Human Retinal Pigment Epithelial Cells Overexpressing the NeuroprotectiveProteins PEDF and GM-CSF to Treat Degeneration of the NeuralRetina
Thais Bascuas, Hajer Zedira, Martina Kropp, Nina Harmening, Mohamed Asrih, Cécile Prat-Souteyrand, Shuwei Tian, Gabriele Thumann
Abstract
Background: Non-viral transposon-mediated gene delivery can overcome viral vectors’ limitations. Transposon gene delivery offers the safe and life-long expression of genes such as Pigment Epithelium-Derived Factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) to counteract retinal degeneration by reducing oxidative stress damage. Objective: The study aimed at using Sleeping Beauty transposon to transfect human Retinal Pigment Epithelial (RPE) cells with the neuroprotective factors PEDF and GM-CSF to investigate the effect of these factors on oxidative stress damage. Methods: Human RPE cells were transfected with PEDF and GM-CSF by electroporation, using the hyperactive Sleeping Beauty transposon gene delivery system (SB100X). Gene expression was determined by RT-qPCR, and protein level by Western Blot as well as ELISA. The cellular stress level and the neuroprotective effect of the proteins were determined by measuring the concentrations of the antioxidant glutathione in human RPE cells, and conducting immunohistochemical examination of retinal integrity, inflammation, and apoptosis of rat Retina-Organotypic Cultures (ROC) exposed to H2O2. Results: Human RPE cells were efficiently transfected showing a significantly augmented gene expression and protein secretion. Human RPE cells overexpressing PEDF and/or GM-CSF or pretreated with recombinant proteins presented significantly increased glutathione levels post-H2O2 incubation than non-transfected/untreated controls. rPEDF and/or rGM-CSF-treated ROC exhibited decreased inflammatory reactions and cell degeneration. Conclusion: GM-CSF and/or PEDF could be delivered successfully to RPE cells with combined use of SB100X and electroporation. PEDF and/or GM-CSF reduced H2O2-mediated oxidative stress damage in RPE cells and ROC offering an encouraging technique to re-establish a cell protective environment to halt age-related retinal degeneration.