Bubble Liposomes and Ultrasound Exposure Improve Localized Morpholino Oligomer Delivery into the Skeletal Muscles of Dystrophic mdx Mice

Duchenne muscular dystrophy (DMD) is a genetic disorder that is caused by mutations in the DMD gene that lead to an absence of functional protein. The mdx dystrophic mouse contains a nonsense mutation in exon 23 of the dystrophin gene; a phosphorodiamidate morpholino oligomer (PMO) designed to skip this mutated exon in the mRNA induces dystrophin expression. However, an efficient PMO delivery method is needed to improve treatment strategies for DMD. We previously developed polyethylene glycol (PEG)-modified liposomes (Bubble liposomes; BLs) that entrap ultrasound (US) contrast gas and demonstrated that the combination of BLs with US exposure is an effective gene delivery tool in vitro and in vivo. In this study, to evaluate the ability of BLs as a PMO delivery tool, we tested the potency of the BLs combined with US exposure to boost the delivery of PMO and increase the skipping of the mutated exon in the mdx mouse. The results indicated that the combination of BLs and US exposure increased the uptake of the PMO targeting a nonsense mutation in exon 23 of the dystrophin gene and consequently increased the PMO-mediated exon-skipping efficiency compared with PMO injection alone, leading to significantly enhanced dystrophin expression. This increased efficiency indicated the potential of the combination of BL with US exposure to enhance PMO delivery for treating DMD. Thus, this US-mediated BL technique may provide an effective, non-invasive, non-viral method for PMO therapy for DMD muscle as well as for other muscular dystrophies.