preferably from the group comprising or consisting of splice acceptor sites of said DUX4 intron 1, intron 1 bis, intron 2, intron 2a or intron 2a his (preferably of intron 1 or 2); even more preferably may be the splice acceptor site of said DUX4 intron 2.
  • Sequence elements to be targeted by anti-DUX4 antisense agents as disclosed herein particularly sequence elements required for polyadenylation of the DUX4 gene and to be targeted by anti-DUX4 antisense agents capable of binding to such elements, preferably include the polyadenylation signal ATTAAA.
  • genomic sequence of the DUX4c gene As shown in an exemplary but non-limiting genomic sequence of the DUX4c gene (Genbank accession no. AY500824, sequence version 1, i.e., AY500824.1), the ORF encoding the DUX4c protein is found at positions 918-2042 of AY500824.1 and is not disrupted by introns.
  • a larger exemplary but non-limiting genomic sequence of the DUX4c gene (Genbank accession no. NC — 000004, sequence version 11, i.e., NC — 000004.11, range 190940254 . . .
  • 190945505 complement indicates that the DUX4c ORF may be included within a larger DUX4c transcript containing six putative exons (denoted as exons 1 to 6) at respectively positions 1-65, 617-741, 966-1160, 1385-2945, 4034-4154 and 4911-5251 of NC — 000004.11 (range 190940254 . . . 190945505, complement) (wherein putative exon 4 contains the DUX4c ORF) and corresponding five putative introns (denoted introns 1 to 5) at positions 66-616, 742-965, 1161-1384, 2946-4033 and 4155-4910 of NC — 000004.11 (range 190940254 . . . 190945505, complement).
  • sequence elements to be targeted by anti-DUX4c antisense agents as disclosed herein particularly sequence elements required for splicing of the DUX4c gene to be targeted by anti-DUX4c antisense agents capable of binding to such elements, preferably include those sequence elements (such as, e.g., splice donor sites, splice acceptor sites, exon-intron boundaries, intron-exon boundaries, pyrimidine-rich or polypyrimidine tracts, branch sites and/or exonic splicing enhancer elements) required for removal of said DUX4c introns, e.g. DUX4c introns 1, 2, 3, 4 or 5 upon splicing of such DUX4c gene.
  • sequence elements such as, e.g., splice donor sites, splice acceptor sites, exon-intron boundaries, intron-exon boundaries, pyrimidine-rich or polypyrimidine tracts, branch sites and/or exonic splicing enhancer elements
  • the targeted DUX4c sequence elements may be chosen from the group comprising or consisting of splice donor sites of said DUX4c introns 1, 2, 3, 4 or 5 and splice acceptor sites of said DUX4c introns 1, 2, 3, 4 or 5: more preferably from the group comprising or consisting of splice acceptor sites of said DUX4c introns 1, 2, 3, 4 or 5.
  • an antisense agent as disclosed herein may be configured to bind to a region in DUX4 or DUX4c sequence corresponding to positions about +30 to about ⁇ 30, preferably about +25 to about ⁇ 25, more preferably about +20 to about ⁇ 20 relative to the respective exon-intron boundary (i.e., position+1 denoting the last base of the preceding exon and position ⁇ 1 denoting the first base of the following intron).
  • an antisense agent may be configured to bind to (anneal with) at least about 10 bases, preferably at least about 15 bases, more preferably at least about 20 bases, even more preferably at least about 25 bases or at least about 30 bases, such as to between about 10 and about 40 bases or to between about 20 and about 30 bases in any one of the above recited regions in DUX4 or DUX4c sequence, wherein said reference to bases may preferably denote consecutive bases.
  • binding (annealing) will involve at least positions ⁇ 1 or ⁇ 2 (more preferably both positions ⁇ 1 and ⁇ 2) and/or positions +1 or +2 (more preferably both positions +1 and +2) relative to the respective exon-intron boundary.
  • binding may involve at least positions +1 to ⁇ 1 or +1 to ⁇ 2 or +2 to ⁇ 1 or +2 to ⁇ 2. These positions denote bases which adjoin the respective exon-intron boundary and which are particularly relevant for splicing.
  • an antisense agent may be configured to bind to any one of the above recited regions in DUX4 or DUX4c sequence such that it anneals over (i.e., spans or crosses) the respective exon-intron boundary and base pairs with at least 1 base, preferably at least 2 bases, more preferably at least 5 bases and even more preferably at least 7 or at least 10 bases on each side of said exon-intron boundary, such as with between 1 and about 20 bases, preferably between 2 and about 15 bases or between 2 and about 10 bases on each side of said exon-intron boundary.
  • an antisense agent as disclosed herein may be configured to bind to a region in DUX4 or DUX4c sequence corresponding to positions about ⁇ 30 to about +30, preferably about ⁇ 25 to about +25, more preferably about ⁇ 20 to about +20 relative to the respective intron-exon boundary (i.e., position ⁇ 1 denoting the last base of the preceding intron and position +1 denoting the first base of the following exon).
  • an antisense agent may be configured to bind to (anneal with) at least about 10 bases, preferably at least about 15 bases, more preferably at least about 20 bases, even more preferably at least about 25 bases or at least about 30 bases, such as to between about 10 and about 40 bases or to between about 20 and about 30 bases in any one of the above recited regions in DUX4 or DUX4c sequence, wherein said reference to bases may preferably denote consecutive bases.
  • binding (annealing) will involve at least positions ⁇ 1 or ⁇ 2 (more preferably both positions ⁇ 1 and ⁇ 2) and/or positions +1 or +2 (more preferably both positions +1 and +2) relative to the respective intron-exon boundary.
  • binding may involve at least positions ⁇ 1 to +1 or ⁇ 1 to +2 or ⁇ 2 to +1 or ⁇ 2 to +2. These positions denote bases which adjoin the respective intron-exon boundary and which are particularly relevant for splicing.
  • an antisense agent may be configured to bind to any one of the above recited regions in DUX4 or DUX4c sequence such that it anneals over (i.e., spans or crosses) the respective intron-exon boundary and base pairs with at least 1 base, preferably at least 2 bases, more preferably at least 5 bases and even more preferably at least 7 or at least 10 bases on each side of said intron-exon boundary, such as with between 1 and about 20 bases, preferably between 2 and about 15 bases or between 2 and about 10 bases on each side of said intron-exon boundary.
  • an anti-DUX4 antisense agent may be configured to bind to (anneal with) at least about 10 bases, preferably at least about 15 bases, more preferably at least about 20 bases, even more preferably at least about 25 bases or at least about 30 bases, such as to between about 10 and about 40 bases or to between about 20 and about 30 bases, preferably wherein said reference to bases denotes consecutive bases, of any one of the following DUX4 sequences (SEQ ID NO: 2 to 9) or of variants thereof having at least about 80% and preferably at least about 90% or at least about 95% sequence identity to the respective sequences:
  • gttgggacggggtcgggtggttcggggggcag (SEQ ID NO: 2; positions +30 to ⁇ 30 of an exemplary DUX4 exon 1-intron 1 boundary; the intron sequence is in italics); gaggagctttaggacgcggg
  • the anti-DUX4 antisense agent is capable of annealing over (i.e., span or cross) the respective exon-intron or intron-exon boundaries found in SEQ ID NO: 2 to 9 or in the variants thereof (indicated by the “
  • the anti-DUX4 antisense agent is capable of annealing with at least one and preferably both of the two intronic bases (indicated above in bold italics) adjacent to the respective exon-intron or intron-exon boundaries and/or (preferably “and”) with at least one and preferably both of the two exonic bases (underlined above) adjacent to the respective exon-intron or intron-exon boundaries.
  • an effective anti-DUX4 antisense agent may be configured to bind to (anneal with) any one of the following DUX4 sequences (SEQ ID NO: 10 to 15, 66) or to variants thereof having at least about 80% and preferably at least about 90% or at least about 95% sequence identity to the respective sequences, or to fragments thereof comprising at least 10 bases, or at least 12 bases, preferably at least about 15 bases, more preferably at least about 20 bases, even more preferably at least about 25 bases, preferably wherein said reference to bases denotes consecutive bases, of the respective sequences or variants.
  • SEQ ID NO: 10 to 15, 66 any one of the following DUX4 sequences (SEQ ID NO: 10 to 15, 66) or to variants thereof having at least about 80% and preferably at least about 90% or at least about 95% sequence identity to the respective sequences, or to fragments thereof comprising at least 10 bases, or at least 12 bases, preferably at least about 15 bases, more preferably at least about 20 bases, even more preferably at least
  • an anti-DUX4 antisense agent may comprise, consist essentially of or consist of a sequence (e.g., a nucleic acid sequence or nucleic acid analogue sequence) complementary to any one of said DUX4 sequences SEQ ID NO: 10 to 15, 66 or to variants thereof having at least about 80% and preferably at least about 90% or at least about 95% sequence identity to the respective sequences, or to fragments thereof comprising at least 10 bases, or at least 12 bases, preferably at least about 15 bases, more preferably at least about 20 bases, even more preferably at least about 25 bases, preferably wherein said reference to bases denotes consecutive bases, of the respective sequences or variants:
  • the intron sequence is in italics); tggctagacctgcgcgcagtgcgca (SEQ ID NO: 11; positions ⁇ 7 to +18 of an exemplary DUX4 intron 2-exon 3 boundary; positions 12678-12702 of AF117653.2; the intron sequence is in italics); cttcctggctagacctgcgcgcagt (SEQ ID NO: 12; positions ⁇ 12 to +13 of an exemplary DUX4 intron 2-exon 3 boundary; positions 12673-12697 of AF117653.2; the intron sequence is in italics); agacctgcgcgcagtgcgcaccccg (SEQ ID NO: 13; positions ⁇ 2 to +23 of an exemplary DUX4 intron 2 exon 3 boundary; positions 12685-12703 of AF117653.2; the intron sequence is in italics); cttcctggctagacctgg
  • acgcggggttgggacggggtcgggt (SEQ ID NO: 66; positions +7 to ⁇ 18 of an exemplary DUX4 exon 1-intron 1 boundary; positions 12105-12129 of AF117653.2; the intron sequence is in italics).
  • anti-DUX4 antisense agents comprising, consisting essentially of or consisting of any one of sequences (e.g., nucleic acid sequences or nucleic acid analogue sequences) SEQ ID NO: 16 to 21, 64 or variants thereof having at least about 80% and preferably at least about 90% or at least about 95% sequence identity to the respective sequences, or fragments thereof comprising at least 10 bases, or at least 12 bases, preferably at least about 15 bases, more preferably at least about 20 bases, even more preferably at least about, 25 bases, preferably wherein said reference to bases denotes consecutive bases, of the respective sequences or variants:
  • the anti-DUX4 antisense agents comprising, consisting essentially of or consisting of any one of sequences SEQ ID NO: 16 to 21, 64 or the variants or fragments thereof display complementarily to, and are hence configured to bind to (anneal with), the above DUX4 sequences SEQ ID NO: 10 to 15, 66 or the variants or fragments thereof.
  • an anti-DUX4c antisense agent may be configured to bind to (anneal with) at least about 10 bases, preferably at least about 15 bases, more preferably at least about 20 bases, even more preferably at least about 25 bases or at least about 30 bases, such as to between about 10 and about 40 bases or to between about 20 and about 30 bases, preferably wherein said reference to bases denotes consecutive bases, of any one of the following DUX4c sequences (SEQ ID NO: 22 to 41) or of variants thereof having at least about 80% and preferably at least about 90% or at least about 95% sequence identity to the respective sequences:
  • the intron sequence is in italics); agcccacagctcttgtca ta
  • the intron sequence is in italics); aagcggtatccgcctccag
  • the intron sequence is in italics); gctccttcgccctctgcaag
  • the intron sequence is in italics); caggaatccgtggtcagg cc
  • the intron sequence is in italics); gggctctgctggaggagc a g
  • the intron sequence is in italics); cgtttattgccctctgcag
  • the anti-DUX4c antisense agent is capable of annealing over (i.e., span or cross) the respective exon-intron or intron-exon boundaries found in SEQ ID NO: 22 to 41 or in the variants thereof (indicated by the “
  • the anti-DUX4c antisense agent is capable of annealing with at least one and preferably both of the two intronic bases (indicated above in bold italics) adjacent to the respective exon-intron or intron-exon boundaries and/or (preferably “and”) with at least one and preferably both of the two exonic bases (underlined above) adjacent to the respective exon-intron or intron-exon boundaries.
  • an antisense agent as disclosed herein may be configured to bind to a region in DUX4 or DUX4c sequence corresponding to positions about ⁇ 30 to about +30, preferably about ⁇ 25 to about +25, more preferably about ⁇ 20 to about +20 relative to said polyadenylation signal (i.e., position ⁇ 1 denoting the last base preceding the polyadenylation signal and position +1 denoting the first base following the polyadenylation signal).
  • such antisense agent may be configured to bind to (anneal with) at least about 10 bases, preferably at least about 15 bases, more preferably at least about 20 bases, even more preferably at least about 25 bases or at least about 30 bases, such as to between about 10 and about 40 bases or to between about 20 and about 30 bases in any one of the above recited regions in the DUX4 or DUX4c sequence, wherein said reference to bases may preferably denote consecutive bases.
  • the antisense agent may be configured to bind such that it anneals with at least a portion of (e.g., ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5 or ⁇ 6 nucleotides) the polyadenylation signal or with the entire polyadenylation signal.
  • a portion of e.g., ⁇ 1, ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5 or ⁇ 6 nucleotides
  • such antisense agent may be configured to anneal over (i.e., to span or cross) the polyadenylation signal and to base pair with at least 1 base, preferably at least 2 bases, more preferably at least 5 bases and even more preferably at least 7 or at least 10 bases on each side of said polyadenylation signal, such as with between 1 and about 20 bases, preferably between 2 and about 15 bases or between 2 and about 10 bases on each side of said polyadenylation signal.
  • an anti-DUX4 antisense agent may be configured to bind to (anneal with) at least about 10 bases, preferably at least about 15 bases, more preferably at least about 20 bases, even more preferably at least about 25 bases or at least about 30 bases, such as to between about 10 and about 40 bases or to between about 20 and about 30 bases, preferably wherein said reference to bases denotes consecutive bases, of the following DUX4 sequences (SEQ ID NO: 67 or 68) or of variants thereof having at least about 80% and preferably at least about 90% or at least about 95% sequence identity to the respective sequences:
  • acatctcctggatgattagttcagagatatattaaaatgcccectccetgtggatcctatagaaga SEQ ID NO: 67; positions ⁇ 30 to +30 of an exemplary DUX4 Polyadenylation signal; the polyadenylation signal is in italics
  • gatgattagttcagagatatattaaaatgccccctcctgtggatc SEQ ID NO: 68; positions ⁇ 20 to +20 of an exemplary DUX4 polyadenylation signal; the polyadenylation signal is in italics);
  • the anti-DUX4 antisense agent may be capable of annealing over (i.e. span or cross) the polyadenylation signal ATTAAA found in SEQ ID NO: 67 or 68 or in the variants thereof.
