In vitro comparison of AAV–mediated eGFP gene transfer on Müller cells and ARPE cells.

Abstract

Abstract: : Purpose : To investigate the efficiency of adeno–associated virus (AAV) –mediated eGFP gene transfer in vitro on ARPE cells and on a new Müller cells line (MIO–M1), in order to better evaluate the ability of AAV to infect these two important cell lines for intra–ocular inflammation applications. Methods : ARPE cells and MIO–M1 (kind gift from A. Limb, UCL, London) were infected in vitro by an AAV2–CMV–eGFP at different MOI (multiplicity of infection) (10, 30, and 100 viral particles per cell) and eGFP expression was measured at different time points (2 and 5 days). Native fluorescence was observed at 2 and 5 days in all samples by microscopic examination and fluorescence was measured by FACS (fluorescence activated cell sorter). Results : We showed that MIO–M1 cells can be efficiently infected by an AAV2–CMV–eGFP but less than ARPE cells. Fluorescence was first detected by microscopy at 36 hours post–infection on cells analysed by FACS at 2 and 5 days. Conclusions : MIO–M1 and ARPE cells are a good candidate for infection by AAV2. This allow us to test in vitro, in future experiments, the effects of different immunosuppressive molecules mediated by AAV on these two important cell lines before an in vivo evaluation.

Keywords: gene transfer/gene therapy • retinal pigment epithelium • Muller cells