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Eric Gabison, Marc Labetoulle, Marine Gailledrat, Jose Sahel, Benoit Chapelier; Meganuclease Targeting HSV-1 Protects Against Corneal Endothelitis ex-vivo and in-vivo. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1685.
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© ARVO (1962-2015); The Authors (2016-present)
Herpetic Keratitis is a leading cause of decreased best corrected visual acuity in developed and developing countries. The aim of this study is to assess the antiviral property of a meganuclease targeting HSV in the prevention of HSV endothelitis ex-vivo and in vivo.
Normal rabbit corneas were placed in organ culture and transduced by a recombinant adeno-associated virus (rAAV) allowing constitutive expression of meganucleases targeting HSV-1 genome or of 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%. 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. Additionnaly, a rabbit model of corneal HSV endothelitis was developed. Intracamerular injection of HSV F(1) were performed in rabbit eyes 2 days following intravitreal injection of steroid. Corneal edema, keratic precipitates, ocular inflammation and infection rate for plaques or cells were analyzed in this model.
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. In vivo, experiments demonstrated a 30% decreased in endothelial plaque formation. The rate of corneal edema and keratic precipitates was reduced in Megnuclease treated eyes as compared to controles.
Our organ culture and in vivo model of herpetic endothelial infection are reproducible and efficient to quantify viral proliferative capacity. Meganuclease transduction confers a significant inhibition of viral pathogenic effect. Meganuclease gene therapy targeting HSV-1 DNA may be an effective treatment to protect against HSV endothelitis
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