Dataset Information

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

ABSTRACT: 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 DMD 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 DMD 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 DMD 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 DMD 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.

SUBMITTER: Greer K

PROVIDER: S-EPMC7350004 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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

Greer Kane K Johnsen Russell R Nevo Yoram Y Fellig Yakov Y Fletcher Susan S Wilton Steve D SD

International journal of molecular sciences 20200625 12

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 ...[more]

PMID: 32630425

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Project description:BackgroundAntisense oligonucleotides (AOs) can interfere with exon recognition and intron removal during pre-mRNA processing, and induce excision of a targeted exon from the mature gene transcript. AOs have been used in vitro and in vivo to redirect dystrophin pre-mRNA processing in human and animal cells. Targeted exon skipping of selected exons in the dystrophin gene transcript can remove nonsense or frame-shifting mutations that would otherwise have lead to Duchenne Muscular Dystrophy, the most common childhood form of muscle wasting.ResultsAlthough many dystrophin exons can be excised using a single AO, several exons require two motifs to be masked for efficient or specific exon skipping. Some AOs were inactive when applied individually, yet pronounced exon excision was induced in transfected cells when the AOs were used in select combinations, clearly indicating synergistic rather than cumulative effects on splicing. The necessity for AO cocktails to induce efficient exon removal was observed with 2 different chemistries, 2'-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers. Similarly, other trends in exon skipping, as a consequence of 2'-O-methyl AO action, such as removal of additional flanking exons or variations in exon skipping efficiency with overlapping AOs, were also seen when the corresponding sequences were prepared as phosphorodiamidate morpholino oligomers.ConclusionThe combination of 2 AOs, directed at appropriate motifs in target exons was found to induce very efficient targeted exon skipping during processing of the dystrophin pre-mRNA. This combinatorial effect is clearly synergistic and is not influenced by the chemistry of the AOs used to induce exon excision. A hierarchy in exon skipping efficiency, observed with overlapping AOs composed of 2'-O-methyl modified bases, was also observed when these same sequences were evaluated as phosphorodiamidate morpholino oligomers, indicating design parameters established with one chemistry may be applied to the other.

Dataset Information

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

Publications

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

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