PURPOSE: The purpose of this study was to investigate the extent, duration, and direction of transgene expression after nonviral gene transfer to differentiated retinal pigment epithelial (RPE) cells. METHODS: Polarized human RPE cells (ARPE-19) were transfected with nonviral vectors [DOTAP/DOPE with and without protamine sulfate (PS), DOTAP, PEI (polyethyleneimine), DHP-12] using secreted alkaline phosphatase (SEAP) as a reporter gene. Cellular uptake was studied by flow cytometry. RESULTS: Up to 80-fold differences were observed in the peak reporter gene expression. The highest peak levels and the longest lifetime of SEAP expression (> 69 days) were obtained with DOTAP/DOPE/PS/pDNA complexes. With PEI, higher expression was seen to the apical side than to the basolateral side. CONCLUSIONS: In contrast to most differentiated epithelial cells, the differentiated RPE cells can be transfected at high and prolonged levels with selected lipoplexes.
PURPOSE: The purpose of this study was to investigate the extent, duration, and direction of transgene expression after nonviral gene transfer to differentiated retinal pigment epithelial (RPE) cells. METHODS: Polarized human RPE cells (ARPE-19) were transfected with nonviral vectors [DOTAP/DOPE with and without protamine sulfate (PS), DOTAP, PEI (polyethyleneimine), DHP-12] using secreted alkaline phosphatase (SEAP) as a reporter gene. Cellular uptake was studied by flow cytometry. RESULTS: Up to 80-fold differences were observed in the peak reporter gene expression. The highest peak levels and the longest lifetime of SEAP expression (> 69 days) were obtained with DOTAP/DOPE/PS/pDNA complexes. With PEI, higher expression was seen to the apical side than to the basolateral side. CONCLUSIONS: In contrast to most differentiated epithelial cells, the differentiated RPE cells can be transfected at high and prolonged levels with selected lipoplexes.
Authors: Bibhudatta Mishra; David R Wilson; Srinivas R Sripathi; Mark P Suprenant; Yuan Rui; Karl J Wahlin; Cynthia A Berlinicke; Jordan J Green; Donald J Zack Journal: Regen Eng Transl Med Date: 2019-07-24