Abstract
Purpose::
Our aim was to define the optimal conditions for plasmid transfection into the ciliary muscle in order to achieve a sustained and controlled secretion of therapeutic proteins in the vitreous cavity.
Methods::
8-10 weeks old Lewis rats were used in these experiments. Injections of 10µl to 30µl containing 10 to 30µg of pVAX2-gLuc (secreted Gaussia luciferase) or pVAX1-LacZ plasmids (driven by a CMV promoter) were performed in the ciliary muscle of the rat eyes. Several parameters were evaluated regarding the variability and efficacy of transfection and the amount of secreted protein: route of injection (corneal tunnel or transscleral), formulation of DNA plasmids, number of injection sites, amount and volume of injected plasmids, electrode shape and electrical parameters. A kinetic of expression was performed with the gLuc encoding plasmid. Finally other therapeutic proteins were dosed in the vitreous cavity (Epo, sTNFR1-Ig).
Results::
Transscleral injections of naked plasmids (versus PEI) in saline (versus distilled water) were more efficient to transfect reproducibly the ciliary muscle. The optimal electrical conditions were 200V/cm, 8 pulses, 5Hz. Multiple injection sites significantly improved the reproducibility of transfection. The amount of transfected plasmid correlated well with the levels of proteins detected in the vitreous. When no current was applied, a pick of secretion was observed in the vitreous but no sustained protein production. On the contrary, using optimized conditions of electrotransfer, a sustained secretion of gLuc was achieved for at least 5 months. All tested proteins were dosed in the vitreous without any detectable levels into the serum. No major side effects resulted from this smooth muscle transfection technique.
Conclusions::
Electrotransfer of plasmids into the ciliary muscle is an easy, reproducible and simple technique to achieve a sustained secretion of any therapeutic proteins into the ocular media. Applications to neurotrophic proteins or anti-angiogenic approaches are currently evaluated.
Keywords: gene transfer/gene therapy • ciliary muscle • vitreous