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Efficient lentiviral gene transfer into corneal stroma cells using a femtosecond laser

Abstract

We investigated a new procedure for gene transfer into the stroma of pig cornea for the delivery of therapeutic factors. A delimited space was created at 110 μm depth with a LDV femtosecond laser in pig corneas, and a HIV1-derived lentiviral vector expressing green fluorescent protein (GFP) (LV-CMV-GFP) was injected into the pocket. Corneas were subsequently dissected and kept in culture as explants. After 5 days, histological analysis of the explants revealed that the corneal pockets had closed and that the gene transfer procedure was efficient over the whole pocket area. Almost all the keratocytes were transduced in this area. Vector diffusion at right angles to the pocket's plane encompasses four (endothelium side) to 10 (epithelium side) layers of keratocytes. After 21 days, the level of transduction was similar to the results obtained after 5 days. The femtosecond laser technique allows a reliable injection and diffusion of lentiviral vectors to efficiently transduce stromal cells in a delimited area. Showing the efficacy of this procedure in vivo could represent an important step toward treatment or prevention of recurrent angiogenesis of the corneal stroma.

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Acknowledgements

This work was supported by Jules-Gonin Eye Hospital, Lausanne, Switzerland. We thank Mr Bernau, CEO of Ziemer Ophthalmic System AG, for giving the opportunity of using the FEMTO LDV. We are also grateful to Dr Corinne Kostic for fruitful discussion, Dana Wanner, Meriem Tekaya, Elga Motta and Corinne de Weck for excellent technical assistance, and to Dr Sylvie Uffer for histological expertise.

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Correspondence to Y Arsenijevic.

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Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)

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Bemelmans, AP., Arsenijevic, Y. & Majo, F. Efficient lentiviral gene transfer into corneal stroma cells using a femtosecond laser. Gene Ther 16, 933–938 (2009). https://doi.org/10.1038/gt.2009.41

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