Cornea - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - CorneaEric E. Gabison 1, 2 , Marc Labetoulle 4 , Marine Gailledrat 5 , Jose A.Sahel 3 , Benoit Chapelier 3 . 1 Cornea Department, Fondation A. deRothschild, Paris, France; 2 Ophthalmology Department, HôpitalBichat, Paris, France; 3 Institut de la vision, Paris, France;4 Ophthalmology Department, Hôpital du Kremlin Bicêtre, Paris,France; 5 Cellectis, Paris, France.Purpose: Herpetic Keratitis is a leading cause of decreased bestcorrected visual acuity in developed and developing countries. Theaim of this study is to assess the antiviral property of a meganucleasetargeting HSV in the prevention of HSV endothelitis ex-vivo and invivo.Methods: Normal rabbit corneas were placed in organ culture andtransduced by a recombinant adeno-associated virus (rAAV)allowing constitutive expression of meganucleases targeting HSV-1genome or of a non-coding sequence. These organs were thensubmitted to infection by recombinant HSV-1 F(1) virus equippedwith a LacZ expressing cassette at M.O.I. 0.001 to 0.1%. Infectionrates for plaques or cells in endothelium were established byimmunostaining of envelope protein gD or X-gal staining after theend of first or second lytic cycle. Additionnaly, a rabbit model ofcorneal HSV endothelitis was developed. Intracamerular injection ofHSV F(1) were performed in rabbit eyes 2 days following intravitrealinjection of steroid. Corneal edema, keratic precipitates, ocularinflammation and infection rate for plaques or cells were analyzed inthis model.Results: Meganuclease targeting the ICP0 gene which encodes an E3ubiquitin ligase involved in viral reactivation and replication did notchange infection rates in the present organ culture model, but reducedthe average size of plaques in endothelium with a decrease of 27-46%. Conversely, the meganuclease directed against the major capsidprotein UL19 lowered the number and size of plaques, both beingreduced by half at M.O.I. 0.001%. Consequently, the expression of ameganuclease in endothelium, evidenced by RT-PCR, could eitherreduce infectious particle production or induce cell resistance toHSV-1. In vivo, experiments demonstrated a 30% decreased inendothelial plaque formation. The rate of corneal edema and keraticprecipitates was reduced in Megnuclease treated eyes as compared tocontroles.Conclusions: Our organ culture and in vivo model of herpeticendothelial infection are reproducible and efficient to quantify viralproliferative capacity. Meganuclease transduction confers asignificant inhibition of viral pathogenic effect. Meganuclease genetherapy targeting HSV-1 DNA may be an effective treatment toprotect against HSV endothelitisCommercial Relationships: Eric E. Gabison, None; MarcLabetoulle, None; Marine Gailledrat, Cellectis SA (E); Jose A.Sahel, UPMC/Essilor (P), Second Sight (F); Benoit Chapelier, NoneSupport: OSEO ACTIVE GRANT FRANCEProgram Number: 1686 Poster Board Number: D0321Presentation Time: 8:30 AM - 10:15 AMComparison of Endothelial Cell Density at the Central andPeripheral Regions in a DSAEK GraftHiroko Nakagawa, Tsutomu Inatomi, Shigeru Kinoshita. KyotoPrefectural Univ of Med, Kyoto, Japan.Purpose: To analyze endothelial remodeling, including the woundhealing, at the host/graft junction after Descemet’s StrippingAutomated Endothelial Keratoplasty (DSAEK), and compare thechange of corneal endothelium at the central and peripheral regionsof the graft using wide-field specular microscopy.Methods: This study involved 10 eyes of 10 patients (mean age: 72years) treated by DSAEK using internationally shipped precut donorcorneas. None of the patients had ocular complications prior tosurgery, and DSAEK was performed via the pull-through techniqueusing a Busin glide. Wide-field contact specular microscopy wasused to evaluate the alteration of endothelium at the central andtemporal peripheral corneal regions (approximately 1mm inside fromthe graft edge) at 1, 6, and 12 months after surgery. Endothelial celldensity (ECD), coefficient of variation (CV), and the frequency ofhexagonal cells (6A) were analyzed to elucidate the post-DSAEKendothelial remodeling pattern.Results: Mean regional endothelial cell density (cells/mm2±SD) inthe center / periphery at 1, 6, and 12 months postoperative were2286±409 / 1893±432, 2152±464 / 1550±284, and 2138±466 /1152±378, respectively. Peripheral ECD was statistically lower thancentral ECD at each follow-up time-point (p