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Antoine Rousseau, Virgine Escriou, Pierre Roy, Nolwenn Poccardi, Julie Takissian, Yves Gaudin, Pascal Bigey, Marc Labetoulle; In vitro evaluation of anti HSV-1 siRNAs and in vivo evaluation of electroporation to transfect siRNAs on murine cornea.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2340.
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© ARVO (1962-2015); The Authors (2016-present)
HSV-1 keratitis (HSK) is a leading cause of infectious blindness in developed countries. Available treatments rely on nucleosidic DNA polymerase inhibitors, which are used curatively or prophylactically. Massive use of these treatments may favor the emergence of resistance. Anti-HSV1 small interfering RNAs (siRNA) may be efficient to overcome this issue, but their transfection into corneal cells remains a challenge. The purpose of this study was to assess the in vitro efficacy of siRNA targeting HSV-1 DNA polymerase to reduce HSV-1 replication, and the in vivo efficacy of electroporation to transfect siRNA into corneal cells on the murine cornea.
Three different anti HSV-1 DNA polymerase siRNAs (S1-S3) and one control siRNA were transfected into vero cells using cationic lipids, which were secondarily infected with the SC16 strain of HSV-1. Efficacy on viral replication was assessed using flow cytometry, quantitative PCR (qPCR) and plaque assay technique. On murine cornea, fluorescent siRNAs were injected subconjunctivally and electroporation was performed with custom made electrodes applied on the conjunctiva. The eyes were enucleated, and observed under fluorescence microscopy.
The three siRNAs were able to inhibit viral replication. Compared to the control siRNA, S3 was the most efficient siRNA, decreasing by 60% the number of infected cells as measured with flow cytometry, by 59% the number of plaques and by 75% the viral load estimated with qPCR. Electroporation improved siRNA penetration into the corneal epithelium compared to subconjunctival injection alone.
These results demonstrate that siRNA directed against HSV-1 DNA polymerase efficiently inhibits HSV-1 replication, suggesting that siRNA based antiviral strategy may be a potential therapeutic alternative to treat HSK. Besides, intracorneal penetration may be facilitated by electroporation.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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