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Haidong Shan, Dan Ji, Alun R. Barnard, Daniel M. Lipinski, Qisheng You, Edward Lee, Tengku A. Tengku Kamalden, Xing-Huai Sun, Robert E. MacLaren; AAV-mediated Gene Transfer of Human X-linked Inhibitor of Apoptosis Protects Against Oxidative Cell Death in Human RPE Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):892.
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Gene delivery of X-linked inhibitor of apoptosis (XIAP) has resulted in long-term neuroprotection of photoreceptors in laboratory models of retinal degeneration. A promising clinical application for XIAP gene therapy might be to slow degeneration of the retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). We therefore developed an in vitro model of RPE death caused by oxidative stress, which is one possible mechanism active in AMD and applied it to quantify the effects of XIAP gene delivery using an adeno-associated viral (AAV) vector.
Oxidative stress was induced by hydrogen peroxide (H2O2) in ARPE-19 cells and was measured by using the dichlorofluorescein assay. Human XIAP was delivered by AAV and confirmed by bicistronic expression of GFP, immunocytochemistry and western blot. The MTT assay was performed to examine cell viability. Cell death was determined by TUNEL assay. A null AAV vector, containing identical regulatory sequences but without XIAP, was used as control.
Exposure of ARPE-19 cells to 0.5mM H2O2 for 1 hour caused a significant increase of cellular oxidative stress and loss of cell viability (29±1.5% of control cells, p<0.001), accompanied by a significant increase of TUNEL positive cells (66±18 vs. 0.6±0.9, p<0.001). Compared with null vector controls, XIAP transduced cells had approximately 4.9-fold higher expression of XIAP, and significantly preserved cell viability after H2O2 exposure (47±1.7% vs. 33±1.8%, p<0.001). A marked reduction in the number of TUNEL positive cells in the XIAP transduced cultures was also observed (3.0±2.6 vs. 57±10, p<0.001).
XIAP gene therapy protects ARPE-19 cells against the oxidative stress of H2O2 and may have clinical potential in preserving RPE cells in diseases where oxidative stress has a key role, such as AMD.
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