Programm                 "Degeneration und Regeneration– Grundlagen, Diagnostik und Therapie"


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Abstract
Abstract

High Efficiency Gene Electrotransfer to the Endothelium of Human Organ Cultured Corneas

Thuret G.1, Manissolle C.1, Bonnefoy R.1, Campos-Guyotat L.1, Maugery J.1, Gain P.1
1Lab. "cell survival and adherence in cancers and grafts". EA 3063. Faculty of Medicine, University Hospital; 2Ophthalrnology department, University Hospital Saint-Etienne/F

Purpose: Gene transfer to the corneal endothelium is a hopeful strategy to modulate cell functions through the induction of newly synthetised proteins with therapeutic purpose. Gene electrotransfer is deemed safer and triggers dramatically less inflammation than viral vectors and is also reputed to be more efficient than other non viral vectors. Our aim was to develop and optimise ex vivo gene electrotransfer to endothelial cells (ECs) of organ cultured human corneas.
Method: Sterilisable non-contact electrodes were custom-designed. Forty five human organ cultured corneas were tranfected using the LacZ reporter gene coding für the b galactosidase (0.5 mg DNA/corneas). Eight pulses of 100 ms and 100 mA were delivered by the GET 42 pulse generatorTM. Negative controls were performed omitting the current pulses. The expression of beta-galactosidase was assessed at 1(n=10), 3(n=20), 5(n=10), 14(n=5) days after electroporation using an image analysis software. EC toxicity was assessed by histological examination after cornea flat mounting and detection of apoptosis by in situ
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