PurposeOur previous results have associated HIF expression in RPE cells to CNV progression, and we have shown that gene transfer of prolyl hydroxylase domain (PHD)2 in the mouse model of nAMD resulted in mitigation of HIF‐mediated angiogenesis. Here, we investigate the function of hypoxia‐regulated molecular elements in modulating the expression of transgenes in retinal pigment epithelium (RPE) cells, with putative use in gene therapy strategies for the treatment of nAMD.MethodsHypoxia‐mediated RPE‐specific expression vectors were generated using the minimum mouse Rpe65 promoter (pRpe65), while hypoxia‐mediated expression was achieved using hypoxia‐response elements (HRE) and oxygen‐dependent degradation peptides (ODP). Hypoxia‐dependent regulation of transgenes in ARPE‐19 cells was determined by green fluorescence protein (GFP) expression. Expression pattern analyses of transgenes in response to hypoxia and reoxygenation were assayed by western blot and RT‐PCR.ResultsUse of pRpe65 resulted in RPE‐specific expression of GFP. Hypoxia‐enhanced expression of GFP was achieved with HRE‐driven pRpe65, and GFP expression patterns were regulated at the transcriptional level. Fusion of ODP to GFP resulted in hypoxia‐mediated expression, under pRpe65 regulation, and GFP protein expression was subject to hypoxia‐dependent protein expression.ConclusionsOur data shows that transgenes can be expressed specifically in RPE cells and be regulated by hypoxia, both by transcriptional and post‐translational regulation, limiting the therapeutic gene expression to pathologic hypoxic RPE cells. These results may have implications for the clinical treatment of ocular vascular pathologies, such as nAMD, particularly regarding the use of gene therapy to negatively regulate neoangiogenesis.