  • an effective anti-DUX4 antisense agent may be configured to bind to (anneal with) the following DUX4 sequence (SEQ ID NO: 69) or to variants thereof having at least about 80% and preferably at least about 90% or at least about 95% sequence identity to said sequence, or to fragments thereof comprising at least 10 bases, or at least 12 bases, preferably at least about 15 bases, more preferably at least about 20 bases, even more preferably at least about 25 bases, preferably wherein said reference to bases denotes consecutive bases, of said sequence or variants.
  • an anti-DUX4 antisense agent may comprise, consist essentially of or consist of a sequence (e.g., a nucleic acid sequence or nucleic acid analogue sequence) complementary to said DUX4 sequences SEQ ID NO: 69 or to variants thereof having at least about 80% and preferably at least about 90% or at least about 95% sequence identity to said sequence, or to fragments thereof comprising at least 10 bases, or at least 12 bases, preferably at least about 15 bases, more preferably at least about 20 bases, even more preferably at least about 25 bases, preferably wherein said reference to bases denotes consecutive bases, of the respective sequences or variants:
  • agttcagagatatattaactatgccc (SEQ ID NO: 69; positions ⁇ 13 to +6 of an exemplary DUX4 polyadenylation signal; positions 12839-12863 of Genbank sequence AF117653.2 (see FIG. 3 ); the polyadenylation signal is in italics);
  • an anti-DUX4 antisense agents comprising, consisting essentially of or consisting of sequence (e.g., nucleic acid sequences or nucleic acid analogue sequences) SEQ ID NO: 65 or variants thereof having at least about 80% and preferably at least about 90% or at least about 95% sequence identity to said sequence, or fragments thereof comprising at least 10 bases, or at least 12 bases, preferably at least about 15 bases, more preferably at least about 20 bases, even more preferably at least about 25 bases, preferably wherein said reference to bases denotes consecutive bases, of said sequence or variants:
  • the anti-DUX4 antisense agents comprising, consisting essentially of or consisting of any one of sequence SEQ ID NO: 65 or the variants or fragments thereof display complementarily to, and are hence configured to bind to (anneal with), the above DUX4 sequences SEQ ID NO: 69 or the variants or fragments thereof.
  • RNA interference RNA interference
  • the RNAi agent may be configured to target DUX4 and/or DUX4c messenger RNA (mRNA), respectively.
  • the RNAi agent may be configured to target any part of DUX4 and/or DUX4c mRNA, such as for example the 5′-untranslated region (5′ UTR).
  • the RNAi agent may be preferably configured to target the 3′ UTR of DUX4 and/or DUX4c mRNA.
  • the inventors realised that targeting the 3′ UTR of DUX4 and/or DUX4c mRNA allows for particularly effective RNAi-mediated downregulation of the production of DUX4 and/or DUX4c proteins.
  • RNAi agents which are highly specific for either DUX4 or DUX4c mRNA, presumably but without limitation due to sequence differences in the distinct 3′ UTRs.
  • DUX4 emerges as particularly implicated in the aetiology of FSHD.
  • an RNAi agent capable of reducing or abolishing the production of DUX4 protein.
  • Such RNAi agent is configured to target DUX4 mRNA.
  • RNAi agent capable of reducing or abolishing the production of DUX4 protein but not of the DUX4c protein.
  • Such RNAi agent is configured to target DUX4 mRNA but not DUX4c mRNA.
  • an RNAi agent capable of reducing or abolishing the production of DUX4c protein but not of the DUX4 protein is configured to target DUX4c mRNA but not DUX4 mRNA.
  • RNAi agents as intended herein may particularly comprise or denote (i.e., may be selected from a group comprising or consisting of) RNAi nucleic acid molecules or RNAi nucleic acid analogue molecules, such as preferably short interfering nucleic acids and short interfering nucleic acid analogues (siNA) such as short interfering RNA and short interfering RNA analogues (siRNA), and may further denote inter alter double-stranded RNA and double-stranded RNA analogues (dsRNA), micro-RNA and micro-RNA analogues (miRNA), and short hairpin RNA and short hairpin RNA analogues (shRNA).
  • siNA short interfering nucleic acid analogues
  • siRNA short interfering RNA and short interfering RNA analogues
  • dsRNA double-stranded RNA analogues
  • miRNA micro-RNA and micro-RNA analogues
  • shRNA short hairpin
  • an RNAi agent as disclosed herein may be conjugated to a cell penetrating peptide (CPP), to enhance the cellular uptake of said RNAi agents.
  • CPP cell penetrating peptide
  • An RNAi agent typically includes a double stranded portion (notwithstanding the optional and potentially preferred presence of any single-stranded overhands) comprising at least 16 bases, preferably at least 17 bases, more preferably at least 18 bases and still more preferably at least 19 bases, and usually between 18 and 35 bases, preferably between 19 and 30 bases, more preferably between 20 and 25 bases and even more preferably between 21 and 23 bases which are identical or almost identical to (e.g., showing 90% or more, e.g., at least 95%, sequence identity to, or showing maximum 2 and preferably only 1 mismatch with) an mRNA whose silencing is desired and which is thus targeted by said RNAi agent (such as, e.g., DUX4 and/or DUX4c mRNAs).
  • a double stranded portion comprising at least 16 bases, preferably at least 17 bases, more preferably at least 18 bases and still more preferably at least 19 bases, and usually between 18 and 35 bases, preferably between 19 and 30 bases, more
  • a DUX4 gene preferably intended for targeting by the RNAi agents as disclosed herein resides in the distal-most D4Z4 unit which extends into the pLAM region flanking the telomeric side of the D4Z4 array.
  • Such DUX4 gene leads to production of comparably stable mRNA(s) (Dixit et al. 2007, supra).
  • mRNA(s) Distal et al. 2007, supra.
  • FIG. 2 with reference to an exemplary but non-limiting genomic sequence as shown in FIG. 3
  • such DUX4 gene comprises two introns which are located in its 3′ UTR, namely intron 1 (or intron A) within the D4Z4 unit and intron 2 (or intron B) provided by the pLAM region.
  • Alternative splicing of intron 1 as schematically captured in FIG. 2 leads to alternative DUX4 mRNAs.
  • FIGS. 4 Exemplary but non-limiting DUX4 cDNA sequences including or not intron 1 are shown in FIGS. 4 (SEQ ID NO: 42) and 5 (SEQ ID NO: 43), respectively.
  • anti-DUX4 RNAi agents as intended herein may be configured to target DUX4 mRNA as represented by the DUX4 cDNA sequence set forth in SEQ ID NO: 42 or variants thereof having at least about 80% and preferably at least about 90% or at least about 95% sequence identity to SEQ ID NO: 42.
  • anti-DUX4 RNAi agents may be configured to target the 3′ UTR of said DUX4 mRNA or variants, such as for example to target 3′ UTR sequences corresponding to or overlapping with exon 1, intron 1, exon 2 and/or axon 3.
  • anti-DUX4 RNAi agents as intended herein may be configured to target DUX4 mRNA as represented by the DUX4 cDNA sequence set forth in SEQ ID NO: 43 or variants thereof having at least about 80% and preferably at least about 90% or at least about 95% sequence identity to SEQ ID NO: 43.
  • anti-DUX4 RNAi agents may be configured to target the 3′ UTR of said DUX4 mRNA or variants, such as for example to target 3′ UTR sequences corresponding to or overlapping with axon 1, exon 2 and/or exon 3.
  • an anti-DUX4 RNAi agent may be configured to target any one of the following DUX4 mRNA sequences (SEQ ID NO: 44 to 46) or variants thereof having at least about 80% and preferably at least about 90% or at least about 95% sequence identity to the respective sequences, or fragments thereof comprising at least 16 bases, preferably at least 17 bases, more preferably at least 18 bases and still more preferably at least 19 bases, and usually between 18 and 35 bases, preferably between 19 and 30 bases, more preferably between 20 and 25 bases and even more preferably between 21 and 23 bases, preferably wherein said reference to bases denotes consecutive bases, of the respective sequences or variants:
  • an anti-DUX4 RNAi agent as disclosed herein may comprise any one of the following sequences (SEQ ID NO: 47 to 49) or variants thereof having at least about 80% and preferably at least about 90% or at least about 95% sequence identity to (e.g., variants showing maximum 2 and preferably only 1 mismatch with) the respective sequences, or fragments thereof comprising at least 16 bases, preferably at least 17 bases and more preferably at least 18 bases, preferably wherein said reference to bases denotes consecutive bases, of the respective sequences or variants:
  • DUX4c cDNA sequences are shown in FIGS. 6 (SEQ ID NO: 50) and 7 (SEQ ID NO: 51), including 3′ UTR regions of distinct lengths.
  • anti-DUX4c RN Ai agents as intended herein may be configured to target DUX4c mRNA as represented by the DUX4c cDNA sequence set forth in SEQ ID NO: 50 or variants thereof having at least about 80% and preferably at least about 90% or at least about 95% sequence identity to SEQ ID NO: 50.
  • anti-DUX4c RNAi agents may be configured to target the 3′ UTR of said DUX4c mRNA or variants.
  • anti-DUX4c RNAi agents as intended herein may be configured to target DUX4c mRNA as represented by the DUX4c cDNA sequence set forth in SEQ ID NO: 51 or variants thereof having at least about 80% and preferably at least about 90% or at least about 95% sequence identity to SEQ ID NO: 51.
  • anti-DUX4c RNAi agents may be configured to target the 3′ UTR of said DUX4c mRNA or variants.
  • a further exemplary but genomic sequence of the DUX4c gene (Genbank accession no. NC — 000004 range 190940254 . . . 190945505, complement, sequence version 11, i.e., NC — 000004.11 range 190940254 . . . 190945505, complement) predicts a longer DUX4c mRNA than those shown in FIGS. 6 and 7 .
  • such further exemplary but non-limiting DUX4c cDNA sequence is available in the Genbank database under accession no. XR — 041199 (sequences version 2, i.e., XR — 041199.2) and reproduced in FIG. 8 (SEQ ID NO: 52).
  • anti-DUX4c RNAi agents as intended herein may be configured to target DUX4c mRNA as represented by the DUX4c cDNA sequence set forth in SEQ ID NO: 52 or variants thereof having at least about 80% and preferably at least about 90% or at least about 95% sequence identity to SEQ ID NO: 52.
  • anti-DUX4c RNAi agents may be configured to target the 3′ UTR of said DUX4c mRNA or variants.
  • an anti-DUX4c RNAi agent may be configured to target any one of the following DUX4c mRNA sequences (SEQ ID NO: 53 to 55) or variants thereof having at least about 80% and preferably at least about 90% or at least about 95% sequence identity to the respective sequences, or fragments thereof comprising at least 16 bases, preferably at least 17 bases, more preferably at least 18 bases and still more preferably at least 19 bases, and usually between 18 and 35 bases, preferably between 19 and 30 bases, more preferably between 20 and 25 bases and even more preferably between 21 and 23 bases, preferably wherein said reference to bases denotes consecutive bases, of the respective sequences or variants:
  • an anti-DUX4c RNAi agent as disclosed herein may comprise any one of the following sequences (SEQ ID NO: 56 to 58) or variants thereof having at least about 80% and preferably at least about 90% or at least about 95% sequence identity to (e.g., variants showing maximum 2 and preferably only 1 mismatch with) the respective sequences, or fragments thereof comprising at least 16 bases, preferably at least 17 bases and more preferably at least 18 bases, preferably wherein said reference to bases denotes consecutive bases, of the respective sequences or variants:
  • any one anti-DUX4 and/or anti-DUX4c antisense agent or RNAi agent as taught herein, particularly wherein said agent comprises, consists essentially of or consists of a nucleic acid molecule or a nucleic acid analogue molecule, comprising synthesising said agent from its constituent nucleotides or nucleotide analogues, and optionally and preferably at least partly purifying the agent from the synthesis reaction.
  • nucleic acid more specifically an isolated nucleic acid, encoding any one or more anti-DUX4 and/or anti-DUX4c antisense agent or RNAi agent as taught herein.
  • the nucleic acid may be operably linked to one or more regulatory sequences allowing for expression of the nucleic acid in an expression system, such as without limitation in vitro (e.g., in a cell-free expression system) or in a host cell or host organism.
  • a recombinant nucleic acid construct i.e., a vector
  • a nucleic acid encoding any one or more anti-DUX4 and/or anti-DUX4c antisense agent or RNAi agent as taught herein.
  • Such construct may allow inter alia to propagate the nucleic acid encoding said agent, e.g., in vitro or in a host cell or host organism.
  • a method for producing said recombinant nucleic acid construct (vector) comprising introducing the nucleic acid encoding said agent to a recipient nucleic acid construct (recipient vector).
  • the recombinant nucleic acid construct may be an expression construct (i.e., an expression vector), hence may be capable of expressing (configured to express) the nucleic acid encoding the one or more anti-DUX4 and/or anti-DUX4c antisense agent or RNAi agent in an expression system, such as without limitation in vitro (e.g., in a cell-free expression system) or in a host cell or host organism.
  • an expression construct expression vector
  • the nucleic acid encoding said agent is operably linked to one or more regulatory sequences allowing for expression of the nucleic acid in said expression system.
  • a method for producing said expression construct (expression vector) comprising introducing the nucleic acid encoding said agent to a recipient expression construct (recipient expression vector).
  • any one or more anti-DUX4 and/or anti-DUX4c antisense agent or RNAi agent as taught herein comprising expressing said agent from an expression construct (expression vector) as taught herein comprising a nucleic acid encoding said agent, in an expression system, and optionally at least partly purifying the agent.
  • a host cell comprising any one or more anti-DUX4 and/or anti-DUX4c antisense agent or RNAi agent, or an isolated nucleic acid encoding such an agent, or a recombinant construct (vector) (preferably an expression construct, expression vector) comprising a nucleic acid encoding such an agent, as taught herein.
  • Also encompassed is a method for producing such a host cell comprising introducing into a recipient host cell the anti-DUX4 and/or anti-DUX4c antisense agent or RNAi agent, or the isolated nucleic acid encoding such an agent, or the recombinant construct (vector) (preferably an expression construct, expression vector) comprising a nucleic acid encoding such an agent.
  • the host cell may be a prokaryotic or eukaryotic cell, more preferably a bacterial, fungal, plant or animal cell, even more preferably a mammal cell or a primate cell, including very preferably human cells, as well as non-human mammal cells and non-human primate cells.
