Abstract
Purpose :
Toinvestigate the function of hypoxia-regulated molecular elements to modulate the expression of transgenes in retinal pigment epithelium (RPE) cells, with putative use in gene therapy strategies for the treatment of neovascular ocular pathologies.
Methods :
RPE-specific expression vectors were generated using the minimum Rpe65 promoter (pRpe65), while hypoxia-mediated expression was achieved using hypoxia-response elements (HRE), and oxygen-dependent degradation peptides (ODP). Hypoxia-dependent expression of transgenes in ARPE-19 cells was determined by green fluorescence protein (GFP). Expression pattern analyses of transgenes in response to hypoxia and reoxygenation were assayed by quantitative immunofluorescence and western blot. Statistical analysis was performed by one-way ANOVA with Bonferoni posthoc tests (P < 0.05 was considered significant).
Results :
Use of pRpe65 resulted in RPE-specific expression of GFP. Statistically significant hypoxia-enhanced expression of GFP was achieved with HRE-driven pRpe65. Fusion of ODP to GFP, under pRpe65 regulation, resulted in hypoxia-dependent protein expression while subject to statistical relevant normoxia-mediated protein degradation.
Conclusions :
The data shows that transgenes can be expressed specifically in RPE cells and regulated by hypoxia, both by transcriptional and post-translational regulation. Such expression patterns would limit the therapeutic gene’s expression to pathologic hypoxic RPE cells. These results may have implications for the clinical treatment of ocular vascular pathologies, particularly regarding the use of gene therapy to negatively regulate hypoxia-driven neoangiogenesis.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.