Abstract
P 069
High efficient non-viral transfection of primary pigment epithelial cells
Sandra Johnen, Christiane Maltusch, Gabriele Thumann
RWTH Aachen, Universitäts-Augenklinik, IZKF Biomat., Aachen
Objective
Previous studies have shown that (a) transplanted autologous pigment epithelial cells in suspension are well tolerated, but do not result in significant functional improvement and (b) a number of growth factors prevent or delay photoreceptor degeneration in animal models of retinal degeneration. Therefore, replacement of degenerated RPE cells and delivery of one or more neurotrophic factors by transplanting monolayers of genetically modified pigment epithelial cells into the subretinal space should be considered as a promising treatment modality for AMD. Since the use of viral vectors is complicated by possible dissemination of the virus as well as severe immune reactions, we have explored the use of non-viral transfection protocols to insert the gene encoding pigment epithelium-derived factor (PEDF) in pigment epithelial cells.
Methods
Primary pigment epithelial cells were transfected by electroporation with plasmids encoding for recombinant PEDF and PEDF-EGFP fusion proteins. The PEDF fusion proteins, which were expressed and secreted into the cell culture medium, were purified by Ni-NTA metal affinity chromatography and subsequently identified by immunoblotting. Their biological activity was determined by examining their ability to up-regulate the mRNA expression level of the zinc transporter protein ZIP2 in cultivated ARPE-19 cells.
Results
Primary pigment epithelial cells were successfully transfected with efficiencies greater than 70%. The recombinant PEDF fusion proteins were expressed and secreted into the cell culture medium for the four months the cultures were followed as well as after 5 passages. The secreted PEDF fusion proteins were biologically active, as determined by the up-regulation of the ZIP2 mRNA expression level in ARPE-19 cells.
Conclusions
Using electroporation, it is possible to genetically modify primary pigment epithelial cells with high efficiency without the aid of viruses. Here we have shown that pigment epithelial cells, transfected with the gene encoding PEDF, express and secrete biologically active PEDF into the cell culture medium for extended periods of time, even after the cells were passaged, suggesting integration of the transfected PEDF gene into the cells’ genome.
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