Meganuclease targeting herpes simplex virus protects against viral endothelitis : An organ culture model

Abstract

AbstractPurpose Despite advances in antiviral therapies, past history of HSV keratitis is associated with poor prognosis of subsequent penetrating keratoplasty.The aim of this study is to assess the antiviral property of a meganuclease targeting HSV in the prevention of HSV endothelitis.Methods Normal rabbit corneas were placed in organ culture using an immersion method and transduced by a recombinant adeno‐associated virus (rAAV) allowing constitutive expression of meganucleases targeting HSV‐1 genome or containing the same expression cassette with a non‐coding sequence. These organs were then submitted to infection by recombinant HSV‐1 F(1) virus equipped with a LacZ expressing cassette at M.O.I. 0.001 to 0.1% in liquid or semi‐solid medium. Infection rates for plaques or cells in endothelium were established by immunostaining of envelope protein gD or X‐gal staining after the end of first or second lytic cycle.Results Meganuclease targeting the ICP0 gene which encodes an E3 ubiquitin ligase involved in viral reactivation and replication did not change infection rates in the present organ culture model, but reduced the average size of plaques in endothelium with a decrease of 27‐46%. Conversely, the meganuclease directed against the major capsid protein UL19 lowered the number and size of plaques, both being reduced by half at M.O.I. 0.001%. Consequently, the expression of a meganuclease in endothelium, evidenced by RT‐PCR, could either reduce infectious particle production or induce cell resistance to HSV‐1.Conclusion These meganucleases are currently checked for their anti‐infective properties in an in vivo model of endothelitis.