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Gabriele Thumann, Christiane Stickelmann, Naredra Armogan, Niklas Plange, Martin Hermel, Peter Walter, Sandra Johnen; Novel Hyperactive Transposons Enable Stable Transfection Of Isolated, Terminally Differentiated Cells Immediately After Isolation From A Biopsy. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5472.
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The safe introduction of a cell based gene therapy into clinical practice is limited due to several factors, such as side effects associated with viral vectors, de-differentiation and possible contamination of cultured cells, limited lifespan of the transfected gene due to non-integration, and low transfection efficiency. Here we detail a procedure for an efficient, non-virally transposon-mediated transfection allowing for a safe autologous cell-based additive gene therapy. The procedure comprises cell isolation from a biopsy, transfection, and transplantation of the transfected cells to the subretinal space, which can be done in the operating room within a 45 - 60 minutes timeline.
Freshly isolated iris pigment epithelial cells were electroporated with an hyperactive SB vector and plasmids encoding the gene for Venus control protein (yellow fluorescent mutant of the green fluorescent protein (GFP) or the gene for human pigment epithelium-derived factor (PEDF) co-expressed with GFP. Fluorescent and light microscopy of the transfected and cultured cells as well as Western Blot (WB) analysis of the culture supernatants were performed to assess transfection efficiency, stability of transgene expression, and secretion of the recombinant proteins.
Immediately following isolation, cell suspensions isolated from iris biopsies were transfected using the plasmid-based SB system. Biopsy, cell isolation, and transfection were accomplished in a period of 20 minutes. Subsequent culture of the transfected cells showed the monolayer development of monolayers of fluorescent, pigmented, and well differentiated cells with cobblestone morphology. Stable integration of the PEDF and GFP genes was demonstrated by the maintenance of fluorescence in proliferating cells after several passages. WB analysis of the culture supernatants showed secretion of PEDF for the three months the transfected cells were followed in culture.
The efficient transfection of freshly isolated cells in a very short time span will allow for a one step cell-based gene therapy for the treatment of neovascular AMD. This procedure will comprise iridectomy, cell isolation, transfection, and subretinal cell transplantation and can be accomplished in the operating room within a 45-60 minutes session.
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