AbstractPurpose Recombinant Adeno‐Associated virus (rAVV) encoding meganucleases specifically designed to address Herpes simplex virus type 1 (HSV1) reduce HSV1 replication both in vitro and ex‐vivo. The main issue of this potential antiviral treatment is the delivery system. To evaluate a vector‐free method, we tested an electroporation process to transfer HSV1‐specific meganucleases (HSV1‐SM) to the murine corneaMethods Corneal gene transfer was achieved by subconjunctival injection (SCI) of recombinant plasmid encoding either GFP (group 1; n=30) or HSV1‐SM (group 2; n=12), followed by electroporation. Mice were sacrified at day (D)1, 3, 7, 13, 35 (group 1) and at D13 and 35 (group 2). Control group consisted in SCI w/o electroporation. Corneal samples were analyzed for the expression of transcripts encoding GFP and HSV1‐SM using real time PCR. The results were compared to those obtained following SCI of rAAV encoding either GFP or HSV1‐SMResults For HSV1‐SM, SCI plus electroporation induced a stronger expression than SCI alone at D13. In group 1, the expression of GFP transcripts decreased from D1 to D35. However, until D13, their levels of expression were higher than those obtained with the rAAV method (p<0.002). Similarly, the expression of HSV1‐SM transcripts at D13 was higher than those obtained with the rAAV method (p<0.002), suggesting that therapeutic concentration could be optimised with this method. At D35, results were not statistically different between the 2 methodsConclusion In our hands, with the method we used, we can conclude that SCI 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