  • said human host cell may be a myoblast or a myoblast precursor derived from a patient, such as for example a myoblast derived from a muscle biopsy of said patient or derived from a mesangioblast of said patient, or said myoblast or myoblast precursor may be differentiated from an adult stem cell or an induced pluripotent stem (iPS) cell of said patient.
  • the isolated nucleic acid or construct (vector) may be integrated, preferably stably integrated, into the genome of the host cell or may remain extra-genomic or extra-chromosomal. Insofar the host cell comprises said agent, isolated nucleic acid or construct (vector), it may be denoted a ‘transgenic’ or ‘transformed’ cell in that regard.
  • a host cell expresses or is under suitable conditions capable of expressing the isolated nucleic acid or vector comprised therein, thereby producing the anti-DUX4 and/or anti-DUX4c antisense agent or RNAi agent encoded thereby.
  • a method for producing any one or more anti-DUX4 and/or anti-DUX4c antisense agent or RNAi agent as taught herein comprising culturing or maintaining a host cell comprising an isolated nucleic acid encoding said agent or an expression construct (expression vector) comprising a nucleic acid encoding said agent, under conditions conducive to expression of said agent from said isolated nucleic acid or expression construct.
  • the so-produced agent may be intended to exert its silencing effect in the host cell expressing it, or may by intended for use elsewhere in which case the method may further optionally and preferably comprise at least partly purifying the agent.
  • a host organism comprising any one or more anti-DUX4 and/or anti-DUX4c antisense agent or RNAi agent, or an isolated nucleic acid encoding such an agent, or a recombinant construct (vector) (preferably an expression construct, expression vector) comprising a nucleic acid encoding such an agent, or a host cell, as taught herein.
  • vector preferably an expression construct, expression vector
  • Also encompassed is a method for producing such a host organism comprising introducing the anti-DUX4 and/or anti-DUX4c antisense agent or RNAi agent, or the isolated nucleic acid encoding such an agent, or the recombinant construct (vector) (preferably an expression construct, expression vector) comprising a nucleic acid encoding such an agent, into a recipient host organism, e.g., to a cell, tissue or organ of said host organism, or introducing the host cell as taught herein to a recipient host organism, or at least partly regenerating an organism from said host cell.
  • a method for producing such a host organism comprising introducing the anti-DUX4 and/or anti-DUX4c antisense agent or RNAi agent, or the isolated nucleic acid encoding such an agent, or the recombinant construct (vector) (preferably an expression construct, expression vector) comprising a nucleic acid encoding such an agent, into a recipient host organism, e.g.
  • the host organism may be a multi-cellular organism, more preferably a plant or animal organism, even more preferably a mammal or primate, particularly including non-human mammals and non-human primates.
  • the isolated nucleic acid or construct (vector) may be integrated, preferably stably integrated, into the genome of the host organism or may remain extra-genomic or extra-chromosomal. Insofar the host organism comprises said agent, isolated nucleic acid or construct (vector), it may be denoted a ‘transgenic’ or ‘transformed’ organism in that regard.
  • a host organism expresses or is under suitable conditions capable of expressing the isolated nucleic acid or vector comprised therein, thereby producing the anti-DUX4 and/or anti-DUX4c antisense agent or RNAi agent encoded thereby.
  • a method for producing any one or more anti-DUX4 and/or anti-DUX4c antisense agent or RNAi agent as taught herein comprising culturing or maintaining a host organism comprising an isolated nucleic acid encoding said agent or an expression construct (expression vector) comprising a nucleic acid encoding said agent, under conditions conducive to expression of said agent from said isolated nucleic acid or expression construct.
  • the so-produced agent may be intended to exert its silencing effect in the host organism expressing it, or may by intended for use elsewhere in which case the method may further optionally and preferably comprise at least partly purifying the agent.
  • progeny of the host cell or host organism as taught herein. Particularly intended is progeny comprising the introduced agent, or isolated nucleic acid encoding the agent, or a construct (vector) comprising a nucleic acid encoding the agent, or comprising a replicated copy of said nucleic acid or construct (vector), i.e., progeny transgenic or transformed with regard to said nucleic acid or construct.
  • compositions and formulations comprising any one or more anti-DUX4 and/or anti-DUX4c antisense agent or RNAi agent as taught herein, or an isolated nucleic acid encoding such an agent, a recombinant construct (vector) (preferably an expression construct, expression vector) comprising a nucleic acid encoding such an agent, or a host cell or host organism as taught herein, and one or more additional components, such as without limitation one or more solvents and/or one or more pharmaceutically acceptable carriers.
  • vector preferably an expression construct, expression vector
  • additional components such as without limitation one or more solvents and/or one or more pharmaceutically acceptable carriers.
  • methods for producing the above compositions or formulations comprising admixing said agent, isolated nucleic acid, construct (vector), host cell or host organism as taught herein with one or more additional components.
  • compositions and formulations comprising any one or more anti-DUX4 and/or anti-DUX4c antisense agent or RNAi agent as taught herein, or an isolated nucleic acid encoding such an agent, a recombinant construct (vector) (preferably an expression construct, expression vector) comprising a nucleic acid encoding such an agent, or a host cell or host organism as taught herein, one or more pharmaceutically acceptable carriers; and methods for producing said pharmaceutical compositions and formulations, comprising admixing said agent, isolated nucleic acid, construct (vector), host cell or host organism as taught herein with said one or more pharmaceutically acceptable carriers.
  • a recombinant construct vector
  • methods for producing said pharmaceutical compositions and formulations comprising admixing said agent, isolated nucleic acid, construct (vector), host cell or host organism as taught herein with said one or more pharmaceutically acceptable carriers.
  • kits of parts comprising any one or more anti-DUX4 and/or anti-DUX4c antisense agent or RNAi agent as taught herein, or an isolated nucleic acid encoding such an agent, a recombinant construct (vector) (preferably an expression construct, expression vector) comprising a nucleic acid encoding such an agent, or a host cell or host organism or progeny thereof as taught herein, or composition(s) or formulation(s) comprising any of such.
  • the components of the kits may be in various forms, such as, e.g., lyophilised, free in solution or immobilised on a solid phase.
  • kits may be, e.g., provided in a multi-well plate or as an array or microarray, or they may be packaged separately and/or individually.
  • a kit will further typically comprise instructions for its use.
  • the kits may be advantageously employed in various applications, such as inter cilia in therapeutic, diagnostic, compound-screening and research applications.
  • the diseases or conditions include ones comprising increased levels and/or increased activity of double homeobox 4 and/or double homeobox 4c, more preferably the disease or condition is facioscapulohumeral muscular dystrophy (FSHD), Double homeobox 4 emerges as particularly implicated in the aetiology of FSHD, Anti-DUX4 antisense and/or RNAi agents and the related or derived reagents are thus preferred.
  • FSHD facioscapulohumeral muscular dystrophy
  • Double homeobox 4 emerges as particularly implicated in the aetiology of FSHD
  • Anti-DUX4 antisense and/or RNAi agents and the related or derived reagents are thus preferred.
  • any one or more anti-DUX4 and/or anti-DUX4c antisense agent or RNAi agent covers not only such single agents, but also any combinations of two or more such agents. Expressly intended are without limitation a combination of two or more anti-DUX4 and/or anti-DUX4c antisense agents; a combination of two or more anti-DUX4 and/or anti-DUX4c RNAi agents; and a combination of one or more anti-DUX4 and/or anti-DUX4c antisense agent and one or more anti-DUX4 and/or anti-DUX4c RNAi agent.
  • Agents in a combination of two or more agents may be typically provided as separate molecules, or may otherwise be covalently or non-covalently conjugated to one another, either directly or via a suitable linker or carrier.
  • a non-limiting example of joined agents includes “weasel” agents of two or more co-joined antisense oligonucleotides as disclosed in WO 2006/000057, or in Aartsma-Rus et al. 2004 (Am J Hum Genet. 74: 83-92).
  • FIG. 1 illustrates a schematic representation of the DUX4 transcripts expressed from an exemplary pathogenic D4Z4 repeat array containing four D4Z4 units (grey arrows) at the 4q35 locus.
  • Each of the four D4Z4 units contains the DUX4 open reading frame (ORF) (white boxes) and a transcription start site (white bended arrows).
  • the repeat array is flanked on its telomeric end by the pLAM region (grey box) which is only present on the 4qA allele uniquely linked to FSHD.
  • the alternative 4qB allele is not linked to FSHD (Lemmers et al. 2004 (Am J Hum Genet. 75(6): 1124-30)).
  • FIG. 2 illustrates a scheme of an EcoRI genomic fragment cloned in pGEM7Z and encompassing the 3′ portion of the DUX4 ORF and its 3′ UTR.
  • the stop codon of the DUX4 ORF, the pLAM region and the poly-A addition signal (ATTAAA) are indicated in the upper panel.
  • the lower panel captures the mapping of the 3′ mRNA ends and illustrates the location of introns 1 and 2. Intron 1 is alternatively spliced.
  • the nucleotide positions are as shown in the sequence in FIG. 3 .
  • FIG. 3 illustrates the sequence (SEQ ID NO: 1) of an exemplary genomic fragment as schematically set out in FIG. 2 , encompassing the 3′ portion of the DUX4 ORF and its 3′ UTR.
  • This particular sequence reproduces positions 12001 to 13080 of the genomic sequence available in the NCBI Genbank database under accession number AF117653 (sequence version no. 2, i.e., AF117653.2).
  • AF117653 sequence version no. 2, i.e., AF117653.2
  • said DUX4 ORF extends from an ATG translation initiation codon at position 10829 (not shown) to the stop codon at positions 12101-12103 (boxed).
  • Exon 1 ends at position 12111, intron 1 extends from position 12112 to 12247 within the D4Z4 unit (italics), exon 2 extends from position 12248 to 12329 (bold), the last D4Z4 unit ends at position 12329 continuing with the pLAM region, intron 2 extends from position 12330 to 12684 (larger) (italics) or alternatively 12338-12682 (smaller), and exon 3 extends from position 12685 to 12873 (bold).
  • FIG. 4 illustrates the sequence (SEQ ID NO: 42) of an exemplary DUX4 cDNA.
  • the DUX4 ORF demarcated by the translation initiation codon (bold, boxed) and the stop codon (boxed), and the 5′ UTR upstream of the ATG codon correspond to these portions in the exemplary DUX4 cDNA sequence available in Genbank under accession no. NM 033178 (sequence version 2, i.e., NM — 033178.2).
  • the 3′ UTR downstream of the stop codon i.e., starting from position 1576 of SEQ ID NO: 42
  • is compiled from the DUX4 genomic sequence AF117653.2 see FIG. 3 and legend thereto) and includes the remainder of exon 1, intron 1 (italics), exon 2 (bold) and exon 3 (underlined).
  • FIG. 5 illustrates the sequence (SEQ ID NO: 43) of an exemplary DUX4 cDNA.
  • the DUX4 ORF demarcated by the translation initiation codon (bold, boxed) and the stop codon (boxed), and the 5′ UTR upstream of the ATG codon correspond to these portions in the exemplary DUX4 cDNA sequence available in Genbank under accession no. NM — 033178 (sequence version 2, i.e., NM — 033178.2).
  • the 3′ UTR downstream of the stop codon i.e., starting from position 1576 of SEQ ID NO: 43 is compiled from the DUX4 genomic sequence AF117653.2 (see FIG. 3 and legend thereto) and includes the remainder of exon 1, exon 2 (bold) and exon 3 (underlined).
  • FIG. 6 illustrates the sequence (SEQ ID NO: 50) of an exemplary putative DUX4c cDNA. This sequence corresponds to positions 727 to 2440 of an exemplary but non-limiting genomic sequence of the DUX4c gene (Genbank accession no. AY500824, sequence version 1, i.e., AY500824.1).
  • the 3′ UTR as experimentally detected extends downstream of the stop codon (i.e., starting from position 1317 of SEQ ID NO: 50; position 2043 of AY500824.1) down to position 1714 of SEQ ID NO: 50 (position 2440 of AY500824.1).
  • FIG. 7 illustrates the sequence (SEQ ID NO: 51) of an exemplary putative DUX4c cDNA. This sequence corresponds to positions 727 to 2629 of an exemplary but non-limiting genomic sequence of the DUX4c gene (Genbank accession no. AY500824, sequence version 1, AY500824.1).
  • the 3′ UTR as experimentally detected in an FSHD patient extends downstream of the stop codon (i.e., starting from position 1317 of SEQ ID NO: 51; position 2043 of AY500824.1) down to position 1903 of SEQ ID NO: 51 (position 2629 of AY500824.1).
  • FIG. 8 illustrates the sequence (SEQ ID NO: 52) of an exemplary putative DUX4c cDNA.
  • This sequence corresponds to predicted DUX4c mRNA as available in the Genbank database under accession no. XR — 041199 (sequences version 2, i.e., XR — 041199.2).
  • Indicated are the DUX4c ORF demarcated by the translation initiation codon (bold, boxed) at positions 688-670 and the stop codon (boxed) at positions 1810-1812.
  • the predicted 3′ UTR extends downstream of the stop codon (i.e., starting from position 1813.
  • FIG. 9 illustrates the inhibitory effect of anti-DUX4 pre-mRNA antisense oligomers on DUX4 protein expression.
  • FIGS. 10 and 11 illustrate that antisense oligomer 1524 can exert a specific inhibitory effect on DUX4 protein expression.
  • FIG. 12 illustrates that antisense oligomers 1524, 1523 and 1522 can exert a specific inhibitory effect on DUX4 protein expression.
  • FIGS. 13 and 14 illustrates evaluation of siRNA targeting DUX4c.
  • FIG. 15 illustrates evaluation of siRNA targeting DUX4.
  • FIG. 16 illustrates evaluation of anti-DUX4c and anti-DUX4 siRNA specificity by Western blot.
  • A anti-DUX4 antibody
  • B anti-DUX4c antibody.
  • FIG. 17 illustrates pLVTH-shRNA expression vector.
  • FIG. 18 schematically illustrates production of shRNA from an shRNA vector and its subsequence processing to siRNA by Dicer.
  • FIG. 19 illustrates efficiency and specificity of shRNA vectors in western blot.
  • FIG. 20 illustrates an exemplary sequence of DUX4 protein.
  • FIG. 21 illustrates an exemplary sequence of DUX4c protein.
  • FIG. 22 illustrates optimal transfection conditions for FSHD primary myoblasts.
  • FIG. 23 illustrates evaluation of siRNA targeting DUX4 in FSHD primary myoblasts.
  • FIG. 24 illustrates a schematic representation of the structure of an oligonucleotide chemically modified with a 2′-O-methylated phosphorothioate backbone.
