title: By-passing the nonsense mutation in the 4 CV mouse model of muscular dystrophy by induced exon skipping.
abstract: BACKGROUND
  Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder, is caused by protein-truncating mutations in the dystrophin gene. Absence of functional dystrophin renders muscle fibres more vulnerable to damage and necrosis. We report antisense oligomer (AO) induced exon skipping in the B6Ros.Cg-Dmd(mdx-4Cv)/J (4(CV)) mouse, a muscular dystrophy model arising from a nonsense mutation in dystrophin exon 53. Both exons 52 and 53 must be excised to remove the mutation and maintain the reading frame.
  
  
  METHODS
  A series of 2'-O-methyl modified oligomers on a phosphorothioate backbone (2OMeAOs) were designed and evaluated for the removal of each exon, and the most effective compounds were then combined to induce dual exon skipping in both myoblast cultures and in vivo. Exon skipping efficiency of 2OMeAOs and phosphorodiamidate morpholino oligomers (PMOs) was evaluated both in vitro and in vivo at the RNA and protein levels.
  
  
  RESULTS
  Compared to the original mdx mouse studies, induction of exon skipping from the 4(CV) dystrophin mRNA was far more challenging. PMO cocktails could restore synthesis of near-full length dystrophin protein in cultured 4(CV) myogenic cells and in vivo, after a single intramuscular injection.
  
  
  CONCLUSIONS
  By-passing the protein-truncating mutation in the 4(CV) mouse model of muscular dystrophy could not be achieved with single oligomers targeting both exons and was only achieved after the application of AO cocktails to remove exons 52 and 53. As in previous studies, the stability and efficiency of PMOs proved superior to 2OMeAOs for consistent and sustained protein induction in vivo.
authors.affiliation: Centre for Neuromuscular and Neurological Disorders, University of Western Australia, QE II Medical Centre, Nedlands, Australia.
authors.forename: Chalermchai
authors.initials: C
authors.lastname: Mitrpant
authors.forename: Sue
authors.initials: S
authors.lastname: Fletcher
authors.forename: Patrick L
authors.initials: PL
authors.lastname: Iversen
authors.forename: Steve D
authors.initials: SD
authors.lastname: Wilton
mesh_terms: D000818:Animals; D018389:Codon, Nonsense / Q000235:genetics*; D016189:Dystrophin / Q000235:genetics; D005091:Exons / Q000235:genetics*; D007273:Injections, Intramuscular; D051379:Mice; D018101:Mice, Inbred mdx; D023421:Models, Animal; D009025:Morpholines / Q000008:administration & dosage / Q000737:chemistry / Q000627:therapeutic use; D060172:Morpholinos; D009137:Muscular Dystrophy, Animal / Q000188:drug therapy / Q000235:genetics*; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000737:chemistry / Q000627:therapeutic use
chemical_list: D018389:Codon, Nonsense; D016189:Dystrophin; D009025:Morpholines; D060172:Morpholinos; D016376:Oligonucleotides, Antisense