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 diseases. To fulfill this promise, plasmid DNA must be delivered to the retinal target cells. We evaluated the efficacy of synthetic DNA complexing compounds in transfecting primary human retinal pigment epithelial (RPE) cells in vitro. Fetal human RPE cells were cultured with or without extracellular matrix (ECM), produced using calf corneal endothelial cells. Plasmids encoding nuclear localizing beta galactosidase or luciferase (pRSVLuc, pCLuc4, pSV2Luc) were complexed in water at various +/- charge ratios using cationic lipids (Lipofectin, DOTAP, DOGS), polyethylene imines (25 and 750 kDa), and with degraded 6th generation starburst polyamidoamine dendrimers. Luciferase was quantified using a luminometric assay and beta galactosidase with X-gal staining. Toxicities of transfections were evaluated with the MTT-assay. Using beta galactosidase as the reporter gene naked DNA did not transfect RPE cells at measurable levels whereas 1-5% of the cells expressed histochemically detectable amounts of the gene after transfection with cationic lipid 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 ratios, expression levels of luciferase were > 10(9) light units/mg protein after 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 molecular weight PEI were less toxic than cationic lipids or PEI25 to RPE cells. Serum and ECM decreased gene expression to the RPE cells with all carriers. Despite low percentage of transfected cells the transgene expression per RPE cell is high, important feature in the retinal tissue with small dimensions, in particular in the case of secreted gene products. Degraded dendrimers and high molecular weight PEI exhibited the best combination of high activity and low toxicity in RPE cell transfection.
Urtti
A
A
Department of Bio-Pharmaceutical Sciences, University of California San Francisco, USA. Arto.Urtti@messi.uku.fi
Polansky
J
J
Lui
G M
GM
Szoka
F C
FC
D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D004847:Epithelial Cells / Q000378:metabolism; D005109:Extracellular Matrix / Q000502:physiology*; D015870:Gene Expression; D015316:Genetic Therapy; D006801:Humans; D008081:Liposomes / Q000008:administration & dosage; D008156:Luciferases / Q000235:genetics; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D011108:Polymers / Q000008:administration & dosage; D014162:Transfection
D008081:Liposomes; D011108:Polymers; D008156:Luciferases