In vivo transfer of a reporter gene into the ocular ciliary muscle mediated by ultrasound and microbubbles

Our goal is to assess the feasibility of low-intensity ultrasound (US) combined with echocontrast agent microbubbles (MB) to transfect the ciliary muscle which has previously been demonstrated to potentially allow the secretion of therapeutic proteins. Reporter plasmid DNA encoding Gaussia luciferase (pCMV-Gluc-1), containing the LacZ gene (pVAXl-LacZ), alone or mixed with 50 % microbubbles (Artison), were injected into the ciliary muscle of rat eyes, with or without ultrasound exposure. Animals were sacrificed 7 and 30 days after injection to assess the secreted luciferase into ocular fluids using luminometry. The localization of gene expression was investigated by β -galactosidase histochemistry, 7 days after transfection. At day 7, luciferase activity in ocular media was significantly higher in eyes injected with plasmid and treated with MB and US, as compared with eyes treated with US alone or with eyes injected with the plasmid only. US plus microbubbles showed a 2.6-fold increase in luminescence (p <; 0.023) compared with the control non-US treated eyes, β-galactosidase expression was observed in the ciliary region in case of US and MB application, whereas no expression was observed in controls eyes without US application. We demonstrate for the first time the feasibility of in vivo gene transfer, mediated by ultrasound and microbubbles in the ciliary muscle for the intraocular production of secreted proteins in the ocular media.