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Sandra Johnen, Christiane Stickelmann, Michael Stöcker, Zsuzsanna Izsvak, Peter Walter, Gabriele Thumann; Sleeping Beauty-based Non-Viral Transfer of PEDF into Primary Pigment Epithelial Cells: A Method Yielding in High Transfection Efficiencies and Long Term Expression. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4379.
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© ARVO (1962-2015); The Authors (2016-present)
Transplantation of pigment epithelial cell suspensions to the subretinal space as a treatment for retinal degenerations has resulted in very limited success. One reason may be that the transplanted cells do not secrete adequate factors necessary to maintain a healthy environment. To address this concern, we have explored the non-viral transfer of the neurogenic and anti-angiogenic pigment epithelium-derived factor (PEDF) into primary pigment epithelial cells using the hyperactive Sleeping Beauty (SB100X) transposon system. The SB100X transposon system is capable of efficient and stable transgene insertion into a host cell genome insuring consistent gene expression.
Using electroporation protocols, ARPE-19 and primary pigment epithelial cells were transfected with a control or a PEDF encoding plasmid, both based on the SB100X system and controlled by either a CMV or a CAG promoter. Transfection efficiency and stability of protein expression were evaluated weekly by flow cytometry (FACS) as well as supernatant analysis using Western Blot (WB) technique for at least 6 months.
SB100X-based transfection resulted in efficiencies of 100% with the control plasmid and 40% with the PEDF encoding plasmid. Subsequent FACS sorting of the PEDF transfected cells enabled establishment of populations containing 99% positive cells. Follow-up showed that gene expression was maintained in all cell types for the 6 months, indicating genomic integration of the transgene. In ARPE-19 cells, WB analysis showed that the synthetic CAG promoter resulted in markedly decreased PEDF transcription compared to cultures transfected with appropriate CMV-controlled plasmids. However, primary pigment epithelial cells exhibited equal PEDF expression with both promoters.
The limitation of non-viral approaches to therapeutic gene transfer can be overcome by using transposon-based vectors. In our studies, SB-mediated gene transfer ensured the stable expression of PEDF in primary pigment epithelial cells, thus providing stably transfected cells that could be used for somatic gene addition therapy to treat AMD.
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