Studies have shown that ultrasound, used either alone or in combination with
microbubble contrast agents, can increase cell membrane permeability to plasmid
DNA. Because ultrasound is a non-painful and well-established tool in clinical medicine, its potential to enhance
DNA uptake into the muscles of patients with
muscular dystrophy is conceptually attractive. Therefore, we evaluated the ability of ultrasound pulses (1 MHz; 1.5 W/cm2) to increase exogenous (LacZ) gene expression in normal wild-type and dystrophic Dmd(mdx/mdx) mice after plasmid
DNA injection into muscle. We also ascertained whether co-injection of
lipid-encapsulated
perfluoropropane microbubbles (
Definity) or pretreatment with
hyaluronidase could further increase the level of gene transfer to ultrasound-treated muscles. The use of ultrasound did not increase transfection efficiency in normal mice. In contrast, dystrophic mice demonstrated an increase in the number of transfected fibers (threefold) as well as the amount of LacZ
protein (22-fold) after ultrasound exposure, provided that
Definity was also co-injected with the
DNA. Pretreatment of muscles with
hyaluronidase before ultrasound exposure was not effective in augmenting the level of gene transfer. Under the optimal conditions for dystrophic muscle transfection (ultrasound +
Definity), there was no associated increase in muscle damage. Hence ultrasound may provide a safe and effective method for enhancing gene transfer to dystrophic muscles, thereby increasing the prospects for therapeutic application of naked
DNA in
muscular dystrophy patients.