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Anna Dobias, Mattia Ronchetti, Ruggero Cadossi, Peter Walter, Gabriele Thumann, Sandra Johnen; Non-Viral Sleeping Beauty-Mediated Transfection of Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1827.
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In neovascular age-related macular degeneration (AMD), which leads to severe vision loss within several months, the growth of choroidal blood vessels into the subretinal space is caused by an increase of the pro-angiogenic vascular endothelial growth factor (VEGF) and a decrease of the anti-angiogenic pigment epithelium-derived factor (PEDF). The current treatment with anti-VEGF antibodies improves vision in about 30% of patients, but may be accompanied by severe side effects and non-compliance. To increase the level of PEDF in the subretinal space, we proposed the subretinal transplantation of pigment epithelial cells genetically modified to stably overexpress PEDF. Since transgene delivery using viral vectors is complicated by immune responses, viral spread, and transgene introduction into unwanted cells, we have developed a transfection protocol that uses the non-viral Sleeping Beauty (SB100X) transposon system.
Primary human retinal pigment epithelial (RPE) cells were transfected with plasmids encoding the SB100X transposase and the Venus reporter gene using the pipette tip-based Neon Transfection System (Life Technologies) or the cuvette-based CliniporatorTM System (IGEA, Italy). Transfection efficiency and transgene expression stability were evaluated by fluorescence microscopy and flow cytometric analysis
Using the Neon Transfection System, increasing electric field parameters ranging from 1100 to 1350 V / 20 to 30 ms / 2 pulses resulted in increased numbers of transfected cells (14.8 ± 20.1% to 28.1 ± 19.7%), stable for the 12 months that the cells have been cultured. With the CliniporatorTM System, best transfection efficiencies were observed with a combination of high (150 V / 0.1 ms / 7 pulses) and low (40 V / 20 ms / 2 pulses) voltage pulses.
SB100X-mediated transfection by electroporation is efficient and stable for primary human RPE cells. In contrast to the Neon Transfection System, the CliniporatorTM System allows for quality control of the electroporation process, which is essential for the development of a transposon-based gene therapeutic approach.
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