Exonic sequences provide better targets for antisense oligonucleotides than splice site sequences in the modulation...

Antisense-mediated exon skipping is currently the most promising therapeutic approach for Duchenne muscular dystrophy (DMD). The rationale is to use antisense oligonucleotides (AONs) to hide exons from the splicing machinery, causing them to be skipped from the mature mRNA. Thus, the mutated, out-of-frame dystrophin transcripts as seen in DMD are reframed, allowing the generation of internally deleted, partly functional dystrophin proteins, rather than prematurely truncated, nonfunctional ones. This approach is mutation specific, so multiple AONs targeting all internal DMD exons have been designed and tested. Here, we have retrospectively compared our own set of 156 exon-internal AONs and 256 AONs as present in patents and publications from Dr. Wilton (Australia), which includes exon-internal as well as splice site-targeting AONs. Effective AONs are significantly more often exon-internal and, as anticipated, have better thermodynamic properties. Comparison of splice site and exon-internal AONs revealed that exon-internal AONs are more efficient and target more predicted exonic splicing enhancer and less predicted exon splicing silencer sites, but also have better thermodynamic properties. This suggests that exons may be better AON targets than introns per se, because of their higher GC content, which generally will result in improved AON binding.