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Gabriele Thumann, Corinne Marie, Zsuzsanna Izsvák, Daniel Scherman, Peter Walter, Sandra Johnen; The Use of Plasmids Free of Antibiotic Resistance Markers for Safe and Efficient Sleeping Beauty-Mediated PEDF Gene Delivery into Primary Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1826.
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Exudative age-related macular degeneration leads to blindness within several months and is characterized by the growth of choroidal blood vessels into the subretinal space, resulting in RPE and photoreceptor degeneration. Since the growth of new blood vessels is caused by the increased expression of the pro-angiogenic vascular endothelial growth factor (VEGF), current treatments are based on the inhibition of VEGF by anti-VEGF antibodies. These treatments improve vision in about 30% of patients, but may be accompanied by severe side effects and non-compliance. We have postulated that subretinal transplantation of pigment epithelial cells genetically modified to stably overexpress pigment epithelium-derived factor (PEDF), a VEGF inhibitor, may be a more effective treatment. Here we report the use of the Sleeping Beauty (SB100X) transposon system to deliver the human PEDF gene, carried by pFAR4-plasmids, an expression vector free of antibiotic resistance markers.
Primary retinal pigment epithelial (RPE) cells from human donor eyes were transfected with pFAR4-plasmids encoding the SB100X transposase and the PEDF gene at a ratio of 1:16. Stability of PEDF gene expression was determined by RT-PCR and stability of PEDF secretion was analysed by immunoblotting and ELISA.
Stable PEDF transgene delivery into as few as 10,000 RPE cells was shown in more than 12 independent experiments. PEDF was continuously secreted for the 7 months that the cells were followed, at levels of 1.78 ± 1.64 ng/hour/10,000 cells, which is approximately 40-times higher than in non-transfected control cells.
The use of pFAR4-plasmids for gene delivery adds a level of safety to gene therapeutic approaches, since it avoids the transfer and potential integration of antibiotic resistance genes. This study demonstrates the feasibility of using the combination of the Sleeping Beauty transposon and the pFAR4-plasmid technology for efficient delivery and sustained expression of the PEDF transgene into as few as 10,000 primary RPE cells, a number that can be obtained from a peripheral retinal biopsy of a patient for later subretinal transplantation into the same patient.
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