Gene delivery and expression in human retinal pigment epithelial cells: Effects of synthetic carriers, serum, extracellular matrix and viral promoters

Non-viral gene therapy is a potential treatment to many incurable retinal d iseases. To fulfill this promise, plasmid DNA must be delivered to the reti nal target cells. We evaluated the efficacy of synthetic DNA complexing com pounds in transfecting primary human retinal pigment epithelial (RPE) cells in vitro. Fetal human RPE cells were cultured with or without extracellular matrix (E CM), produced using calf corneal endothelial cells. Plasmids encoding nucle ar localizing beta galactosidase or luciferase (pRSVLuc, pCLuc4, pSV2Luc) w ere complexed in water at various +/-charge ratios using cationic lipids (L ipofectin, DOTAP, DOGS), polyethylene imines (25 and 750 kDa), and with deg raded 6th generation starburst polyamido-amine dendrimers. Luciferase was q uantified using a luminometric assay and beta galactosidase with X-gal stai ning. Toxicities of transfections were evaluated with the MTT-assay. Using beta galactosidase as the reporter gene naked DNA did not transfect R PE cells at measurable levels whereas 1-5% of the cells expressed histochem ically detectable amounts of the gene after transfection with cationic lipi d-DNA complexes. In RPE cells. Rous sarcoma virus and cytomegalovirus (CMV) were more efficient promoters than SV40 in driving luciferase expression, and CMV was chosen for further experiments. At optimal complex charge ratio s, expression levels of luciferase were > 10(9) light units/mg protein afte r transfection using dendrimers and PEI25, while transfection mediated with the other carriers resulted in luciferase expression levels of 10(7) - 10( 9) light units/mg protein or less. In general, dendrimers and large molecul ar weight PEI were less toxic than cationic lipids or PEI25 to RPE cells. S erum and ECM decreased gene expression to the RPE cells with all carriers. Despite low percentage of transfected cells the transgene expression per RP E cell is high, important feature in the retinal tissue with small dimensio ns, in particular in the case of secreted gene products. Degraded dendrimer s and high molecular weight PEI exhibited the best combination of high acti vity and low toxicity in RPE cell transfection.