Comparative analysis of the uptake and expression of plasmid vectors in human ciliary and retinal pigment epithelial cells in vitro.
The retinal pigment epithelium is uniquely suited to gene therapy that uses lipid-mediated DNA transfer due to its high phagocytic activity in situ. We compared the relative efficacy of phagocytosis on the uptake of labeled plasmid vectors by retinal pigment epithelial and ciliary epithelial cells in vitro. Relative levels of endocytosis were then compared with the efficiency of marker transgene expression in these cells. Human retinal pigment epithelial and ciliary epithelial cells from a single donor were isolated and expanded in vitro. Polyplex-mediated transfections were performed using a rhodamine-labeled expression vector for green fluorescent protein. Rhodamine-labeled endosomes were examined by fluorescence microscopy at different time points. Rhodamine labeling and green fluorescent protein expression were analyzed by flow cytometry 48 h after transfection. These gene transfer studies showed that expression of transgenes does occur in both human retinal pigment epithelial and ciliary epithelial cells in vitro. Endocytosis of labeled plasmid vectors occurs at a significantly higher number and density in retinal pigment epithelial cells than in ciliary epithelial cells (P < 0.04). However, the efficiency of marker transgene expression is similar in the two cell types. These studies demonstrate that the higher intrinsic phagocytic activity does not enhance the efficacy of transgene expression in retinal pigment epithelial cells in vitro. Both human retinal pigment epithelial and ciliary epithelial cells are competent recipients for lipid-mediated gene transfer, and transgene expression occurs at similar levels in both cell types.
Chaum
E
E
Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA. echaum@mail.eye.utmem.edu
D000818:Animals; D002478:Cells, Cultured; D002924:Ciliary Body / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D005819:Genetic Markers; D005822:Genetic Vectors / Q000096:biosynthesis / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis; D008856:Microscopy, Fluorescence; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D010957:Plasmids / Q000096:biosynthesis / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism; D014162:Transfection / Q000379:methods
D005456:Fluorescent Dyes; D005819:Genetic Markers; D008164:Luminescent Proteins; D012235:Rhodamines; D049452:Green Fluorescent Proteins