884. Anti-Oxidant Gene Delivery to Retinal Epithelial Cells: A Gene Therapy Approach for Macular Degeneration

Age related macular degeneration (AMD) is a progressive degenerative disorder that affects the retina, retinal pigments and the choriocapillaries of the eye. Oxidative stress has been implicated in AMD through the production of reactive oxygen species (ROS) in the retinal pigment epithelium (RPE), which provides structural and nutritional support to the retina. Blue light exposure, elevated oxygen tension and high cellular lipid content contribute to oxidative stress in the RPE.The aim of this study was to determine if anti-oxidant gene expression by adenoviral vectors could protect a retinal epithelial cell line, APRE-19 from acute oxidative stress with glucose oxidase treatment. The APRE-19 retinal epithelial cells were transfected separately with adenoviral vectors expressing Manganese Super oxide dismutase (MnSOD), Catalase, Copper Zinc Super oxide dismutase (CuZnSOD), Heme-oxygenase (HO-1) or the heat shock protein HSP-27. 48 hours following transfection the cells were exposed to a range of glucose oxidase concentrations from 0.157 mu to 2.5 mu/ml over a time period ranging from 2hrs to 72hrs. The production of ROS was assessed by the fluorogenic marker carboxy- H2DCFDA and cell viability was measured by the MTT assay and the annexin apoptosis assay. The cellular cytoskeleton was stained by Phalloidin-FITC.At a concentration of 0.313mu/ml glucose oxidase, which dramatically reduced the viability of non transfected APRE-19 cells, Adenoviral expression of MnSOD and Catalase showed a level of protection, which increased the viability ratio almost two-fold, the greatest protection with an anti-oxidant gene was observed with CuZnSOD expression. A similar trend was also observed in the reduction of apoptotic cells with CuZnSOD demonstrating increased numbers of viable cells. No protection was observed with HO-1 treatment but interestingly HSP-27 treatment demonstrated a level of protection significantly greater than any anti-oxidant treatment. These in-vitro results demonstrate a potential therapeutic benefit from expression of either CuZnSOD or HSP27 in AMD.