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E. Touchard, C. Bloquel, P. Bigey, C. Gandolphe, D. BenEzra, D. Scherman, F. Behar-Cohen; Ciliary Muscle Electrotransfer Allows for Controlled and Sustained Production of Therapeutic Proteins in Ocular Media. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5811.
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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.
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).
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.
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.
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