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G. Thumann, C. Maltusch, M. Stöcker, A. K. Salz, P. Walter, S. Johnen; Efficient Non-Viral Transfection of Primary Pigment Epithelial Cells With Human PEDF. Invest. Ophthalmol. Vis. Sci. 2009;50(13):6231.
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Studies in animal models of retinal degeneration have shown that a number of growth factors prevent or delay photoreceptor degeneration. To replace degenerated RPE cells and deliver growth factors to the subretinal space transplantation of retinal (RPE) and iris pigment epithelial (IPE) cells has been advocated as a treatment modality for AMD. Since transplantation of pigment cells has not resulted in significant functional improvement, transplantation of encapsulated, homologous RPE cells transfected with growth factors are now in progress. To avoid the immunologic complications associated with viral transfection or the use of a homologous cell line we have explored the use of non-viral protocols to insert genetic material into primary pigment epithelial cells.
Using nucleofection primary RPE and IPE cells were transfected with plasmids encoding GFP, EGFP-PEDF, PEDF-EGFP fusion proteins, PEDF and an integrated zeocin resistance gene. Transfection efficiency and stability of PEDF expression were evaluated by fluorescence microscopy and immunoblotting of the purified PEDF-variants respectively.
IPE and RPE cells were successfully transfected with GFP and PEDF encoding plasmids with efficiencies greater than 70%. Expression of GFP has been observed for 15 months in IPE cells and expression of PEDF fusion proteins and PEDF has been stable for the 4 months since transfection in IPE and RPE cells.
Since autologous IPE cells, contrary to RPE cells, are easily obtained with minimally invasive surgery, the ability to stably, non-virally transfect human IPE cells with the PEDF or other appropriate gene, followed by transplantation of the modified cells subretinally in AMD patients may prove an important first step in the application of tissue engineering in the treatment of neurodegenerative retinal diseases.
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