Exons 45-55 skipping using mutation-tailored cocktails of antisense morpholinos in the DMD gene

Mutations in the dystrophin (DMD) gene and consequent loss of dystrophin cause Duchenne muscular dystrophy (DMD). A promising therapy for DMD, single-exon skipping using antisense phosphorodiamidate morpholino oligomers (PMOs), currently confronts major issues in that an antisense drug induces the production of functionally undefined dystrophin and may not be similarly efficacious among patients with different mutations. Accordingly, the applicability of this approach is limited to out-of-frame mutations. Here, using an exon-skipping efficiency predictive tool, we designed three different PMO-cocktail sets for exons 45-55 skipping aiming to produce a dystrophin variant with preserved functionality as seen in milder/asymptomatic individuals with an in-frame exons 45-55 deletion. Of them, the most effective set was composed of select PMOs that each efficiently skips an assigned exon in cell-based screening. These combinational PMOs fitted to different deletions of immortalized DMD patient-muscle cells significantly induced exons 45-55 skipping with removing three, eight or ten exons and dystrophin restoration as represented by Western blotting. In vivo skipping of the maximum eleven human DMD exons was confirmed in humanized mice. The finding indicates that our PMO set can be used to create mutation-tailored cocktails for exons 45-55 skipping and treat over 65% DMD patients carrying out-of- or in-frame deletions.