Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal blood vessels growing into the subretinal space, leading to retinal pigment epithelial (RPE) cell degeneration and vision loss. Vessel growth results from an imbalance of pro-angiogenic (e.g., vascular endothelial growth factor [VEGF]) and anti-angiogenic factors (e.g., pigment epithelium-derived factor [PEDF]). Current treatment using intravitreal injections of anti-VEGF antibodies improves vision in about 30% of patients but may be accompanied by side effects and non-compliance. To avoid the difficulties posed by frequent intravitreal injections, we have proposed the transplantation of pigment epithelial cells modified to overexpress human PEDF. Stable transgene integration and expression is ensured by the hyperactive Sleeping Beauty transposon system delivered by pFAR4 miniplasmids, which have a backbone free of antibiotic resistance markers. We demonstrated efficient expression of the PEDF gene and an optimized PEDF cDNA sequence in as few as 5 × 103 primary cells. At 3 weeks post-transfection, PEDF secretion was significantly elevated and long-term follow-up indicated a more stable secretion by cells transfected with the optimized PEDF transgene. Analysis of transgene insertion sites in human RPE cells showed an almost random genomic distribution. The results represent an important contribution toward a clinical trial aiming at a non-viral gene therapy of nvAMD.

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: Use 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 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 pre-treated 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 by combining the 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.