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F. Majo, A.-P. Bemelmans, Y. Arsenijevic; Creating a Pocket in the Cornea With a Femtosecond Laser to Enhance Lentiviral Gene Transfer Into Stromal Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1499.
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We investigated a new procedure for gene transfer into the stroma of pig cornea for the delivery of therapeutic factors.
A pocket was created with the Femtosecond laser LDV (Ziemer Ophthalmic System AG, Switzerland) in 6 pig corneas. The cut was performed at 140 µm deep. The tip of a 31 gauge was then immediately inserted into the pocket to inject 100 µl of HBSS containing a HIV1-derived lentiviral vector expressing GFP (LV-CMV-GFP) at a dose of 150 ng of p24. The corneas were then conserved in CorneaMax medium (Eurobio SA, France) at +31°C for 5 days. After fixation and cryosectioning, a standard coloration was performed on the sections with hematoxylin-eosin reagents. Gene transfer in corneal cells was analyzed by GFP immunohistochemistry using the PAP method.
Creation of the corneal pocket and subsequent injection of the vector solution was performed easily. The histological analysis revealed that corneal pockets were located at the expected deepness and healed after the culture period. All the 6 injected corneas were well positive for transgene expression in an area encompassing the whole pocket. Around the pockets almost all the keratocytes were transduced. Vector diffusion was more effective in the direction of the epithelium side (9+/-0.8 layers of keratocytes were transduced, n=4) than to the endothelium side (4.5+/-0.75 layers were transduced, n=4, p=0.0033).
Intrastromal vector injection, without the creation of a pocket, would lead to cornea injury and no control of vector diffusion. In the present study, we show that the Femtosecond laser procedure allows efficient gene transfer by controlling the size of the transduced area. Angiogenesis in the cornea is a major cause of visual impairment in human. Recurrences after the therapy are the limit of the current strategy and the development of the present technique of gene transfer could be an attractive strategy for anti-angiogenic gene transfer.The Femtosecond laser technique allows a reliable injection and diffusion of lentiviral vectors to efficiently transduce stromal cells in a delimited area. Demonstrating the efficiency of this procedure in vivo could be an important step to cure or prevent angiogenesis of the corneal stroma.
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