  • FIG. 25 illustrates a schematic representation of the structure of an oligonucleotide chemically modified with a phosphorodiamidate morpholino backbone.
  • FIG. 26 illustrates exemplary transcripts derived from one or more D4Z4 units and comprising the pLAM region or not.
  • the upper panel schematically shows mimic fragments of a D4Z4 unit and the pLAM region.
  • the DUX4 ORF is represented in black with the two homeobox regions in grey. The positions of the different introns are indicated (dark grey boxes).
  • the pLAM region encompasses an intron (dark grey box) and the poly-A signal (ATTAAA).
  • the lower panels illustrate the location of introns 1, 2, 2a, 1bis and 2a bis.
  • the first transcript published begins in the last D4Z4 unit with alternative splicing of intron 1 in the D4Z4 sequence, then extending into the pLAM region where intron 2 is always spliced out, and ending 6 to 16 bp after the poly-A signal.
  • a second transcript (Coppée et al., unpublished) that begins in a D4Z4 unit (adjacent or not to the last unit) that has the same intron 1 as reported above.
  • the transcript continues in the adjacent D4Z4 unit where another intron is found that is named either 2a if the splice acceptor site is in the D4Z4 unit or 2a bis if this splice acceptor site is in the pLAM region. No poly-A signals were reported at proximity. Snider et al., supra found a DUX4 transcript corresponding to those described in Dixit et al. and a DUX4 transcript with 2 copies of exon 2.
  • FIG. 27 illustrates exemplary genomic sequence of the DUX4 transcripts of FIG. 26 .
  • Snider et al. reported a different sequence for the beginning of the pLAM region that contains the intron 2 donor splicing site (boxed sequence GGTACC). Sequence comparison revealed that this sequence is identical to those in the beginning of a D4Z4 unit surrounding the intron 2a splice donor site (Coppée et al.).
  • FIG. 28 schematically shows the DUX4 gene structure by aligning D4Z4 and pLAM variants.
  • Two adjacent D4Z4 units are represented to scale from GenBank accession number AF117653.1 (first and second line) as well as the flanking pLAM region (fourth line). This region differs from that represented in the third line (GenBank accession number U74497.1) by a deletion of a 1609-bp segment (vertical stripes).
  • This pLAM region (third line) is nearly identical to a D4Z4 unit over 1890 bp (grey stripes) and diverges in further distal sequences.
  • the 1609-bp deletion in the pLAM region of the fourth line is found in the region nearly identical to D4Z4.
  • the DUX4 ORF is represented in black with the two homeobox regions in grey. The positions of the different introns are indicated (dark grey boxes).
  • the DUX4 mRNA start sites are indicated by black upward arrows for the 1 st mRNA (Dixit et al. 2007) and the 2 nd mRNA (Coppée et al., unpublished).
  • the DUX4 mRNA ends are indicated by black downwards arrows. Different ends were found: 6 to 16 bp downstream from the poly-A signal (Dixit et al., 2007) for the mRNA, and two possible ends (either in D4Z4 or in pLAM) for the 2 nd mRNA.
  • FIG. 29 illustrates the efficiency of the antisense oligonucleotides 1521 (a) and 1523 (b) in decreasing endogenous DUX4 mRNA amount in FSHD primary myotubes.
  • FIG. 30 illustrates the efficiency of anti-DUX4 siRNA3 in decreasing endogenous DUX4 mRNA amount in FSHD primary myotubes.
  • FIG. 31 schematically shows the position of the antisense oligonucleotides 2245 and 2250 on the DUX4 genomic sequence fragment available in Genbank under accession no. AF117653 (sequence version 2, i.e., AF117653.2). (see FIG. 3 and legend thereto).
  • This sequence fragment includes intron 1 (italics), exon 2 (bold), exon 3 (italics, bold) and the stop codon of the DUX4 ORF (boxed), antisense oligonucleotides 2245 (underlined) and 2250 (underlined, bold).
  • FIG. 32 illustrates the inhibitory effect of the antisense oligomers 2245 and 2250 on DUX4 protein expression.
  • FIG. 33 illustrates optimal concentration of antisense oligomer 2245 for specific inhibition of DUX4 protein expression.
  • double homeobox 4 and “DUX4” are synonymous and refer to genes, gene products, nucleic acids, proteins and polypeptides commonly known under these designations in the art.
  • the terms encompass such genes, gene products, nucleic acids, proteins and polypeptides of any organism where found, and particularly of animals, preferably vertebrates, more preferably mammals, including humans and non-human mammals, even more preferably of humans.
  • native sequences of DUX4 may differ between different species due to genetic divergence between such species.
  • native sequences of DUX4 may differ between or within different individuals of the same species due to normal genetic diversity (genetic variation) or due to mutation within a given species.
  • native sequences of DUX4 may differ between or even within different individuals' of the same species due to post-transcriptional or post-translational modifications. Accordingly, all DUX4 sequences found in or derived from nature are considered “native”.
  • the terms encompass DUX4 genes, gene products, nucleic acids, proteins and polypeptides when forming a part of a living organism, organ, tissue or cell, when forming a part of a biological sample, as well as when at least partly isolated from such sources.
  • the terms also encompass genes, gene products, nucleic acids, proteins and polypeptides when produced by recombinant or synthetic means.
  • DUX4 gene as intended herein may particularly denote a DUX4 gene present in the distal-most unit of a D4Z4 array on chromosome 4q35; particularly wherein the DUX4 gene extends into the pLAM region flanking the telomeric side of the D4Z4 array, more particularly wherein said pLAM region provides a polyadenylation signal, such as preferably ATTAAA, Such DUX4 gene leads to production of comparably stable mRNA(s) (Dixie et al. 2007, supra).
  • DUX4 gene as intended herein may also particularly denote such D4Z4-resident transcription units, particularly ones that give rise to a transcript leading to production of comparably stable mRNA comprising DUX4 sequences, even more particularly wherein the transcript comprises the pLAM region, still more particularly wherein said pLAM region provides a polyadenylation signal, such as preferably ATTAAA.
  • DUX4 transcripts and mRNA are schematically illustrated in FIGS. 26 and 28 with reference to an exemplary but non-limiting genomic sequence as shown in FIG. 27 .
  • the pLAM region may display polymorphisms, such as without limitation the presence or absence of a 1.6-kb sequence within its intron (Gabri ⁇ ls et al. 1999, supra; van Deutekom et al. 2009, supra).
  • DUX4 gene as intended herein denotes DUX4 gene as above as present in a pathogenic D4Z4 array associated with facioscapulohumeral muscular dystrophy (FSHD).
  • FSHD facioscapulohumeral muscular dystrophy
  • Exemplary DUX4 gene includes without limitation human DUX4 gene having nucleic acid sequence as annotated under NCBI Genbank (http://www.ncbi.nlm.nih.gov/) accession number AF117653 (sequence version no. 2 revised on Nov. 30, 2009, i.e., AF117653.2), more particularly the DUX4 gene at positions about 10650 to about 12873 of AF117653.2, also particularly at positions about 10829 to about 12873 of AF117653 2.
  • Exemplary but non-limiting DUX4 cDNA includes without limitation human DUX4 cDNA having nucleic acid sequence as annotated under Genbank accession number NM — 033178 (sequence version 2 revised on Feb. 28, 2010, i.e., NM — 033178.2).
  • Further exemplary but non-limiting DUX4 cDNA (and respective mRNA) include without limitation human DUX4 cDNA having nucleic acid sequence as set out in SEQ ID NO: 42 ( FIG. 4 ) or SEQ ID NO: 43 ( FIG. 5 ).
  • Exemplary DUX4 protein or polypeptide includes without limitation human DUX4 protein or polypeptide having primary amino acid sequence as annotated under Genbank accession no. NP — 149418 (sequence version 3 revised on Feb. 28, 2010, i.e., NP — 149418.3), also reproduced in FIG. 20 as SEQ ID NO: 59.
  • double homeobox 4c and DUX4c are synonymous and refer to genes, gene products, nucleic acids, proteins and polypeptides commonly known under these designations in the art.
  • the terms encompass such genes, gene products, nucleic acids, proteins and polypeptides of any organism where found, and particularly of animals, preferably vertebrates, more preferably mammals, including humans and non-human mammals, even more preferably of humans.
  • native sequences of DUX4c may differ between different species due to genetic divergence between such species.
  • native sequences of DUX4c may differ between or within different individuals of the same species due to normal genetic diversity (genetic variation) or due to mutation within a given species.
  • native sequences of DUX4c may differ between or even within different individuals of the same species due to post-transcriptional or post-translational modifications. Accordingly, all DUX4c sequences found in or derived from nature are considered “native”.
  • the terms encompass DUX4c genes, gene products, nucleic acids, proteins and polypeptides when forming a part of a living organism, organ, tissue or cell, when forming a part of a biological sample, as well as when at least partly isolated from such sources.
  • the terms also encompass genes, gene products, nucleic acids, proteins and polypeptides when produced by recombinant or synthetic means.
  • Exemplary DUX4c gene includes without limitation human DUX4c gene having nucleic acid sequence as annotated under Genbank accession number AY500824 (sequence version 1 revised on Dec. 1, 2009, i.e., AY500824.1).
  • a further exemplary DUX4c gene includes without limitation human DUX4c gene having nucleic acid sequence as annotated under Genbank accession number NC — 000004 range 190940254 . . . 190945505, complement (sequence version 11 revised on Jun. 10, 2009, i.e., NC — 000004.14
  • Exemplary but non-limiting DUX4c cDNA includes without limitation human DUX4c cDNA having nucleic acid sequence as set out in SEQ ID NO: 50 ( FIG. 6 ) or SEQ ID NO: 51 ( FIG. 7 ).
  • a further exemplary but non-limiting DUX4c cDNA (and respective mRNA) includes without limitation human DUX4c cDNA having nucleic acid sequence as annotated under Genbank accession no. XR — 041199 (sequences version 2 revised on Jun. 10, 2009.1.e. XR — 041199.2) also reproduced in FIG. 8 .
  • Exemplary DUX4c protein or polypeptide includes without limitation human DUX4c protein or polypeptide having primary amino acid sequence as annotated under Genbank accession no. AAS15569 (sequence version 1 revised on Dec. 1, 2009, i.e., AAS15569.1), also reproduced in FIG. 21 as SEQ ID NO: 60.
  • antisense and siRNA agents as intended herein preferably target DUX4 and/or DUX4c genes specifically, i.e., substantially to the exclusion of other DUX genes.
  • specific agents may display adequate sequence identity to DUX4 and/or DUX4c sequences but not to said other DUX genes.
  • the particular antisense and siRNA agents as taught herein are highly advantageous in this respect.
  • DUX4 and DUX4c genes genes, gene products, nucleic acids, proteins and polypeptides also encompasses fragments and/or variants of the respective substances.
  • fragment with reference to a protein or polypeptide generally denotes a N- and/or C-terminally truncated form of a protein or polypeptide.
  • a fragment may comprise at least about 30%, e.g., at least about 50% or at least about 70%, preferably at least about 80%, e.g., at least about 85%, more preferably at least about 90%, and yet more preferably at least about 95% or even about 99% of the amino acid sequence length of said protein or polypeptide.
  • fragment with reference to a nucleic acid (polynucleotide) generally denotes a 5′- and/or 3′-truncated form of a nucleic acid.
  • a fragment may comprise at least about 30%, e.g., at least about 50% or at least about 70%, preferably at least about 80%, e.g., at least about 85%, more preferably at least about 90%, and yet more preferably at least about 95% or even about 99% of the nucleic acid sequence length of said nucleic acid.
  • nucleic acid polynucleotide
  • protein or polypeptide refers to nucleic acids, proteins or polypeptides the sequence (i.e., nucleotide sequence or amino acid sequence, respectively) of which is substantially identical (i.e., largely but not wholly identical) to the sequence of said recited nucleic acid, protein or polypeptide, e.g., at least about 80% identical or at least about 85% identical, e.g., preferably at least about 90% identical, e.g., at least 91% identical, 92% identical, more preferably at least about 93% identical, e.g., at least 94% identical, even more preferably at least about 95% identical, e.g., at least 96% identical, yet more preferably at least about 97% identical, e.g., at least 98% identical, and most preferably at least 99% identical.
  • a variant may display such degrees of identity to a recited nucleic acid, protein or polypeptide when the whole sequence of the recited nucleic acid, protein or polypeptide is queried in the sequence alignment (i.e., overall sequence identity).
  • fragments and variants of a given recited nucleic acid, protein or polypeptide are fusion products of said nucleic acid, protein or polypeptide with another, usually unrelated, nucleic acid, protein or polypeptide, respectively.
  • Particularly included among fragments and variants as intended herein are thus fusion genes between the DUX4 or DUX4c gene and other genes, leading to the expression of fusion (i.e., chimeric) proteins. More specifically included are such fusion genes arising through chromosomal rearrangements, even more specifically wherein said fusion genes and their chimeric proteins cause or contribute to a pathology.
  • examples of DUX4 or DUX4c fragments and variants which are encompassed herein and may benefit from targeting by the antisense or RNAi agents of the present invention include fusions between CIC, a human homolog of Drosophila capicua, and DUX4, as seen in Ewing's family tumours (EFTs) (Kawamura-Saito et al, 2006, supra) and paediatric undifferentiated soft tissue sarcomas (LISTS) (Yoshimoto et al. 2009, supra), and fusions between EWSR1 and DUX4, as seen in rhabdomyosarcomas (RMS) (Sirvent et al. 2009, supra). More generally, fusions containing the C-terminal fragment of DUX4 are intended, since the resultant chimeric proteins acquire an enhanced transcriptional activity, which may lead to tumour formation.
  • EFTs Ewing's family tumours
  • LISTS paediatric undifferentiated soft
  • Sequence identity may be determined using suitable algorithms for performing sequence alignments and determination of sequence identity as know per se.
  • BLAST Basic Local Alignment Search Tool
  • Blast 2 sequences algorithm described by Tatusova and Madden
  • a variant of a given nucleic acid (polynucleotide), protein or polypeptide may be a homologue (e.g., orthologue or paralogue) of said nucleic acid, protein or polypeptide.
  • homologue e.g., orthologue or paralogue
  • the term “homology” generally denotes structural similarity between two macromolecules, particularly between two nucleic acids, proteins or polypeptides, from same or different taxons, wherein said similarity is due to shared ancestry.
  • variants and/or fragments of substances such as of antisense or RNAi agents, nucleic acids, proteins or polypeptides
  • a functional variant and/or fragment of a DUX4 or DUX4c gene, gene product, nucleic acid, protein or polypeptide shall at least partly retain the biological activity of DUX4 or DUX4c, respectively.
