Targeted Exon Skipping to Correct Exon Duplications in the Dystrophin Gene

• Main Authors: Kane L Greer, Hanns Lochmüller, Kevin Flanigan, Susan Fletcher, Steve D Wilton
• Format: Article
• Language: English
• Published: Elsevier 2014-01-01
• Series: Molecular Therapy: Nucleic Acids
• Subjects: antisense oligomers; Duchenne muscular dystrophy; duplication mutations; dystrophin; exon skipping
• Online Access: http://www.sciencedirect.com/science/article/pii/S2162253116302955

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.