Dosing regimen has a significant impact on the efficiency of morpholino oligomer-induced exon skipping in mdx mice

Duchenne Muscular Dystrophy (DMD) is a myodegenerative disorder caused primarily by mutations that create premature termination of dystrophin translation. The antisense oligonucleotide (AO) approach for skipping dystrophin exons allows restoration of the correct reading frame in the dystrophin transcript thus producing a shorter protein. A similar approach in humans would result in the conversion of DMD to the milder Becker Muscular Dystrophy. It has been demonstrated previously that repeated intravascular injection of phosphorodiamidate morpholino oligomers (PMOs) in the mdx mouse induces more dystrophin expression than a single injection, but this approach is very costly, and data demonstrating the safety of high doses of systemically-injected PMO are unavailable. Furthermore, several recent publications have demonstrated the efficacy of peptide-conjugated PMOs, but the clinical applicability of such compounds is unclear at this stage. Here, we report that multiple intravascular injections of low doses of naked PMO show significantly more dystrophin positive fibres in a variety of muscle groups, 8 weeks after administration compared to a single dose of the same total amount. After administration of a total of 200mg/kg of PMO, histological features, such as the cross sectional area, centronucleation index and expression of the dystrophin-associated protein complex, showed a significant improvement in mice treated by repeated injection. Furthermore, four administrations of just 5mg/kg induced a significant amount of dystrophin expression. These results clearly demonstrate the key role of the optimisation of dosing regimen for the systemic administration of PMO in patients, and support the clinical feasibility of this approach with naked PMO.