  • such functional variant and/or fragment may retain one or more aspects of the biological activity of DUX4 or DUX4c, such as, e.g., ability to participate in one or more cellular pathways, ability to regulate transcription of one or more genes, etc.
  • a functional variant and/or fragment of an anti-DUX4 and/or anti-DUX4c antisense agent or RNAi agent shall at least partly retain the functionality of said agent, i.e., its ability to reduce or abolish the expression of the target molecule such as DUX4 and/or DUX4c.
  • a functional variant and/or fragment may retain at least about 20%, e.g., at least 30%, or at least about 40%, or at least about 50%, e.g., at least 60%, more preferably at least about 70%, e.g., at least 80%, yet more preferably at least about 85%, still more preferably at least about 90%, and most preferably at least about 95% or even about 100% or higher of the intended biological activity or functionality compared to the corresponding recited substance such as an agent, gene, gene product, nucleic acid, protein or polypeptide.
  • nucleic acid typically refers to a polymer (preferably a linear polymer) of any length composed essentially of nucleoside units.
  • a nucleoside unit commonly includes a heterocyclic base and a sugar group.
  • Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) which are widespread in naturally-occurring nucleic acids, other naturally-occurring bases (e.g., xanthine, inosine, hypoxanthine) as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatised bases.
  • A adenine
  • G guanine
  • C cytosine
  • T thymine
  • U uracil
  • modified nucleobases include without limitation 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability and may be preferred base substitutions in for example antisense agents, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
  • Sugar groups may include inter alia pentose (pentofuranose) groups such as preferably ribose and/or 2-deoxyribose common in naturally-occurring nucleic acids, or arabinose, 2-deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups (such as without limitation 2′-O-alkylated, e.g., 2′-O-methylated or 2′-O-ethylated sugars such as ribose; 2′-O-alkyloxyalkylated, 2′-O-methoxyethylated sugars such as ribose; or 2′-O,4′-C-alkylene-linked, e.g., 2′-O,4′-C-methylene-linked or 2′-O,4′-C-ethylene-linked sugars such as ribose; 2′-fluoro-arabinose, etc.).
  • pentose
  • Nucleoside units may be linked to one another by any one of numerous known inter-nucleoside linkages, including inter alia phosphodiester linkages common in naturally-occurring nucleic acids, and further modified phosphate- or phosphonate-based linkages such as phosphorothioate, alkyl phosphorothioate such as methyl phosphorothioate, phosphorodithioate, alkylphosphonate such as methylphosphonate, alkylphosphonothioate, phosphotriester such as alkylphosphotriester, phosphoramidate, phosphoropiperazidate, phosphoromorpholidate, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate; and further siloxane, carbonate, sulfamate, carboalkoxy, acetamidate, carbamate such as 3′-N-carbamate, morpholino, borano, thioether, 3′-thi
  • inter-nucleoside linkages may be phosphate-based linkages including modified phosphate-based linkages, such as more preferably phosphodiester, phosphorothioate or phosphorodithioate linkages or combinations thereof.
  • nucleic acid also encompasses any other nucleobase containing polymers such as nucleic acid mimetics, including, without limitation, peptide nucleic acids (PNA), peptide nucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA), morpholino phosphorodiamidate-backbone nucleic acids (PMO), cyclohexane nucleic acids (CeNA), tricyclo-DNA (tcDNA), and nucleic acids having backbone sections with alkyl linkers or amino linkers (see, e.g., Kurreck 2003 (Eur J Biochem 270: 1628-1644)).
  • Alkyl as used herein particularly encompasses lower hydrocarbon moieties, e.g., C1-C4 linear or branched, saturated or unsaturated hydrocarbon, such as methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl.
  • Nucleic acids as intended herein may include naturally occurring nucleosides, modified nucleosides or mixtures thereof.
  • a modified nucleoside may include a modified heterocyclic base, a modified sugar moiety, a modified inter-nucleoside linkage or a combination thereof.
  • nucleic acid further preferably encompasses DNA, RNA and DNA/RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic (e.g. chemically synthesised) DNA, RNA or DNA/RNA hybrids.
  • a nucleic acid can be naturally occurring, e.g., present in or isolated from nature, can be recombinant, i.e., produced by recombinant DNA technology, and/or can be, partly or entirely, chemically or biochemically synthesised.
  • a “nucleic acid” can be double-stranded, partly double stranded, or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.
  • Nucleic acids and particularly antisense oligonucleotides or RNAi agents may be herein denoted as comprising uracil (U) bases. It shall be appreciated that U may be optionally substituted by thymine (T) in (at least some such nucleic acids and agents. For example, as 2′-O-methyl phosphorothioate antisense oligonucleotides are more ‘RNA-like’, U may be used and denoted in such molecules. With other antisense chemistries, such as peptide nucleic acids or morpholino backbones. T bases may be preferably denoted and used.
  • oligonucleotide refers to a nucleic acid (including nucleic acid analogues and mimetics) oligomer or polymer as defined herein.
  • an oligonucleotide such as more particularly an antisense oligonucleotide, is (substantially) single-stranded.
  • Oligonucleotides as intended herein may be preferably between about 10 and about 100 nucleoside units (i.e., nucleotides or nucleotide analogues) in length, preferably between about 15 and about 50, more preferably between about 20 and about 40, also preferably between about 20 and about 30.
  • oligonucleotides as intended herein may comprise one or more or all non-naturally occurring heterocyclic bases and/or one or more or all non-naturally occurring sugar groups and/or one or more or all non-naturally occurring inter-nucleoside linkages, the inclusion of which may improve properties such as, for example, enhanced cellular uptake, increased stability in the presence of nucleases and increased hybridization affinity, increased tolerance for mismatches, etc.
  • oligonucleotides as intended herein may be configured to not activate RNAse H, accordance with known techniques (see, e.g., U.S. Pat. No. 5,149,797).
  • Antisense agents such as oligonucleotides as taught herein may be further conjugated (e.g., covalently or non-covalently, directly or via a suitable linker) to one or more moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.
  • Such moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.
  • lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.
  • antisense compounds that are chimeric compounds. “Chimeric” antisense compounds or “chimeras” are antisense molecules, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound.
  • oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the increased resistance to nuclease degradation, increased cellular uptake, and an additional region for increased binding affinity for the target nucleic acid.
  • antisense generally refers to an agent (e.g., an oligonucleotide) configured to specifically anneal with (hybridise to) a given sequence in a target nucleic acid, such as for example in a target DNA, hnRNA, pre-mRNA or mRNA, and typically comprises, consist essentially of or consist of a nucleic acid sequence that is complementary or substantially complementary to said target nucleic acid sequence.
  • Antisense agents suitable for use herein may typically be capable of annealing with (hybridising to) the respective target nucleic acid sequences at high stringency conditions, and capable of hybridising specifically to the target under physiological conditions.
  • complementary or “complementarity” as used herein with reference to nucleic acids refer to the normal binding of single-stranded nucleic acids under permissive salt (ionic strength) and temperature conditions by base pairing, preferably Watson-Crick base pairing.
  • base pairing preferably Watson-Crick base pairing.
  • complementary Watson-Crick base pairing occurs between the bases A and T, A and U or G and C.
  • sequence 5′-A-G-U-3′ is complementary to sequence 5′-A-C-U-3′.
  • binding or “binding” as used herein preferably refers to specific binding, i.e., where an agent binds to (anneals with) one or more targets of interest, such as to one or more pre-mRNA molecules or fragments or variants thereof, substantially to the exclusion of other molecules which are random or unrelated, and optionally substantially to the exclusion of other molecules that are structurally related. Binding of an agent to a target may be evaluated inter alia using conventional interaction-querying methods, such as in silico sequence analysis or nucleic acid hybridisation experiments, e.g., to verify specific hybridisation, e.g., under high stringency conditions.
  • an agent may be said to specifically bind to a given pre-mRNA of interest or fragments or variants thereof if its affinity for such intended target(s) under the conditions of binding is at least about 2-fold greater, preferably at least about 5-fold greater, more preferably at least about 10-fold greater, yet more preferably at least about 25-fold greater, still more preferably at least about 50-fold greater, and even more preferably at least about 100-fold or at least about 1000-fold or more greater, than its affinity for a non-target molecule, such as non-target other DUX genes.
  • an antisense agent need not be 100% complementary to that of its target sequence to bind or hybridise specifically with the latter.
  • An antisense agent may be said to be specifically hybridisable when binding of the agent to a target nucleic acid molecule interferes with the normal function of the target nucleic acid such as to attain an intended outcome (e.g., loss of utility), and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense agent to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.
  • “specifically hybridisable” and “complementary” may indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between an antisense agent and a nucleic acid target.
  • Agents as intended herein preferably specifically bind to the desired DUX4 and/or DUX4c targets substantially to the exclusion of other DUX genes.
  • the sequence of said antisense agents may be at least about 80% identical, preferably at least about 90% identical, more preferably at least about 95% identical, such as, e.g., about 96%, about 97%, about 98%, about 99% and up to 100% identical to the respective target DUX4 and/or DUX4c sequence.
  • reduce generally denotes a qualitative and/or quantitative alteration, change or variation leading to decrease of that which is being reduced (e.g., production and/or level of a given protein).
  • the term covers any extent of such reduction.
  • reduction may encompass a decrease in the value of said variable by at least about 10%, e.g., by at least about 20%, by at least about 30%, e.g., by at least about 40%, by at least about 50%, e.g., by at least about 60%, by at least about 70%, e.g., by at least about 80%, by at least about 90%, e.g., by at least about 95%, such as by at least about 96%, 97%, 98%, 99% or even by 100% (abolishment), compared to a reference situation without said reduction.
  • reduction of the production and/or level of intended target(s) may be specific or selective, i.e. the production and/or level of the production and/or level of the production and/or level of the production and/or level of
  • Agents such as antisense or RNAi agents as taught herein may without limitation reduce or abolish the production and/or level of DUX4 and/or DUX4c pre-mRNA and/or mRNA, whereby such agents may be capable of reducing or abolishing the production of DUX4 and/or DUX4c proteins.
  • Reference to the “level” of a target may preferably encompass the quantity and/or the availability (e.g., availability for performing its biological activity) of the target, e.g., within a cell, tissue, organ or an organism.
  • splicing denotes the process and means of removing intervening sequences (introns) from pre-mRNA in the process of producing mature mRNA.
  • the reference to splicing particularly aims at native splicing such as occurs under normal physiological conditions.
  • pre-mRNA and “transcript” are used herein to denote RNA species that precede mature mRNA, such as in particular a primary RNA transcript and any partially processed forms thereof.
  • Sequence elements required for splicing refer particularly to cis elements in the sequence of pre-mRNA which direct the cellular splicing machinery (spliceosome) towards correct and precise removal of introns from the pre-mRNA. Sequence elements involved in splicing are generally known per se and can be further determined by known techniques including infer alia mutation or deletion analysis.
  • “splice donor site” or “5′ splice site” generally refer to a conserved sequence immediately adjacent to an exon-intron boundary at the 5′ end of an intron. Commonly, a splice donor site may contain a dinucleotide GU, and may involve a consensus sequence of about 8 bases at about positions +2 to ⁇ 6.
  • “Splice acceptor site” or “3′ splice site” generally refers to a conserved sequence immediately adjacent to an intron-exon boundary at the 3′ end of an intron. Commonly, a splice acceptor site may contain a dinucleotide AG, and may involve a consensus sequence of about 16 bases at about positions ⁇ 14 to +2 (see, e.g., FIG. 1 of WO 2006/00057 for illustrative consensus sequences of splice donor and splice acceptor sites).
  • polyadenylation denotes the process and means of adding a polyadenylic acid (poly(A)) tail, i.e., multiple adenosine monophosphates, to an RNA molecule.
  • polyadenylation may denote the process and means of adding a poly(A) tail to a pre-mRNA molecule, in the process of producing mature mRNA.
  • the reference to polyadenylation particularly aims at native polyadenylation such as occurs under normal physiological conditions.
  • Sequence elements required for polyadenylation refer particularly to cis elements in the sequence of pre-mRNA which the cellular polyadenylation machinery recognises such as to which it binds, such as for example the polyadenylation signal.
  • sequence such as the polyadenylation signal may vary between groups of eukaryotes.
  • the polyadenylation signal sequence may typically be AATAAA (i.e., AAUAAA in RNA such as pre-mRNA), but variants of it exist, such as ATTAAA.
  • CPP cell-penetrating peptide
  • HIV-1 Tat-derived CPP see, e.g., Frankel et al. 1988 (Science 240: 70-73)
  • Antennapedia peptides or penetratins see, e.g., Derossi et al. 1994 (J Biol Chem 269: 10444-10450)
  • peptides derived from HSV-1 VP22 see, e.g., Aints et al.
  • CPP can be of any length.
  • CPP may be less than or equal to 500, 250, 150, 100, 50, 25, 10 or 6 amino acids in length.
  • CPP may be greater than or equal to 4, 5, 6, 10, 25, 50, 100, 150 or 250 amino acids in length.
  • a CPP may be between 4 and 25 amino acids in length.
  • the suitable length and design of the CPP will be easily determined by those skilled in the art.
  • CPPs can serve inter alga “Cell penetrating peptides: processes and applications” (ed. Ulo Langel, 1st ed., CRC Press 2002); Advanced Drug Delivery Reviews 57: 489-660 (2005); Dietz & Bahr 2004 (Moll Cell Neurosci 27: 85-131)).
  • RNA interference or “RNAi” technology is known in the art, and refers generally to the process and means of sequence-specific post-transcriptional gene silencing mediated particularly by short interfering nucleic acids (siNA).
  • siNA short interfering nucleic acids
  • RNAi agent typically comprises, consists essentially of or consists of a double-stranded portion or region (notwithstanding the optional and potentially preferred presence of single-stranded overhangs) of annealed complementary strands, one of which has a sequence corresponding to a target nucleotide sequence (hence, to at least a portion of an mRNA) of the target gene to be down-regulated.
  • the other strand of the RNAi agent is complementary to said target nucleotide sequence.
  • the number of mismatches between a target sequence and a nucleotide sequence of the RNAi agent is preferably no more than 1 in 5 bases, or 1 in 10 bases, or 1 in 20 bases, or 1 in 50 bases.
  • the sequence of said RNAi agents may be at least about 80% identical, preferably at least about 90% identical, more preferably at least about 95% identical, such as, e.g., about 96%, about 97%, about 98%, about 99% and up to 100% identical to the respective target DUX4 and/or DUX4c sequence.
  • RNAi agent may be formed by separate sense and antisense strands or, alternatively, by a common strand providing for fold-back stem-loop or hairpin design where the two annealed strands of an RNAi agent are covalently linked.
