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Anna Dobias, Corinne Marie, Daniel Scherman, Zsuzsanna Izsvák, Zoltan Ivics, Peter Walter, Gabriele Thumann, Sandra Johnen; Analysis of Potential Tumorigenicity of PEDF-Transfected Primary Human Retinal Pigment Epithelial Cells Using Soft Agar Assay. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2348.
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© ARVO (1962-2015); The Authors (2016-present)
Exudative age-related macular degeneration leads to blindness within several months. It is characterized by the growth of choroidal blood vessels into the subretinal space, whose new formation is due to the increased expression of the pro-angiogenic vascular endothelial growth factor (VEGF). Unlike the current treatment based on anti-VEGF antibodies, we developed a method to transplant genetically modified pigment epithelial cells that overexpress the anti-angiogenic pigment epithelium-derived factor (PEDF). Stability of PEDF transgene expression was ensured by the Sleeping Beauty (SB100X) transposon system, carried by pFAR4-plasmids, an expression vector free of antibiotic resistance markers. Here we report the analysis of potential tumorigenicity of the transfected cells by means of a soft agar assay.
Retinal pigment epithelial (RPE) cells from human donor eyes were electroporated with pFAR4-plasmids encoding the SB100X transposase and the PEDF gene at a ratio of 1:16. Stability of PEDF secretion was analyzed by immunoblotting. Up to 31.250 cells/cm2 were applied to a two-layer soft agar system. Colony development of the transfected cells was analyzed after 1, 5, 7, and 14 days via phase contrast microscopy and compared to the growth patterns of HeLa cells, which were used as positive control.
pFAR4/SB100X-mediated PEDF gene delivery into as few as 10,000 primary RPE cells led to an increase in PEDF secretion. No colony formation was observed for primary transfected RPE cells, whereas for HeLa cells, 89.4 ± 26.4 soft agar colonies per microscopic field were detected.
The results shown here give a first evidence that the PEDF-transfected primary RPE cells have no tumorigenic potential. Further safety studies analyzing the integration loci of the PEDF transgene as well as the biodistribution of the genetically modified cells will be conducted. This study is just one of many aspects demonstrating the feasibility of our approach based on ex vivo transfection of a low number of primary autologous pigment epithelial cells followed by subretinal transplantation into the same patient.
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