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

Abstract

AbstractDuchenne muscular dystrophy is a common and devastating genetic disease that is 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, which restores dystrophin expression, but creates 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, of possibly different properties and therapeutic consequences. Here, we examined three such dystrophin exon skipped edits, comprising two pairs of alternative repairs of the same underlying DMD defect. We found that in both cases, one member of each alternative repair was more stable than the other by a variety of thermodynamic and biochemical measures. We also examined the origin of these differences by 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 which caused domain-localized perturbations, while in another there was unfolding at the protein domain junctions distal to the edit site which 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.