  • siRNA molecule may be typically produced, e.g., synthesised, as a double stranded molecule of separate, substantially complementary strands, wherein each strand is about 18 to about 35 bases long, preferably about 19 to about 30 bases, more preferably about 20 to about 25 bases and even more preferably about 21 to about 23 bases.
  • shRNA is in the form of a hairpin structure.
  • shRNA can be synthesized exogenously or can be formed by transcribing from RNA polymerase III promoters in vivo.
  • shRNAs can be engineered in host cells or organisms to ensure continuous and stable suppression of a desired gene. It is known that siRNA can be produced by processing a hairpin RNA in cells.
  • RNAi agents as intended herein may include any modifications as set out herein for nucleic acids and oligonucleotides, in order to improve their therapeutic properties.
  • At least one strand of an RNAi molecules may have a 3′ overhang from about 1 to about 6 bases in length, e.g., from 2 to 4 bases, more preferably from 1 to 3 bases.
  • one strand may have a 3′ overhang and the other strand may be either blunt-ended or may also have a 3′ overhang.
  • the length of the overhangs may be the same or different for each strand.
  • the 3′ overhangs can be stabilised against degradation.
  • the RNA may be stabilised by including purine nucleotides, such as A or G nucleotides.
  • substitution of pyrimidine nucleotides by modified analogues e.g., substitution of U 3′ overhangs by 2′-deoxythymidine is tolerated and does not affect the efficiency of RNAi.
  • An exemplary but non-limiting siRNA molecule may by characterized by any one or more, and preferably by all of the following criteria:
  • agents intended herein can be carried out by any processes known in the art, such as inter alia partly or entirely by chemical synthesis (e.g., routinely known solid phase synthesis; an exemplary an non-limiting method for synthesising oligonucleotides on a modified solid support is described in U.S. Pat. No. 4,458,066; in another example, diethyl-phosphoramidites are used as starting materials and may be synthesised as described by Beaucage et al.
  • chemical synthesis e.g., routinely known solid phase synthesis; an exemplary an non-limiting method for synthesising oligonucleotides on a modified solid support is described in U.S. Pat. No. 4,458,066; in another example, diethyl-phosphoramidites are used as starting materials and may be synthesised as described by Beaucage et al.
  • biochemical (enzymatic) synthesis e.g., by in vitro transcription from a nucleic acid construct (template) using a suitable polymerase such as a T7 or SP6 RNA polymerase, or by recombinant nucleic acid techniques, e.g., expression from a vector in a host cell or host organism.
  • Nucleotide analogues can be introduced by in vitro chemical or biochemical synthesis.
  • the antisense agents of the invention are synthesised in vitro and do not include antisense compositions of biological origin, or genetic vector constructs designed to direct the in vivo synthesis of antisense molecules.
  • isolated with reference to a particular component (such as for instance a nucleic acid) generally denotes that such component exists in separation from—for example, has been separated from or prepared and/or maintained in separation from—one or more other components of its natural environment.
  • a particular component such as for instance a nucleic acid
  • an isolated human or animal nucleic acid may exist in separation from a human or animal body where it naturally occurs.
  • isolated may preferably also encompass the qualifier “purified”.
  • purified with reference to a substance (e.g., an agent or a nucleic acid) does not require absolute purity. Instead, it denotes that such substances are in a discrete environment in which their abundance (conveniently expressed in terms of mass or weight or concentration) relative to other relevant substances is greater than in a biological sample.
  • a discrete environment denotes a single medium, such as for example a single solution, gel, precipitate, lyophilisate, etc.
  • Purified substances may be obtained by known methods including, for example, laboratory or recombinant synthesis, chromatography, preparative electrophoresis, centrifugation, precipitation, affinity purification, etc.
  • purified nucleic acids may preferably constitute by weight ⁇ about 10%, more preferably ⁇ about 50%, such as ⁇ about 60%, yet more preferably ⁇ about 70%, such as ⁇ about 80%, and still more preferably ⁇ about 90%, such as ⁇ about 95%, ⁇ about 96%, ⁇ about 97%, ⁇ about 98%, ⁇ about 99% or even 100%, of the nucleic acid content of the discrete environment.
  • purity of a nucleic acid may be determined by measuring absorbance A 260 /A 280 .
  • an isolated nucleic acid may be purified to homogeneity as determined by agarose- or polyacrylamide-gel electrophoresis and ethidium bromide or similar staining.
  • nucleic acid sequence or part(s) thereof corresponds to another nucleic acid sequence in a template—transcription product (e.g., RNA or RNA analogue) relationship, or corresponds, by virtue of the genetic code of an organism in question, to a particular amino acid sequence, e.g., die amino acid sequence of one or more desired proteins or polypeptides.
  • a template—transcription product e.g., RNA or RNA analogue
  • a nucleic acid encoding one or more proteins or polypeptides may comprise an open reading frame (ORF) encoding said protein or polypeptide.
  • ORF open reading frame
  • An “open reading frame” or “ORF” refers to a succession of coding nucleotide triplets (codons) starting with a translation initiation codon and closing with a translation termination codon known per se, and not containing any internal in-frame translation termination codon, and potentially capable of encoding a protein or polypeptide.
  • the term may be synonymous with “coding sequence” as used in the art.
  • transcription products or proteins and polypeptides can be achieved through operably linking nucleic acid sequences or ORFs encoding the intended transcription products or proteins and polypeptides with regulatory sequences allowing for expression of the nucleic acids or ORFs, e.g., in vitro, in a host cell, host organ and/or host organism. Such expression may be achieved, e.g., under suitable (culture) conditions or upon addition of inducers (e.g., where inducible regulatory sequences are used).
  • operably linkage is a linkage in which regulatory sequences and sequences sought to be expressed are connected in such a way as to permit said expression.
  • sequences such as, e.g., a promoter and an ORF, may be said to be operably linked if the nature of the linkage between said sequences does not: (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter to direct the transcription of the ORF, (3) interfere with the ability of the ORF to be transcribed from the promoter sequence.
  • regulatory sequences or elements required for expression may vary between expression environments, but may typically include a promoter and a transcription terminator, and optionally an enhancer, as known per se.
  • vector generally refers to a nucleic acid molecule, typically DNA, to which nucleic acid segments may be inserted and cloned, i.e., propagated.
  • a vector will typically contain one or more unique restriction sites, and may be capable of autonomous replication in a defined host or vehicle organism such that the cloned sequence is reproducible.
  • Vectors may include, without limitation, plasmids, phagemids, bacteriophages, bacteriophage-derived vectors, PAC, BAC, linear nucleic acids, e.g., linear DNA, viral vectors, etc., as appropriate.
  • Expression vectors are generally configured to allow for and/or effect the expression of nucleic acids or ORFs introduced thereto in a desired expression system, e.g., in vitro, in a host cell, host organ and/or host organism.
  • expression vectors may advantageously comprise suitable regulatory sequences.
  • viral vectors which are well known and include vectors derived from for example, but without limitation, retroviruses, vaccinia viruses, poxviruses, adenoviruses, and adeno-associated viruses (AAV).
  • retroviruses vaccinia viruses
  • poxviruses poxviruses
  • adenoviruses adeno-associated viruses
  • AAV adeno-associated viruses
  • Such viral vectors may me be engineered by recombinant techniques as known per se to introduce thereto nucleic acid sequence(s) encoding any one of the antisense or RNAi agents disclosed herein.
  • retroviral vectors may be used herein.
  • retroviral vectors may comprise the retroviral gnomic sequences encoding components necessary for the integration of the recombinant viral genome (randomly) into the host cell genome and the nucleic acid sequence(s) of interest, such as in particular the nucleic acid sequence(s) encoding any one of the antisense or RNAi agents disclosed herein.
  • retroviral vectors may be readily constructed using standard recombinant techniques (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989) from a wide variety of retroviruses, including for example, B, C, and D type retroviruses as well as spumaviruses and lentiviruses (see RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985).
  • standard recombinant techniques e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989
  • retroviruses including for example, B, C, and D type retroviruses as well as spumaviruses and lentiviruses (see RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985).
  • Recombinant adenoviral vectors may also be contemplated for delivery and expression of antisense or RNAi agents as disclosed herein in a host cell.
  • Adenovirus-based viral vectors have the advantage of being capable of infecting non-dividing host cells, but the recombinant viral genome is not integrated into the host cell genome.
  • a suitable adenoviral vector, a method for constructing a recombinant adenoviral vector thereof, and a method for delivering the recombinant vector into host cells are described in Xia H et al. (2002) (Nat. Biotech. 20: 1006-1010).
  • Use of recombinant AAV (RAAV) vectors is also contemplated herein.
  • RAAV vectors can infect both dividing and non-dividing cells and may incorporate its recombinant viral genome into that of the host cell.
  • RAAV vectors may be generated from a variety of adeno-associated viruses, including for example, serotypes 1 through 6.
  • RAAV vectors may comprise, in order, a 5′ adeno-associated virus inverted terminal repeat (ITR), a nucleic acid of interest, such as in particular a nucleic acid sequence encoding any one of the antisense or RNAi agents disclosed herein, operatively linked to a sequence which regulates its expression in a host cell or host organism, and a 3′ adeno-associated virus ITR.
  • ITR 5′ adeno-associated virus inverted terminal repeat
  • the rAAV vector may preferably have a polyadenylation signal.
  • Suitable RAAV vectors are described inter alga in WO 1994/13788, WO 1993/24641 and in Goyenvalle et al. 2004 (Science 306: 1796-1799) where antisense sequences are linked to a modified U7 small nuclear RNA.
  • viral vectors for use herein are vectors derived from a pox virus such as a vaccinia virus, for example an attenuated vaccinia virus such as Modified Virus Ankara (MVA) or NYVAC, an avipox virus such as fowl pox virus or canary pox virus.
  • a pox virus such as a vaccinia virus
  • an attenuated vaccinia virus such as Modified Virus Ankara (MVA) or NYVAC
  • an avipox virus such as fowl pox virus or canary pox virus.
  • host cell and “host organism” may suitably refer to cells or organisms encompassing both prokaryotes, such as bacteria, and eukaryotes, such as yeast, fungi, protozoan, plants and animals. Contemplated as host cells are inter alia unicellular organisms, such as bacteria (e.g., E.
  • yeast e.g., Saccharomyces cerevisiae or Pichia pastoris
  • plant cells e.g., from Arabidopsis thaliana or Nicotiana tobaccum
  • animal cells e.g., vertebrate animal cells, mammalian cells, primate cells, human cells or insect cells.
  • Contemplated as host organisms are inter alia multi-cellular organisms, such as plants and animals, preferably animals, more preferably warm-blooded animals, even more preferably vertebrate animals, still more preferably mammals, yet more preferably primates; particularly contemplated are such animals and animal categories which are non-human.
  • antisense agents and RNAi agents as used herein also encompasses any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
  • pro-drugs and pharmaceutically acceptable salts of the compounds of the invention pharmaceutically acceptable salts of such pro-drugs, and other bio-equivalents.
  • pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the agents disclosed herein, wherein said salts retain the desired biological activity of the parent agent and do not impart undesired toxicological effects thereto.
  • preferred examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, poly
  • compositions or formulations may be formulated into pharmaceutical compositions or formulations with one or more pharmaceutically acceptable carriers/excipients.
  • pharmaceutically acceptable as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.
  • carrier or “excipient” includes any and all solvents, diluents, buffers (such as, e.g., neutral buffered saline, phosphate buffered saline, or optionally Tris-HCl, acetate or phosphate buffers), solubilisers (such as, e.g., Tween 80, Polysorbate 80), colloids, dispersion media, vehicles, fillers, chelating agents (such as, e.g., EDTA or glutathione), amino acids (such as, e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavourings, aromatisers, thickeners, agents for achieving a depot effect, coatings, antifungal agents, preservatives (such as, e.g., ThimerosalTM, benzyl alcohol), antioxidants (such as, e
  • Illustrative, non-limiting carriers for use in formulating the pharmaceutical compositions include, for example, oil-in-water or water-in-oil emulsions, aqueous compositions with or without inclusion of organic co-solvents suitable for intravenous (IV) use, liposomes or surfactant-containing vesicles, particulate preparations with polymeric compounds such as inter alia polylactic acid or polyglycolic acid, microspheres, microbeads and microsomes, powders, tablets, capsules, suppositories, aqueous suspensions, aerosols, and other carriers apparent to one of ordinary skill in the art.
  • Pharmaceutical carriers may comprise sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • compositions of the invention may be formulated for essentially any route of administration, such as without limitation, oral administration (such as, e.g., oral ingestion or inhalation), intranasal administration (such as, e.g., intranasal inhalation or intranasal mucosal application), pulmonary (such as, e.g., by inhalation or insufflation of powders or aerosols), parenteral administration (such as, e.g., subcutaneous, intravenous, intra-arterial, intramuscular, intraperitoneal or intrasternal injection or infusion, or intracranial, e.g., intrathecal or intraventricular administration), epidermal and transdermal, or transmucosal (such as, e.g., oral, sublingual, intranasal) administration, topical administration (including inter alia ophthalmic administration), rectal, vaginal or intra-tracheal instillation, and the like.
  • oral administration such as, e.g.
  • the therapeutic effects attainable by the methods and compositions of the invention can be, for example, systemic, local, tissue-specific, etc., depending of the specific needs of a given application of the invention.
  • Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.
  • compositions may be formulated in the form of pills, tablets, lacquered tablets, coated (e.g., sugar-coated) tablets, granules, hard and soft gelatin capsules, aqueous, alcoholic or oily solutions, syrups, emulsions or suspensions.
  • preparation of oral dosage forms may be is suitably accomplished by uniformly and intimately blending together a suitable amount of the active compound in the form of a powder, optionally also including finely divided one or more solid carrier, and formulating the blend in a pill, tablet or a capsule.
  • Exemplary but non-limiting solid carriers include calcium phosphate, magnesium stearate, talc, sugars (such as, e.g., glucose, mannose, lactose or sucrose), sugar alcohols (such as, e.g., mannitol), dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
  • Compressed tablets containing the pharmaceutical composition can be prepared by uniformly and intimately mixing the active ingredient with a solid carrier such as described above to provide a mixture having the necessary compression properties, and then compacting the mixture in a suitable machine to the shape and size desired.
  • Moulded tablets maybe made by moulding in a suitable machine, a mixture of powdered compound moistened with an inert liquid diluent.
  • Suitable carriers for soft gelatin capsules and suppositories are, for example, fats, waxes, semisolid and liquid polyols, natural or hardened oils, etc.
  • compositions may be formulated with illustrative carriers, such as, e.g., as in solution with saline, polyethylene glycol or glycols, DPPC, methylcellulose, or in mixture with powdered dispersing agents, further employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilising or dispersing agents known in the art.
