Design of Phosphorodiamidate Morpholino Oligomers ( PMOs) for the Induction of Exon Skipping of the Human DMD Gene

Duchenne muscular dystrophy (DMD) is caused by out-of-frame mutations of the human DMD gene. Antisense oligonucleotides (AOs) have previously been used to skip additional exons that border the deletions such that the reading frame is restored and internally truncated, but functional, dystrophin expressed. We have designed phosphorodiamidate morpholino oligomer (PMO) AOs to various exons of the human dystrophin gene. PMOs were designed to have their target sites overlapping areas of open RNA structure, as defined by hybridization-array analysis, and likely exonic splicing enhancer (ESE)/silencer sites on the target RNA. The ability of each PMO to produce exon skipping was tested in vitro in normal human skeletal muscle cells. Retrospective analysis of design parameters used and PMO variables revealed that active PMOs were longer, bound to their targets more strongly, had their target sites closer to the acceptor splice site of the exon, overlapped areas of open conformation (as defined by the hybridization or the RNA secondary structure prediction software), and could interfere with the binding of certain SR proteins. No other parameter appeared to show significant association to PMO-skipping efficacy. No design tool is strong enough in isolation; however, if used in conjunction with other significant parameters it can aid AO design.