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S. Johnen, M. Stöcker, P. Walter, G. Thumann; Long Term Expression of rPEDF in Primary Pigment Epithelial Cells After Non-Viral Transfection With Linearized Plasmids. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5255.
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© ARVO (1962-2015); The Authors (2016-present)
Transplantation of pigment epithelial cells in suspension into the subretinal space as a treatment modality for AMD did not result in significant functional improvement. One cause for the lack of functional rehabilitation may be the fact that the transplanted cells do not produce and secrete sufficient neurogenic factors. To address this problem, we have transfected pigment epithelial cells with the neurogenic and anti-angiogenic factor pigment epithelium-derived factor (PEDF) using non-viral protocols. In this study we show that the configuration of the inserted plasmid DNA affects transfection efficiency as well as stability of expression of rPEDF in primary pigment epithelial cells.
Using electroporation protocols primary pigment epithelial cells were transfected with circular and linearized plasmids encoding PEDF and PEDF-EGFP fusion proteins. For selection purpose the plasmids also encoded a Zeocin resistance gene. Plasmid linearization was carried out by restriction enzyme mediated cleavage beyond the coding regions. To evaluate transfection efficiency and protein expression stability, secreted rPEDF was purified by Ni-NTA affinity chromatography and identified by immunoblotting at various times (weekly) after transfection.
Primary retinal (RPE) and iris (IPE) pigment epithelial cells were successfully transfected with PEDF encoding plasmids. However, cells transfected with circular plasmids showed only weak and transient secretion of rPEDF fusion proteins, whereas cells transfected with linearized plasmids resulted in stable expression of rPEDF. Cells transfected with linearized plasmids expressed and secreted rPEDF fusion proteins into the cell culture medium for the 7 months and through the 5 passages that the cultures were followed.
The ability to non-virally transfect primary pigment epithelial cells with PEDF and the subsequent stable expression of rPEDF are first important steps in the development of a non-viral, ex vivo somatic gene addition therapy for AMD using autologous IPE or RPE cells.
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