  • illustrative carriers such as, e.g., as in solution with saline, polyethylene glycol or glycols, DPPC, methylcellulose, or in mixture with powdered dispersing agents, further employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilising or dispersing agents known in the art.
  • Suitable pharmaceutical formulations for administration in the form of aerosols or sprays are, for example, solutions, suspensions or emulsions of the compounds of the invention or their physiologically tolerable salts in a pharmaceutically acceptable solvent, such as ethanol or water, or a mixture of such solvents.
  • a pharmaceutically acceptable solvent such as ethanol or water, or a mixture of such solvents.
  • the formulation can also additionally contain other pharmaceutical auxiliaries such as surfactants, emulsifiers and stabilizers as well as a propellant.
  • delivery may be by use of a single-use delivery device, a mist nebuliser, a breath-activated powder inhaler, an aerosol metered-dose inhaler (MDT) or any other of the numerous nebuliser delivery devices available in the art.
  • mist tents or direct administration through endotracheal tubes may also be used.
  • Examples of carriers for administration via mucosal surfaces depend upon the particular route, e.g., oral, sublingual, intranasal, etc.
  • illustrative examples include pharmaceutical grades of mannitol, starch, lactose, magnesium stearate, sodium saccharide, cellulose, magnesium carbonate and the like, with mannitol being preferred.
  • illustrative examples include polyethylene glycol, phospholipids, glycols and glycolipids, sucrose, and/or methylcellulose, powder suspensions with or without bulking agents such as lactose and preservatives such as benzalkonium chloride, EDTA.
  • the phospholipid 1,2 dipalmitoyl-sn-glycero-3-phosphocholine is used as an isotonic aqueous carrier at about 0.01-0.2% for intranasal administration of the compound of the subject invention at a concentration of about 0.1 to 3.0 mg/ml.
  • compositions may be advantageously formulated as solutions, suspensions or emulsions with suitable solvents, diluents, solubilisers or emulsifiers, etc.
  • suitable solvents are, without limitation, water, physiological saline solution or alcohols, e.g. ethanol, propanol, glycerol, in addition also sugar solutions such as glucose, invert sugar, sucrose or mannitol solutions, or alternatively mixtures of the various solvents mentioned.
  • the injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water.
  • Ringer's solution or isotonic sodium chloride solution or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • suitable dispersing or wetting and suspending agents such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • the compounds and pharmaceutically acceptable salts thereof of the invention can also be lyophilised and the lyophilisates obtained used, for example, for the production of injection or infusion preparations.
  • a carrier for intravenous use includes a mixture of 10% USP ethanol, 40% USP propylene glycol or polyethylene glycol 600 and the balance USP Water for Injection (WFI).
  • WFI Water for Injection
  • illustrative carriers for intravenous use include 10% USP ethanol and USP WFI; 0.01-0.1% triethanolamine in USP WFI; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI; and 1-10% squalene or parenteral vegetable oil-in-water emulsion.
  • Water or saline solutions and aqueous dextrose and glycerol solutions may be preferably employed as carriers, particularly for injectable solutions.
  • Illustrative examples of carriers for subcutaneous or intramuscular use include phosphate buffered saline (PBS) solution, 5% dextrose in WFI and 0.01-0.1% triethanolamine in 5% dextrose or 0.9% sodium chloride in USP WFI, or a 1 to 2 or 1 to 4 mixture of 10% USP ethanol, 40% propylene glycol and the balance an acceptable isotonic solution such as 5% dextrose or 0.9% sodium chloride; or 0.01-0.2% dipalmitoyl diphosphatidylcholine in USP WFI and 1 to 10% squalene or parenteral vegetable oil-in-water emulsions.
  • PBS phosphate buffered saline
  • aqueous formulations may comprise one or more surfactants.
  • the composition can be in the form of a micellar dispersion comprising at least one suitable surfactant, e.g., a phospholipid surfactant
  • suitable surfactant e.g., a phospholipid surfactant
  • phospholipids include diacyl phosphatidyl glycerols, such as dimyristoyl phosphatidyl glycerol (DPMG), dipalmitoyl phosphatidyl glycerol (DPPG), and distearoyl phosphatidyl glycerol (DSPG), diacyl phosphatidyl cholines, such as dimyristoyl phosphatidylcholine (DPMC), dipalmitoyl phosphatidylcholine (DPPC), and distearoyl phosphatidylcholine (DSPC); diacyl phosphatidic acids, such as dimyristoyl phosphatidic acid (
  • a surfactant:active substance molar ratio in an aqueous formulation will be from about 10:1 to about 1:10, more typically from about 5:1 to about 1:5, however any effective amount of surfactant may be used in an aqueous formulation to best suit the specific objectives of interest.
  • these formulations When rectally administered in the form of suppositories, these formulations may be prepared by mixing the compounds according to the invention with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify and/or dissolve in the rectal cavity to release the drug.
  • a suitable non-irritating excipient such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify and/or dissolve in the rectal cavity to release the drug.
  • Suitable carriers for microcapsules, implants or rods are, for example, copolymers of glycolic acid and lactic acid.
  • nucleic acids e.g., antisense and RNAi agents
  • the nucleic acid can be directly injected into the target cell/target tissue.
  • Other methods include fusion of the recipient cell with bacterial protoplasts containing the nucleic acid, the use of compositions like calcium chloride, rubidium chloride, lithium chloride, calcium phosphate, DEAE dextran, cationic lipids or liposomes or methods like receptor-mediated endocytosis, biolistic particle bombardment (“gene gun” method), infection with viral vectors, for example such as taught herein, electroporation, and the like.
  • nucleic acid molecules to target cells
  • techniques or methods which are suitable for delivering nucleic acid molecules to target cells include the continuous delivery of an NA molecule from poly(lactic-Co-Glycolic Acid) polymeric microspheres or the direct injection of protected (stabilized) NA molecule(s) into micropumps delivering the product.
  • Another possibility is the use of implantable drug-releasing biodegradable microspheres.
  • NA in various types of liposomes (immunoliposomes, PEGylated (immuno) liposomes), cationic lipids and polymers, nanoparticules or dendrimers, poly(lactic-Co-Glycolic Acid) polymeric microspheres, implantable drug-releasing biodegradable microspheres, etc; and co-injection of NA with protective agent like the nuclease inhibitor aurintricarboxylic acid. It shall be clear that also a combination of different above-mentioned delivery modes or methods may be used.
  • a preferred method of intracellular delivery of the antisense agents and RNAi agents disclosed herein may include infection with viral vectors as taught herein.
  • a recombinant viral vector as taught herein is brought in contact with a host cell, such as introduced (e.g., locally or systemically) to a host organism, and incubated at conditions favourable to viral infection and hence, makes use of the natural ability of a virus to infect a cell.
  • a retrovirus obtains entry to a host cell via the interaction of a retroviral protein with a transmembrane protein acting as a receptor on the surface of the host cell.
  • Another approach of viral vector-mediated delivery of antisense and RNAi agents as disclosed herein may encompass a physical cell entry-based technique, such as for example the use of ultrasound and microbubbles, in combination with viral vector-mediated delivery as described in WO 2006/129080.
  • nucleic acids such as antisense agents and RNAi agents may employ previously published methods.
  • intracellular delivery of the nucleic acids may be via a composition comprising an admixture of the nucleic acid molecule and an effective amount of a block copolymer.
  • An example of this method is described in US 2004/0248833.
  • colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes or liposome formulations.
  • Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in viva. These formulations may have net cationic, anionic or neutral charge characteristics and are useful characteristics with in vitro, in vivo and ex viva delivery methods.
  • RNA, and DNA can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley et al. 1981 (Trends Biochem ScL 6: 77).
  • a liposome In order for a liposome to be an efficient gene transfer vehicle, the following characteristics should be present: (1) encapsulation of the nucleic acid molecule of interest at high efficiency while not compromising their biological activity; (2) preferential and substantial binding to a target cell in comparison to non-target cells; (3) delivery of the aqueous contents of the vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino et al. 1988 (Biotechniques 6: 682).
  • the composition of the liposome is usually a combination of phospholipids, particularly high-phase-transition-temperature phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used.
  • the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
  • the nucleic acid molecule may be combined with other pharmaceutically acceptable carriers or diluents to produce a pharmaceutical composition.
  • suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline.
  • the composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral or transdermal administration.
  • the pharmaceutical formulations as disclosed herein may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques may generally include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the present active substances may be used alone or in combination with any other pharmaceutically or biologically active ingredient, particularly which is suitable for the treatment of diseases as taught herein (“combination therapy”).
  • Combination therapies as contemplated herein may comprise the administration of at least one active substance of the present invention and at least one other pharmaceutically or biologically active ingredient.
  • Said present active substance(s) and said pharmaceutically or biologically active ingredient(s) may be administered in either the same or different pharmaceutical formulation(s), simultaneously or sequentially in any order.
  • the dosage or amount of the present active substances used, optionally in combination with one or more other active compound to be administered, depends on the individual case and is, as is customary, to be adapted to the individual circumstances to achieve an optimum effect. Thus, it depends on the nature and the severity of the disorder to be treated, and also on the sex, age, body weight, general health, diet, mode and time of administration, and individual responsiveness of the human or animal to be treated, on the route of administration, efficacy, metabolic stability and duration of action of the compounds used, on whether the therapy is acute or chronic or prophylactic, or on whether other active compounds are administered in addition to the agent(s) of the invention.
  • a typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg of body weight or more, depending on the factors mentioned above.
  • the treatment is sustained until a desired suppression of disease symptoms occurs.
  • a preferred dosage of the active substance of the invention may be in the range from about 0.05 mg/kg to about 10 mg/kg of body weight.
  • one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient.
  • Such doses may be administered intermittently, e.g., every week or every two or three weeks.
  • a pharmaceutical composition may comprise between about 10 nM and about 1 ⁇ M, preferably between about 20 nM and about 600 nM, such as, e.g., about 100 nM or about 200 nM, or about 300 nM, or about 400 nM or about 500 nM of antisense agent or RNAi agent as taught herein.
  • subject or “patient” are used interchangeably and refer to animals, preferably warm-blooded animals, more preferably vertebrates, even more preferably mammals, still more preferably primates, and specifically includes human patients and non-human mammals and primates.
  • Preferred patients are human subjects.
  • a phrase such as “a subject in need of treatment” includes subjects that would benefit from treatment of a given condition, particularly of facioscapulohumeral muscular dystrophy (FSHD) or a tumour, such as preferably a sarcoma, such as more preferably a sarcoma selected from Ewing's family tumours, paediatric undifferentiated soft tissue sarcomas and rhabdomyosarcomas.
  • FSHD facioscapulohumeral muscular dystrophy
  • a tumour such as preferably a sarcoma, such as more preferably a sarcoma selected from Ewing's family tumours, paediatric undifferentiated soft tissue sarcomas and rhabdomyosarcomas.
  • Such subjects may include, without limitation, those that have been diagnosed with said condition, (hose prone to contract or develop said condition and/or those in whom said condition is to be prevented.
  • treat or “treatment” encompass both the therapeutic treatment of an already developed disease or condition, such as the therapy of an already developed FSHD or tumour, such as preferably a sarcoma, such as more preferably a sarcoma selected from Ewing's family tumours, paediatric undifferentiated soft tissue sarcomas and rhabdomyosarcomas, as well as prophylactic or preventative measures, wherein the aim is to prevent or lessen the chances of incidence of an undesired affliction, such as to prevent the chances of contraction and progression of FSHD or a tumour, such as preferably a sarcoma, such as more preferably a sarcoma selected from Ewing's family tumours, paediatric undifferentiated soft tissue sarcomas and rhabdomyosarcomas.
  • an already developed disease or condition such as the therapy of an already developed FSHD or tumour
  • a sarcoma such as more preferably a sarcoma selected
  • Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms or one or more biological markers, diminishment of extent of disease, stabilised (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and the like. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • prophylactically effective amount refers to an amount of an active compound or pharmaceutical agent that inhibits or delays in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician.
  • therapeutically effective amount refers to an amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a subject that is being sought by a researcher, veterinarian, medical doctor or other clinician, which may include inter cilia alleviation of the symptoms of the disease or condition being treated. Methods are known in the art for determining therapeutically and prophylactically effective doses for the present compounds.
  • Reference to “diseases or conditions comprising increased levels and/or increased activity of double homeobox 4 and/or double homeobox 4c” generally covers diseases and conditions in which the level and/or activity of DUX4 and/or DUX4c is increased by any (measurable) extent compared to a reference non-disease state, such as without limitation is increased by at least about 10%, e.g., by at least about 20%, preferably by at least about 30%, e.g., by at least about 40%, more preferably by at least about 50%, e.g., by at least about 75%, even more preferably by at least about 100%, e.g., by at least about 150%, 200%, 250%, 300%, 400% or by at least about 500%, compared to a reference non-disease state.
  • the term also covers situations in which a reference non-disease state comprises no demonstrable level and/or activity of DUX4 and/or DUX4c whereas a disease state comprises some demonstrable level and/or activity of DUX4 and/or DUX4c.
  • the level and/or activity of DUX4 and/or DUX4c may be increased in any cells, tissues and/or organs of a patient, preferably in cells, tissues and/or organs relevant to or affected in a disease or condition.
  • the level and/or activity of DUX4 and/or DUX4c may be increased in muscle cells and muscle tissues, such as, e.g., in myoblasts and/or in myocytes, in smooth muscles and/or in striated muscles such as in skeletal and or cardiac muscles, etc.
  • the level of DUX4 and/or DUX4c may be measured at any one or more stages, such as at the stage of pre-mRNA or mRNA (e.g., by qualitative or quantitative RT-PCR) and/or protein (e.g., immunoassay methods).
  • the activity of DUX4 and/or DUX4c protein may be measured by any suitable biochemical or cellular assays. e.g., measuring its trans-activation potential on known targets.
  • Facioscapulohumeral muscular dystrophy also known as Landouzy-Dejerine muscular dystrophy encompasses all diseases and condition known under these designations in the art.
  • FSHD as intended herein is an autosomal dominant muscle disorder genetically linked to contractions of the D4Z4 repeat array on the 4q35 subtelomeric region, more particularly where FSHD patients have between 1 and 10 D4Z4 copies, such as e.g., 2, 3, 4, 5, 6, 7, 8 or 9 copies.
  • FSHD may be linked to the 4qA allele, even more particularly to the permissive alleles 411161, 4A161 L, 4A159 or 4A168.
