Single Exon Skipping Can Address a Multi-Exon Duplication in the Dystrophin Gene

• Main Authors: Kane Greer, Russell Johnsen, Yoram Nevo, Yakov Fellig, Susan Fletcher, Steve D. Wilton
• Format: Article
• Language: English
• Published: MDPI AG 2020-06-01
• Series: International Journal of Molecular Sciences
• Subjects: Duchenne muscular dystrophy; dystrophin; antisense oligomers; duplication mutations; exon skipping; splicing
• Online Access: https://www.mdpi.com/1422-0067/21/12/4511
• ISSN: 1661-6596; 1422-0067

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease typically caused by protein-truncating mutations that preclude synthesis of a functional dystrophin. Exonic deletions are the most common type of <i>DMD</i> lesion, however, whole exon duplications account for between 10–15% of all reported mutations. Here, we describe in vitro evaluation of antisense oligonucleotide-induced splice switching strategies to re-frame the transcript disrupted by a multi-exon duplication within the <i>DMD</i> gene. Phosphorodiamidate morpholino oligomers and phosphorodiamidate morpholino oligomers coupled to a cell penetrating peptide were evaluated in a Duchenne muscular dystrophy patient cell strain carrying an exon 14–17 duplication. Two strategies were employed; the conventional approach was to remove both copies of exon 17 in addition to exon 18, and the second strategy was to remove only the first copy of exon 17. Both approaches result in a larger than normal but in-frame <i>DMD</i> transcript, but surprisingly, the removal of only the first exon 17 appeared to be more efficient in restoring dystrophin, as determined using western blotting. The emergence of a normal sized <i>DMD</i> mRNA transcript that was not apparent in untreated samples may have arisen from back splicing and could also account for some of the dystrophin protein being produced.