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Nina Harmening, Martina Kropp, Sandra Johnen, Corinne Marie, Daniel Scherman, Zsuzsanna Izsvák, Gabriele Thumann; The use of mini-plasmids free of antibiotic resistance markers for a gene therapeutical approach to treat AMD. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2325.
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© ARVO (1962-2015); The Authors (2016-present)
For the treatment of exudative age-related macular degeneration (AMD), we developed a protocol to electroporate pigment epithelial cells with the anti-angiogenic and neuroprotective pigment epithelium-derived factor (PEDF). The ultimate goal of our investigations is the transplantation of autologous, transfected cells into the subretinal space, where the secreted PEDF inhibits neovascularization. For stable and efficient transfection, we use the non-viral Sleeping Beauty (SB100X) transposon system, which enables integration of the gene into the host cell’s genome. To further enhance the transfection efficiency and safety characteristics of the procedure, a new generation of vectors free of antibiotic resistance markers (pFAR4) was evaluated.
Primary pigment epithelial cells were co-transfected with a SB100X transposase-encoding plasmid and a transposon plasmid encoding the PEDF or the Venus gene using electroporation protocols. Analogous pT2 transposon plasmids carrying an ampicillin-resistance gene were compared to pFAR4-plasmids. Fluorescence of Venus-transfected cells was measured by image-based cytometry and PEDF secretion was evaluated by Western blot analysis and quantified by ELISA.
Transfection of 10’000 cells with the Venus-encoding plasmids resulted in higher transfection efficiencies using pFAR4-ITRs-CAGGS-Venus (29.1%±5.28) compared to pT2-CAGGS-Venus (9.7%±2.00). Non-transfected control cells secreted 0.3±0.05 ng PEDF/h/104 cells, cells transfected with the pT2-CMV-PEDF transposon plasmid secreted 4.3±4.05 ng PEDF/h/104 cells, and cells transfected with pFAR4-ITRs-CMV-PEDF secreted 11.1±8.12 ng PEDF/h/104 cells, a 2.5-fold increase compared to cells transfected with pT2-CMV-PEDF. Western Blot analysis highlighted these results and showed higher PEDF secretion in the pFAR4-transfected cells.
The present work demonstrates that using pFAR4 plasmids augments PEDF secretion while simultaneously enhancing the safety of the approach. The avoidance of antibiotic resistance markers is a crucial step towards the clinical use of the SB100X transposon system; in a phase Ib/IIa clinical trial, planned for 2015, autologous pigment epithelial cells will be transfected with the PEDF gene and re-transplanted subretinally as novel therapy for exudative AMD using pFAR4 miniplasmids together with the SB100X transposon system.
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