  • Double homeobox 4 and/or double homeobox 4c also specifically encompass those which comprise expression of DUX4 and/or DUX4c fragments or variants, more particularly expression of fusion proteins between DUX4 or DUX4c (preferably DUX4) and other, unrelated proteins, more preferably, such fusion proteins comprising the C-terminal fragment of DUX4 or DUX4c (preferably DUX4), even more preferably such fusion proteins with CIC, a human homolog of Drosophila capicua, or with EWSR1.
  • fusion proteins are commonly expressed as a result of chromosomal rearrangements and favour cell proliferation and hence, may cause tumours, such as in particular Ewing's family tumours (EFTS) (Kawamura-Saito et al. 2006, supra) and paediatric undifferentiated soft tissue sarcomas (Yoshimoto et al. 2009, supra) and rhabdomyosarcomas (Sirvent et al. 2009, supra), respectively.
  • EFTS Ewing's family tumours
  • RNAs include the sequence elements of the DUX4 or DUX4c genes (preferably the DUX4 gene) that are targeted by the antisense agents and/or RNAi agents as described herein, these tools may reduce the expression of said fusion proteins similarly as they reduce the expression of the full length DUX4 or DUX4c protein.
  • diseases and conditions intended herein also include tumours, more particularly sarcomas, even more particularly the aforementioned tumour types.
  • the term “tumour” refers to an abnormal mass of tissue that results from excessive cell division.
  • a tumour comprises “tumour cells” which are neoplastic cells with abnormal growth properties and may also comprise “tumour-associated non-tumour cells”, e.g., vascular cells which form blood vessels to supply the tumour.
  • a tumour may be benign or malignant.
  • the term “sarcoma” encompasses tumour types involving connective tissue cells, such as for example but without limitation bone, cartilage, fat cells, muscles and blood vessels.
  • Antisense oligomers were designed based on the sequence of the DUX4 gene 3′ UTR ( FIG. 1 ).
  • the 7 AO include one (JSR 1521 pLAM2A) directed against positions ⁇ 7 +18 of DUX4 intron 1-exon 2 boundary (incl. intron 1 splice acceptor site), and six AO directed against DUX4 introit 2-exon 3 boundary (incl.
  • An AO targeting dystrophin was used as negative control.
  • JSR 1662 mGMCSF3A ( ⁇ 05 + 20): UCCCACAGAAGCUAACAUGUGUGCAGAC
  • All AO used in this example had 2′-O-methyl-phosphorothioate backbone.
  • AO having phosphorodiamidate morpholino backbone are used with at least comparable or superior results.
  • AO conjugated to a cell penetrating peptide (CPP) are used with at least comparable or superior results.
  • AO antisense oligonucleotides
  • the cells were lysed 24 hours after transfection, and total protein extracts were prepared in NuPAGE® LDS sample buffer (Invitrogen). 15 ⁇ g of protein extracts were separated by electrophoresis (SOS-PAGE 12%), and transferred to a nitrocellulose membrane.
  • DUX4 52 kDa was detected on this Western blot with the 9A12 monoclonal antibody followed by anti-mouse IgG antibodies coupled to peroxidase (HRP), and revealed with Lumi-Light kit (Roche) detected on a film. After striping these antibodies, the same membrane was incubated with an anti-actin antibody to provide a loading control.
  • the mouse monoclonal antibody (MAb 9A12) was raised as described in Dixit et al., 2007 (supra) directed against the carboxyl terminal part of DUX4. Whereas this antibody cross reacts with DUX4c, the two proteins can be readily distinguished in Western blot based on their different apparent molecular weights, i.e., 52 kDa for DUX4 and 47 kDa for DUX4c.
  • RT Reverse transcription
  • 500 ng of DNase-treated myotube total RNA using the FirstChoice® RLM-RACE kit (Ambion).
  • 5 ⁇ l of the resulting cDNA were amplified by nested PCR with primers previously shown to be specific of the DUX4 mRNA 3′ UTR. (Dixit et al. 2007. supra).
  • GAPDH mRNA amplification was used as an internal control.
  • the RT-PCR products were analysed by electrophoresis on a 1% agarose gel. A densitometry of the bands was performed for quantification. Data were normalized to GAPDH mRNA levels.
  • FIG. 29 shows that the expected 550 bp DNA fragment was detected in FSHD myotubes treated with nc-AO and at a 30% and 50% reduced intensity in cells treated with AOs 1521 pLAM2A( ⁇ 7 +18) ( FIG. 29 a ) or 1523 pLAM3A( ⁇ 12 +13) ( FIG. 29 b ), respectively.
  • This amplicon was also observed in the positive control, i.e. C2C12 cells transfected with pGEM42, but not in the negative controls, i.e. either C2C12 cells transfected with the empty pGEM vector, or primary myoblasts from a healthy donor, or upon omission of reverse transcriptase.
  • the RT-PCR products were cloned and sequenced to confirm DUX4 mRNA amplification (data not shown).
  • siRNA Small Interfering RNA
  • Anti-DUX4 siRNA agents were developed based on and comprising the following DUX4 mRNA sequences:
  • siRNA-DUX41 acaccuggcuggcuacgga (SEQ ID NO: 48)
  • siRNA-DUX42 ggucuaggcccggugagag (SEQ ID NO: 49)
  • siRNA-DUX43 ccuggauuagaguuacauc.
  • Anti-DUX4c siRNA agents were developed based on the following DUX4c mRNA sequences:
  • siRNA-DUX4c1 ccagaguuucagcaaagg
  • siRNA-DUX4c2 ggagggcugucauucuuuuc
  • siRNA-DUX4c3 gcguucuucagucgaguug.
  • TE671 cells (cells derived from a human alveolar rhabdomyosarcoma) were used for transfections, using the “reverse” method recommended by the supplier, in which the transfection reagent is introduced into the culture dish before seeding the cells. This method was three times superior than the traditional method in our hands.
  • Transfection conditions were optimised using control anti-GAPDH siRNA supplied with the above kit. The optimised conditions included 2 ⁇ l SiPORTTM NeoFXTM reagent, 10 nM siRNA, and 5 ⁇ 10 4 cells/ml cell density.
  • siRNA directed against the DUX4c mRNA was tested using stable TE671-DUX4c lines established previously. These cells have incorporated the pAC1M2-DUX4c expression vector in which DUX4c transcription is inducible by doxycycline (DOX).
  • DOX doxycycline
  • the cells were first transfected using the above conditions, which lead to only a weak DUX4c inhibition. Therefore the siRNA concentration was increased to 20 nM that was not toxic to the cells.
  • the cells were seeded at a density of 1 ⁇ 10 5 cells/well of a 6 well culture dish and transfected with siRNA-DUX4cI 20 nm (“si”) by the reverse transfection method (Ambion), 4 hours after transfection, the expression of DUX4c was induced (“I”) by adding 1 mg/ml of doxycycline in the culture medium.
  • siRNA-DUX4cI 20 nm (“si”) by the reverse transfection method (Ambion)
  • I siRNA-DUX4cI 20 nm
  • the 3rd or 5th day after induction the cells were lysed and 20 ⁇ g of protein extracts were analyzed by SDS-PAGE electrophoresis (10%), and transferred to a nitrocellulose membrane.
  • DUX4c protein expression was also analysed by immunohistochemistry in TE-DUX4c cells transfected with siRNA-DUX4c1. The cells were transfected and DUX4c expression was induced as explained above.
  • the cells were transfected with siRNA-DUX4c 20 nM (“si1”, “si2” and “si3”) or negative control siRNA (“SiCN”) using reverse transfection (Ambion) and 4 hours later with the pCIneo-DUX4c vector (DUX4c).
  • the protein extracts were prepared and cells were fixed the third day after transfection of the pCIneo-DUX4c vector.
  • the methodologies for the Western blot and immunofluorescence were as set out above.
  • TE671 cells were transfected with siRNA-DUX4c (“siDUX4c”) or siRNA-DUX4 (“siDUX4”) (20 nM) using reverse transfection and 4 hours later with the pCIneo-DUX4 (“DUX4”) or pCIneo-DUX4c (“DUX4c”) expression vector.
  • the protein extracts were prepared on the third day after pCIneo vectors transfection and revealed by Western blot with the 9A12 monoclonal antibody and secondary antibodies coupled to HRP. The antibodies were then stripped, and the same membrane developed with an anti-actin serum(internal control).
  • siRNA specificity was confirmed by the disappearance, in western blot, of bands corresponding to the molecular weight of DUX4 or DUX4c following the addition of their respective siRNA and not with the siRNA of their homologue ( FIG. 16 ).
  • siRNAs that are processed in the cell to yield identical siRNAs to those that we selected ( FIG. 17 ). It has been previously demonstrated that myoblasts and myotubes could be efficiently transduced by such vectors.
  • synthetic DNA corresponding to the sequences of selected siRNA (sense sequence+loop (CTCGAG)+antisense sequence) were inserted into an expression vector containing the promoter of the histone H1 gene.
  • the transcription unit contains the following element: H1 promoter - - - CCGG(sense strand)loop(anti-sense strand)TTTTT - - - . Transcription produces shRNA which is processed to siRNA by Dicer (see FIG. 18 ).
  • the H1-shRNA gene was sub-cloned in a pLVTH vector containing all the necessary elements for encapsidation.
  • FIG. 19 shows Western blot analysis of DUX4 protein expression on extracts of TE671 cells transfected with the pCIneo-DUX4 expression vector alone (“TE+DUX4”) or with shRNA-DUX4 (“TE DUX4+shDUX4”) or shRNA-DUX4c (“TE DUX4+shDUX4c”) (Fugene 6, Roche Molecular Biochemical). 48 hours after transfection, proteins were extracted and 20 ⁇ g of these extracts were analyzed by SDS-PAGE electrophoresis (12%), then transferred to a nitrocellulose membrane. The membrane was incubated with 9A12 MAb followed by a secondary antibody coupled to peroxidase and revealed with the LiteABlot® kit (Euroclone).
  • Human FSHD primary myoblasts which are difficult to transfect, were transfected following the reverse transfection method as described above using the “Silencer siRNA Starter Kit” (Ambion).
  • Optimal transfection conditions defining an effective transfection reagent with low cytotoxicity for human primary myoblasts, were set up using control anti-GAPDH siRNA supplied with the above kit.
  • 72 hours after transfection cells were harvested and 10 ⁇ g of protein extracts were separated by SDS-PAGE (12%) and transferred to a nitrocellulose membrane. The protein transfer was confirmed by staining the membrane with Ponceau red.
  • Optimal transfection conditions included 4 ⁇ l SiPORTTM NeoFXTM reagent. 20 nM siRNA, and a cell density of 10 5 cells in a 35 mm culture dish ( FIG. 22 ).
  • siRNA directed against the DUX4 mRNA we transfected FSHD primary myoblasts with the siRNA using the transfection conditions specified above.
  • the cells were seeded at a density of 10 5 cells in a 35 mm culture dish and transfected with control siRNA or DUX4-siRNA3 following the reverse transfection method using 4 ⁇ l SiPORTTM NeoFXTM reagent. 3 different DUX4-siRNA3 concentrations were tested (10 nM, 20 nM and 30 nM) to determine the best concentration to use to reduce the endogenous DUX4 expression. Since the DUX4 protein is only detectable in myotubes, 4 hours after transfection, myoblasts differentiation was induced by replacing the culture medium by a medium without serum. Cells were harvested 72 h after differentiation and nuclear protein extracts were realised.
  • FSHD primary myoblasts were transfected with 10 aryl DUX4-siRNA 3 or control siRNA (30 nM) using the transfection conditions specified above. 4 hours after transfection, myoblast differentiation was induced as specified above. Following differentiation for 3 days, total RNA was extracted from myotubes. Reverse transcription (RT) was performed on 500 ng of DNase-treated myotube total RNA using the FirstChoice® RLM-RACE kit (Ambion).
  • the expected 550 bp DNA fragment was detected in FSHD myotubes transfected with the control siRNA (nc-siRNA) and at a 80% reduced intensity in cells treated with the DUX4-siRNA 3.
  • This amplicon was also observed in the positive control i.e. C2C12 cells transfected with the pGEM42 vector containing two D4Z4 units (Gabri ⁇ ls et al. 1999. supra) but not in primary myoblasts from a healthy donor (Cont), or upon omission of reverse transcriptase.
  • the RT-PCR products were cloned and sequenced to confirm DUX4 mRNA amplification (data not shown).
  • JSR 2245 pLAM polyA ( ⁇ 13 +6)
  • JSR 2250 pLAM1D (+7 ⁇ 18 around exon-1 intron-1 boundary
  • JSR 2250 pLAM1D binds to an exon-intron boundary, it does not in an initial experiment appear to interfere with splicing of DUX4.
  • JSR 2245 pLAM polyA ( ⁇ 13 + 6): GGGCAUUUUAAUAUAUCUCUGAACU (SEQ ID NO: 65)
  • Both AOs had 2′-O-methyl-phosphorothioate backbone.
  • the negative control was AO 1662 that targets the dystrophin mRNA and was used at a concentration of 600 nM.
  • the transfection reagent was LipofectamineTM 2000 (Invitrogen) used at a ratio of 1 ⁇ g AO/1 ⁇ l reagent.
  • the cells were lysed 24 hours after transfection, and total protein extracts were prepared in NuPAGE® LDS sample buffer (Invitrogen), 15 ⁇ g of protein extracts were separated by electrophoresis (SDS-PAGE 12%), and transferred to a nitrocellulose membrane.
  • DUX4 (52 kDa) was detected on this Western blot with the 9A12 monoclonal antibody followed by anti-mouse IgG antibodies coupled to peroxidase (HRP), and revealed with Lumi-Light kit (Roche) detected on a film. After striping these antibodies, the same membrane was incubated with an anti-actin antibody to provide a loading control.
  • AO 2245 was used at a concentration of 10, 25, 50, 100 or 150 nM.
  • the optimal concentration for specifically inhibiting DUX4 protein expression was found to be 50 nM ( FIG. 33 ).

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US11638761B2 (en) 2021-07-09 2023-05-02 Dyne Therapeutics, Inc. Muscle targeting complexes and uses thereof for treating Facioscapulohumeral muscular dystrophy
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US11898143B2 (en) 2024-02-13
US20140105873A1 (en) 2014-04-17
US20180265870A1 (en) 2018-09-20
US20210163942A1 (en) 2021-06-03
EP2426203B1 (en) 2016-07-13
EP2426203A3 (en) 2012-03-14
DK2426203T3 (en) 2016-11-07
US9988628B2 (en) 2018-06-05
US20170029814A1 (en) 2017-02-02
ES2596977T3 (es) 2017-01-13
EP2426203A2 (en) 2012-03-07
US20210163941A1 (en) 2021-06-03
US20200231966A1 (en) 2020-07-23
EP3118316A1 (en) 2017-01-18
US10907157B2 (en) 2021-02-02

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