Search

US-12624396-B2 - Methods for alleviating facioscapulohumeral dystrophy (FSHD) by an antisense nucleic acid molecule inhibiting the expression of double homeobox 4 (DUX4)

US12624396B2US 12624396 B2US12624396 B2US 12624396B2US-12624396-B2

Abstract

In one aspect, the invention provides a method of screening a human subject to determine if said subject has a genetic predisposition to develop, or is suffering from Facioscapulohumeral Dystrophy (FSHD), said method comprising: (a) providing a biological sample comprising genomic DNA from the subject; and (b) analyzing the portion of the genomic DNA in the sample corresponding to the distal D4Z4-pLAM region on chromosome 4 and determining the presence or absence of a polymorphism resulting in a functional polyadenylation sequence operationally linked to exon 3 of the DUX4 gene.

Inventors

  • Silvere M. van der Maarel
  • Stephen J. Tapscott
  • Rabi Tawil
  • Richard J.L.F. Lemmers
  • Linda Geng
  • Lauren Snider

Assignees

  • FRED HUTCHINSON CANCER CENTER
  • UNIVERSITY OF ROCHESTER
  • LEIDEN UNIVERSITY MEDICAL CENTER

Dates

Publication Date
20260512
Application Date
20201207

Claims (16)

  1. 1 . A method for alleviating Facioscapulohumeral Dystrophy (FSHD) in a subject having FSHD, the method comprising administering to the subject a Double homeobox, 4 full length (DUX4-fl) inhibitor agent, wherein the DUX4-fl inhibitor agent comprises a Double homeobox, 4 (DUX4) antisense nucleic acid molecule that is identical to a portion of the DUX4 gene and hybridizes to DUX4 mRNA, and wherein the DUX4 antisense nucleic acid molecule inhibits production of DUX4.
  2. 2 . The method of claim 1 , wherein the subject has a functional polyadenylation sequence operationally linked to exon 3 of the DUX4 gene at the distal D4Z4-pLAM region of their genome.
  3. 3 . The method of claim 2 , wherein the polyadenylation sequence is present at the pLAM region located at nucleotides 8046-8051 of SEQ ID NO:5.
  4. 4 . The method of claim 1 , wherein the antisense nucleic acid molecule is an antisense oligonucleotide.
  5. 5 . The method of claim 1 , wherein the antisense nucleic acid molecule is identical to non-coding segments of the DUX4 gene.
  6. 6 . The method of claim 1 , wherein the antisense nucleic acid molecule is identical to at least 8 nucleotides of the DUX4 gene.
  7. 7 . The method of claim 1 , wherein the antisense nucleic acid molecule is administered systemically.
  8. 8 . The method of claim 1 , wherein the subject is a human subject.
  9. 9 . The method of claim 7 , wherein the subject is administered a DUX4 antisense oligonucleotide in a composition and wherein the composition comprises a fatty acid, and wherein the composition is administered parenterally.
  10. 10 . A method for alleviating Facioscapulohumeral Dystrophy (FSHD) in a subject having FSHD, the method comprising administering to the subject a composition comprising Double homeobox, 4 full length (DUX4-fl) inhibitor agent and a fatty acid, wherein the DUX4-fl inhibitor agent comprises a Double homeobox, 4 (DUX4) antisense nucleic acid molecule that is identical to a portion of the DUX4 gene and hybridizes to DUX4 m RNA, and wherein the DUX4 antisense nucleic acid molecule inhibits production of DUX4; and wherein the composition is administered parenterally.
  11. 11 . The method of claim 10 , wherein the subject has a functional polyadenylation sequence operationally linked to exon 3 of the DUX4 gene at the distal D4Z4-pLAM region of their genome.
  12. 12 . The method of claim 11 , wherein the polyadenylation sequence is present at the pLAM region located at nucleotides 8046-8051 of SEQ ID NO:5.
  13. 13 . The method of claim 10 , wherein the antisense nucleic acid molecule is an antisense oligonucleotide.
  14. 14 . The method of claim 10 , wherein the antisense nucleic acid molecule is identical to non-coding segments of the DUX4 gene.
  15. 15 . The method of claim 10 , wherein the antisense nucleic acid molecule is identical to at least 8 nucleotides of the DUX4 gene.
  16. 16 . The method of claim 10 , wherein the subject is a human subject.

