Abstract
Abstract: :
Purpose: Most of the previous gene transfer strategies applied to the retina have used viral vectors. Here, our purpose was to evaluate a nonviral, noninvasive, and safe strategy for retinal delivery of genes into the retina of mice. A 4.7 kb plasmid DNA, driven by a cytomegalovirus promoter that expressed green fluorescent protein (GFP), was transferred into 40 eyes of 20 healthy mice, using transscleral iontophoresis. Methods: Transcleral iontophoresis, 15 minutes at 500µA, delivered plasmid diluted in BSS (balance salt solution). Iontophoresis was tested using different dilutions of the plasmid (200 µg/ml, 1000 µg/ml), different times between iontophoresis and the sacrifice of the animals (d4, d14, d28), and after repeating 4 times the application in the same eyes. Frozen sections, 30µm thick, were prepared for observation under a fluorescence microscope. The fluorescence was quantified using the luminosity channel of the histogram function in the Adobe PhotoShop 5.5 on the single optical section without any image modification, and compared with control eyes (6 eyes). GFP was also revealed by anti-GFP antibodies. Results: No damage to the cornea, retina, RPE or choroid was observed in any of the eyes. No functional impairment was observed on ERG analysis. Fluorescence showing GFP gene expression was found in the outer and inner segments of photoreceptors, in the nuclear layer, and the ganglion cell layer. The highest fluorescence was observed in the group of the mice that received 1000 µg/ml, compared with controls (182%; p<0.00001), and in the group of mice with 4-times repeated iontophoresis, compared with controls (177%; p<0.0002). These results were confirmed using Antibodies anti-GFP. Conclusions: Our findings show that iontophoresis is an effective method to transfer large genes into the retina and that it can be repeated many times. Iontophoresis could be used as a nonviral, noninvasive and safe method for gene therapy in retinal dystrophies.
Keywords: 419 gene transfer/gene therapy • 562 retinal degenerations: hereditary • 561 retinal degenerations: cell biology