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Antoine Rousseau, Ayla Ergani, Eric E. Gabison, Marisol Corral, Nicolas Huot, Marine Gailledrat, Carole Desseaux, Benoit Chapellier, Jr., Pierre Roy, Marc Labetoulle; Gene Transfer Of Hsv1-specific Meganuclease To The Murine Cornea Using Electroporation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6148.
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© ARVO (1962-2015); The Authors (2016-present)
Recombinant Adeno-Associated virus (rAVV) encoding meganucleases specifically designed to address Herpes simplex virus type 1 (HSV1) reduce HSV1 replication both in vitro (ARVO 2011, abstract 5801) and in the rabbit cornea ex-vivo (ARVO 2011, abstract 6655). The main issue of this potential antiviral treatment is the delivery system. In order to evaluate a vector-free method, we tested an electroporation process to transfer HSV1-specific meganucleases (HSV1-SM) to the murine cornea.
Corneal gene transfer was achieved by subconjunctival injection of 6µg of recombinant plasmid encoding either GFP (group 1; n=30) or HSV1-SM (group 2; n=12), followed by electroporation with 8 pulses of 10msec duration at a field strength of 200 V/cm. Mice were sacrified at day 1, 3, 7, 13, 35 (group 1) and at day 13 and 35 (group 2). Subconjunctival injections with no electroporation were also performed, as control. Corneal samples were analyzed for the expression of transcripts encoding GFP and HSV1-SM using real time polymerase chain reaction (RT-PCR). The results were compared to those obtained following the subconjunctival inoculation of rAAV encoding either GFP of HSV1-SM.
For HSV1-SM, injection plus electroporation induced a stronger expression of genes than injection of plasmid alone at day 13 (control group). In group 1, the expression of GFP transcripts progressively decreased from day 1 to day 35. However, until day 13, their levels of expression were higher than those obtained with the rAAV method (p<0.002). Similarly, the expression of transcripts encoding HSV1-SM at days 13 was higher than those obtained with the rAAV method (p<0.002), suggesting that therapeutic concentration could be optimised with this method. At day 35, results were not statistically different between the two methods.
In our hands, with the method we used, we can conclude that subconjunctival injection of plasmid encoding HSV1-SM with immediate electroporation is an efficient method to improve delivery in short-term but not in long-term when compared to rAAV.
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