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G. Thuret, C. Manissolle, A. Durieux, D. Freyssenet, L. Campos, J. Maugery, P. Gain; Ex Vivo Gene Electrotransfer to Human Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4717.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Gene electrotransfer to human cells is a hopeful strategy to modulate cell functions through the induction of newly synthetised proteins with therapeutic purpose. It is safer and triggers dramatically less inflammation than viral vectors and is also reputed to be more efficient than lipofection. Our aim was to develop and optimise ex vivo gene electrotransfer to endothelial cells (ECs) of organ cultured human corneas. Methods: Sterilisable non-contact electrodes were custom-designed. The GET 42 pulse generatorTM, allowed modification of all current parameters such as pulse duration, pulse number, pulse frequency, pulse combination and current intensity. Expression of beta-galactosidase under different pulse types was monitored in 20 human scientific corneas. EC toxicity was assessed by calculation of EC loss using an automated analyser of the endothelial mosaic. Results: After 5 days, transfected ECs were found in all electroporated corneas in variable numbers depending on the current characteristics. No positive ECs were found in the absence of current. The distribution of transfected ECs was heterogeneous, indicating either different cell susceptibility to gene transfer or inconsistent current passage. Gene electrotransfer triggered no extra mortality under these conditions. Conclusions: We demonstrated for the first time the possibility of gene electrotransfer to ECs of organ- cultured human corneas, by mean of newly designed electrodes and an innovative pulse generator. Further studies are ongoing to determine the duration of the transgene expression.
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