Long-lasting secretion of transgene product from differentiated and filter-grown retinal pigment epithelial cells after nonviral gene transfer.
OBJECTIVE
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.
Mannermaa
Eliisa
E
Department of Pharmaceutics, University of Kuopio, Kuopio, Finland.
Rönkkö
Seppo
S
Ruponen
Marika
M
Reinisalo
Mika
M
Urtti
Arto
A
D000469:Alkaline Phosphatase / Q000235:genetics*; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D005229:Fatty Acids, Monounsaturated / Q000378:metabolism; D005434:Flow Cytometry; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000201:enzymology*; D010957:Plasmids; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D019076:Transgenes
C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D005229:Fatty Acids, Monounsaturated; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D000469:Alkaline Phosphatase; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane