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2000
Volume 22, Issue 2
  • ISSN: 1566-5232
  • E-ISSN: 1875-5631

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 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 and by electroporation, using the hyperactive transposon gene delivery system (). 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 HO. 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- HO 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 and electroporation. PEDF and/or GM-CSF reduced HO-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.

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/content/journals/cgt/10.2174/1566523221666210707123809
2022-04-01
2025-06-12
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