Description

CROSS-REFERENCE(S) TO RELATED APPLICATIONS This application is a continuation of U.S. application Ser. No. 13/817,531, filed May 23, 2013, now U.S. Pat. No. 10,865,445, which is a national phase application under 35 U.S.C. § 371 of International Application No. PCT/US2011/048318 filed Aug. 18, 2011, which claims priority to U.S. Provisional Application No. 61/374,967, filed Aug. 18, 2010; U.S. Provisional Application No. 61/384,609, filed Sep. 20, 2010; U.S. Provisional Application No. 61/513,456, filed Jul. 29, 2011; and U.S. Provisional Application No. 61/513,467, filed Jul. 29, 2011, the disclosures of which are incorporated herein by reference. STATEMENT OF GOVERNMENT LICENSE RIGHTS This invention was made with Government support under grant NS069539, AR045203, and AR045113, awarded by National Institutes of Health. The Government has certain rights in the invention. STATEMENT REGARDING SEQUENCE LISTING The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is: FHCCP0039USC1_ST25.txt. The text file is 65866 bytes and was created on Jan. 18, 2023. BACKGROUND Facioscapulohumeral dystrophy (FSHD) is the third most common muscular dystrophy. The mutation that causes FSHD was identified nearly 20 years ago (Wijmenga et al., Nat. Genet. 2:26-30 (1992)), yet the molecular mechanism(s) of the disease remains elusive. The most prevalent form of FSHD (FSHD1) is caused by the deletion of a subset of D4Z4 macrosatellite repeats in the subtelomeric region of chromosome 4q. Unaffected individuals have 11-100 of the 3.3 kb D4Z4 repeat units, whereas FSHD1 individuals have 10 or fewer repeats. At least one repeat unit appears necessary for FSHD because no case has been identified with a complete deletion of D4Z4 repeats (Tuplet et al., J. Med. Genet. 33:366-370 (1996)). Each repeat unit contains a copy of the double homeobox retrogene DUX4 (Clapp et al., Am. J. Hum. Genet. 81:264-279 (2007); Gabriels et al., Gene 236:25-32 (1999); Lyle et al., Genomics 28:389-397 (1995)), and inappropriate expression of DUX4 was initially proposed as a possible cause of FSHD. This was supported by the observations that repeat contraction is associated with decreased repressive epigenetic marks in the remaining D4Z4 units (van Overveld et al., Nat. Genet. 35:315-317 (2003); Zeng et al., PloS Genet 5, e1000559 (2009)), and that overexpression of the DUX4 protein in a variety of cells, including skeletal muscle, causes apoptotic cell death (Kowaljow et al., Neuromuscul. Disord. 17:611-623 (2007); Wallace et al., Ann. Neurol. 69:540-552 (2011); Wuebbles et al., Int. J. Clin. Exp. Pathol. 3:386-400 (2010)). However, initial attempts to identify DUX4 mRNA transcripts in FSHD muscle were unsuccessful, leading to the suggestion that other genes in the region were causative for FSHD (Gabellini et al., Cell 110:339-348 (2002); Klooster et al., Eur. J. Hum. Genet. 17:1615-1624 (2009); Laoudj-Chenivesse et al., J. Mol. Med. 83:216-224 (2005); Reed et al., Exp. Neurol. 205: 583-586 (2007)). Currently, the diagnostic test for FSHD1 requires pulse-field gel electrophoresis and Southern blotting to detect the contraction of the D4Z4 repeats, and there are no commercially available diagnostic tests for FSHD2. FSHD1 can be genetically diagnosed by the contraction of the number of D4Z4 repeats to 10 or fewer repeats on a specific 4qA haplotype. FSHD2 is associated with decreased DNA methylation of the D4Z4 repeats on the same 4qA haplotype. The methods described herein provide an improved method for screening for the presence or risk of developing FSHD and for the treatment of FSHD, and apply both to FSHD1 and FSHD2. SUMMARY This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. In one aspect, the invention provides a method of screening a human subject to determine if said subject has a genetic predisposition to develop, or is suffering from Facioscapulohumeral Dystrophy (FSHD). The method according to this aspect of the invention comprises: (a) providing a biological sample comprising genomic DNA from the subject; and (b) analyzing the portion of the genomic DNA in the sample corresponding to the distal D4Z4-pLAM region on chromosome 4 and determining the presence or absence of a polymorphism resulting in a functional polyadenylation sequence operationally linked to exon 3 of the DUX4 gene, wherein a determination of the absence of a functional polyadenylation sequence operationally linked to exon 3 of the DUX4 gene indicates that the subject does not have a genetic predisposition to develop, and is not suffering from