title: Targeted exon skipping to correct exon duplications in the dystrophin gene.
abstract: Duchenne muscular dystrophy is a severe muscle-wasting disease caused by mutations in the dystrophin gene that ablate functional protein expression. Although exonic deletions are the most common Duchenne muscular dystrophy lesion, duplications account for 10-15% of reported disease-causing mutations, and exon 2 is the most commonly duplicated exon. Here, we describe the in vitro evaluation of phosphorodiamidate morpholino oligomers coupled to a cell-penetrating peptide and 2'-O-methyl phosphorothioate oligonucleotides, using three distinct strategies to reframe the dystrophin transcript in patient cells carrying an exon 2 duplication. Differences in exon-skipping efficiencies in vitro were observed between oligomer analogues of the same sequence, with the phosphorodiamidate morpholino oligomer coupled to a cell-penetrating peptide proving the most effective. Differences in exon 2 excision efficiency between normal and exon 2 duplication cells, were apparent, indicating that exon context influences oligomer-induced splice switching. Skipping of a single copy of exon 2 was induced in the cells carrying an exon 2 duplication, the simplest strategy to restore the reading frame and generate a normal dystrophin transcript. In contrast, multiexon skipping of exons 2-7 to generate a Becker muscular dystrophy-like dystrophin transcript was more challenging and could only be induced efficiently with the phosphorodiamidate morpholino oligomer chemistry.
authors.affiliation: 1] Australian Neuromuscular Research Institute, Murdoch University, Perth, Western Australia, Australia [2] Centre for Neuromuscular & Neurological Disorders, University of Western Australia, Crawley, Western Australia, Australia.
authors.forename: Kane L
authors.initials: KL
authors.lastname: Greer
authors.affiliation: Institute of Genetic Medicine, International Centre for Life, University of Newcastle, Newcastle, UK.
authors.forename: Hanns
authors.initials: H
authors.lastname: Lochmüller
authors.affiliation: Nationwide Children's Hospital, Columbus, Ohio, USA.
authors.forename: Kevin
authors.initials: K
authors.lastname: Flanigan
authors.affiliation: 1] Australian Neuromuscular Research Institute, Murdoch University, Perth, Western Australia, Australia [2] Centre for Neuromuscular & Neurological Disorders, University of Western Australia, Crawley, Western Australia, Australia [3] Centre for Comparative Genomics, Murdoch University, Murdoch, Western Australia, Australia.
authors.forename: Susan
authors.initials: S
authors.lastname: Fletcher
authors.affiliation: 1] Australian Neuromuscular Research Institute, Murdoch University, Perth, Western Australia, Australia [2] Centre for Neuromuscular & Neurological Disorders, University of Western Australia, Crawley, Western Australia, Australia [3] Centre for Comparative Genomics, Murdoch University, Murdoch, Western Australia, Australia.
authors.forename: Steve D
authors.initials: SD
authors.lastname: Wilton