Induced exon skipping: AO design is more important than chemistry

Duchenne muscular dystrophy (DMD), a genetic disorder that arises from protein truncating mutations in the dystrophin gene, is characterised by severe and progressive muscle wasting. Mutations causing DMD can occur in any of the 79 exons or introns of this massive gene (2.4 Mb), and antisense oligonucleotide (AO) induced exon skipping is emerging as a novel potential therapy, with two separate trials underway. Our rationale in AO design to address protein truncating dystrophin mutations by induced exon skipping is to prepare 2_-O-methyl modified bases on a phosphorothioate backbone that will anneal to potential splicemotifs and alter dystrophin pre-mRNA processing. These compounds can be prepared in-house and, when transfected as a cationic lipoplexes, are readily taken up in vitro. Overlapping 2OMeAOs are prepared to pre-mRNA motifs shown to influence splicing, in an attempt to optimise AO design. Titration studies then allow identification of the most effective AOs, those that induce pronounced exon excision at low transfection concentrations. Although the 2OMeAOs are being used in clinical trials in the Netherlands, we have found that phosphorodiamidate morpholino oligomers (PMOs) are better suited for in vivo applications. However, PMOs are not taken up efficiently in vitro, impeding design of optimal compounds. We present data showing that the hierarchy of exon skipping efficiency generated with overlapping 2OMeAOs was identical to that observed with the corresponding PMOs