Targeted Non-Viral Gene Transfer into RPE Cells

Abstract

Abstract: : Purpose: To evaluate the ability to transfer Green Fluorescent Protein (GFP) reporter gene into RPE cells using poly (DL-lactide-co-glycolide)(PLGA) nanoparticles. Methods: Biodegradable and biocompatible PLGA nanoparticles (size range 600nm) loaded with plasmid encoding the gene for GFP under the control of a cytomegalovirus promoter were constructed (7.36µg DNA/1mg polyplex). In vitro, bovine and human RPE cells at 70% confluency were exposed to various concentrations of the polyplex nanoparticles. Transfection efficiency was evaluated in vitro after various incubation intervals. In vivo, intravitreal injections of 5µl of nanoparticles (polyplex 1 mg/ml) were carried out in Lewis rat eyes. The rats were sacrificed at different time points after the injection. Gene expression was evaluated by fluorescence and confocal microscopy. Naked GFP plasmids and blank nanoparticles served as control in all experiments. Results: In vitro, optimal transfection efficiency was observed with 0.1 mg/ml of polyplex nanoparticles. At this concentration, 15 to 20% of the RPE cells showed fluorescence. The intracellular intensity of GFP fluorescence varied, with 1 to 5% of the cells demonstrating an intense fluorescence. Around 80% of the cells did not demonstrate any fluorescence despite an incubation period of up to 5 days. Bovine RPE cells in vitro demonstrated similar patterns of fluorescence. In cultures incubated with naked plasmid, occasional GFP positive cells were observed only during the first day of culture. In vivo, a single intravitreal injection of GFP loaded particles yielded GFP expression which was preferentially localized in the RPE cell layer. Fluorescence at this retinal level was observed up to 4 weeks after injection. Conclusion: Non viral gene transfer using GFP loaded nanoparticles into RPE cells is feasible both in vitro and in vivo. This method of vectorization could possibly be assimilated for other genes transfer to be targeted to the RPE cells.

Keywords: retinal pigment epithelium • gene transfer/gene therapy • vitreous