Polyplex-mediated gene transfer into human retinal pigment epithelial cells in vitro.
The human retinal pigment epithelium (RPE) is a potential target tissue for directed transfer of candidate genes to treat age-related macular degeneration (AMD). The RPE is uniquely suited to gene therapy protocols that use liposome-mediated DNA transfer because of its high intrinsic phagocytic function in vivo. In these studies, we examined the efficacy of human RPE cell uptake and expression of the green fluorescent protein (GFP) and neomycin resistance marker genes by polyplex-mediated gene transfer in vitro. The effects of varying DNA and polyplex concentration and ratios on GFP transgene expression were examined. A narrow range of experimental conditions were found to maximize transgene expression; most important were the DNA concentration and the DNA:polyplex ratio. The transfection efficiency for human RPE cells was reproducibly 20% in vitro by this method and reached a maximum level of expression after 48 h. There was a rapid decline in gene expression over 2 weeks following polyplex-mediated gene transfer, but stable integration does occur at low frequencies with and without selection.
Chaum
E
E
Division of Ophthalmology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655, USA. Edward.Chaum@banyan.ummed.edu
Hatton
M P
MP
Stein
G
G
D004247:DNA; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006801:Humans; D066298:In Vitro Techniques; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D019076:Transgenes
D004247:DNA