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Benoit Chapellier, Jr., Marc Labetoulle, Sylvain Arnould, Stephanie Grosse, Julianne Smith, Jose A. Sahel, Eric E. Gabison; Meganuclease Targeting Herpes Simplex Virus type I protects Against Viral Endothelitis: an organ culture model. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6655.
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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. Despite advances in antiviral therapies, past history of HSV keratitis is associated with poor prognosis of subsequent penetrating keratoplasty (PK). HSV infection of the grafted cornea is highly prevalent in primary graft failure following PK, furthermore HSV recurrence may favor secondary graft rejection. The aim of this study is to assess the antiviral property of a meganuclease targeting HSV in the prevention of HSV endothelitis.
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.
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.
These meganucleases are currently checked for their anti-infective properties in an in vivo model of endothelitis.Our organ culture model of herpetic endothelial infection is 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 following PK.
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