Empirical and Computational Comparison of Alternative Therapeutic Exon Skip Repairs for Duchenne Muscular Dystrophy.

ABSTRACT

Duchenne muscular dystrophy (DMD) is a common and devastating genetic disease primarily caused by exon deletions that create a genetic frameshift in dystrophin. Exon skipping therapy seeks to correct this by masking an exon during the mRNA maturation process, restoring dystrophin expression, but creating an edited protein missing both the original defect and the therapeutically skipped region. Crucially, it is possible to correct many defects in alternative ways, by skipping an exon either before or after the patient's defect. This results in alternatively edited, hybrid proteins that might have different properties and therapeutic consequences. We examined three such dystrophin exon-skipped edits, Δe45-53, Δe46-54, and Δe47-55, comprising two pairs of alternative repairs of Δe46-53 and Δe47-54 DMD defects. We found that in both cases, Δe46-54 was the more stable repair as determined by a variety of thermodynamic and biochemical measurements. We also examined the origin of these differences with molecular dynamics simulations, which showed that these stability differences were the result of different types of structural perturbations. For example, in one edit there was partial unfolding at the edit site that caused domain-localized perturbations while in another there was unfolding at the protein domain junctions distal to the edit site that increased molecular flexibility. These results demonstrate that alternative exon skip repairs of the same underlying defect can have very different consequences at the level of protein structure and stability and furthermore that these can arise by different mechanisms, either locally or by more subtle long-range perturbations.