Development of a Gene and Nucleic Acid Delivery System for Skeletal Muscle Administration via Limb Perfusion Using Nanobubbles and Ultrasound.

Strategies for gene and nucleic acid delivery to skeletal muscles have been extensively explored to treat Duchenne muscular dystrophy (DMD) and other neuromuscular diseases. Of these, effective intravascular delivery of naked plasmid DNA (pDNA) and nucleic acids into muscles is an attractive approach, given the high capillary density in close contact with myofibers. We developed lipid-based nanobubbles (NBs) using polyethylene-glycol-modified liposomes and an echo-contrast gas and found that these NBs could improve tissue permeability by ultrasound (US)-induced cavitation. Herein, we delivered naked pDNA or antisense phosphorodiamidate morpholino oligomers (PMOs) into the regional hindlimb muscle via limb perfusion using NBs and US exposure. pDNA encoding the luciferase gene was injected with NBs via limb perfusion into normal mice with application of US. High luciferase activity was achieved in a wide area of the limb muscle. DMD model mice were administered PMOs, designed to skip the mutated exon 23 of the dystrophin gene, with NBs via intravenous limb perfusion, followed by US exposure. The number of dystrophin-positive fibers increased in the muscles of mdx mice. Combining NBs and US exposure, which can be widely delivered to the hind limb muscles via the limb vein, could be an effective therapeutic approach for DMD and other neuromuscular disorders.

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) that entrap ultrasound contrast gas and demonstrated that the combination of Bubble liposomes with ultrasound exposure is an effective gene delivery tool in vitro and in vivo. In this study, to evaluate the ability of Bubble liposomes as a PMO delivery tool, we tested the potency of the Bubble liposomes combined with ultrasound 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 Bubble liposomes and ultrasound 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 Bubble liposomes with ultrasound exposure to enhance PMO delivery for treating DMD. Thus, this ultrasound-mediated Bubble liposome technique may provide an effective, noninvasive, nonviral method for PMO therapy for DMD muscle as well as for other muscular dystrophies.