Rational design of antisense oligomers to induce dystrophin exon skipping.

Duchenne muscular dystrophy (DMD), one of the most severe neuromuscular disorders of childhood, is caused by the absence of a functional dystrophin. Antisense oligomer (AO) induced exon skipping is being investigated to restore functional dystrophin expression in models of muscular dystrophy and DMD patients. One of the major challenges will be in the development of clinically relevant oligomers and exon skipping strategies to address many different mutations. Various models, including cell-free extracts, cells transfected with artificial constructs, or mice with a human transgene, have been proposed as tools to facilitate oligomer design. Despite strong sequence homology between the human and mouse dystrophin genes, directing an oligomer to the same motifs in both species does not always induce comparable exon skipping. We report substantially different levels of exon skipping induced in normal and dystrophic human myogenic cell lines and propose that animal models or artificial assay systems useful in initial studies may be of limited relevance in designing the most efficient compounds to induce targeted skipping of human dystrophin exons for therapeutic outcomes.

Antisense suppression of donor splice site mutations in the dystrophin gene transcript.

We describe two donor splice site mutations, affecting dystrophin exons 16 and 45 that led to Duchenne muscular dystrophy (DMD), through catastrophic inactivation of the mRNA. These gene lesions unexpectedly resulted in the retention of the downstream introns, thereby increasing the length of the dystrophin mRNA by 20.2 and 36 kb, respectively. Splice-switching antisense oligomers targeted to exon 16 excised this in-frame exon and the following intron from the patient dystrophin transcript very efficiently in vitro, thereby restoring the reading frame and allowing synthesis of near-normal levels of a putatively functional dystrophin isoform. In contrast, targeting splice-switching oligomers to exon 45 in patient cells promoted only modest levels of an out-of-frame dystrophin transcript after transfection at high oligomer concentrations, whereas dual targeting of exons 44 and 45 or 45 and 46 resulted in more efficient exon skipping, with concomitant removal of intron 45. The splice site mutations reported here appear highly amenable to antisense oligomer intervention. We suggest that other splice site mutations may need to be evaluated for oligomer interventions on a case-by-case basis.

Personalized exon skipping strategies to address clustered non-deletion dystrophin mutations.

Antisense oligomer induced exon skipping is showing promise as a therapy to reduce the severity of Duchenne muscular dystrophy. To date, the focus has been on excluding single exons flanking frame-shifting deletions in the dystrophin gene. However, a third of all Duchenne muscular dystrophy causing mutations are more subtle DNA changes. Thirty nine dystrophin exons are potentially frame-shifting and mutations in these will require the targeted removal of exon blocks to generate in-frame transcripts. We report that clustered non-deletion mutations in the dystrophin gene respond differently to different antisense oligomer preparations targeting the same dual exon block, the removal of which bypasses the mutation and restores the open reading-frame. The personalized nature of the responses to antisense oligomer application presents additional challenges to the induction of multi-exon skipping with a single oligomer preparation.