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Paulette M. Robinson, Sriniwas Sriram, William Hauswirth, Alfred S. Lewin, Gregory S. Schultz; Gene Transfer of Self Complimentary Adeno-associated Virus into Rabbit Corneas After Photorefractive Keratectomy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1914. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Self complementary (double stranded) adeno-associated viruses (scAAVs) have been shown to have a faster onset of gene expression because the scAAV DNA is transcribed rapidly. In addition, scAAV generally have higher transduction efficiency than conventional rAAV vectors. In this study, we investigated scAAV-mediated gene transfer in rabbit cornea after photorefractive keratectomy (PRK). Our goal was to characterize the time of first detectable expression, determine the duration of transgene expression, and identify what cell types were transfected.
Self complementary AAV expressing green fluorescent protein (scAAV-GFP) was applied following PRK for two minutes. Corneas were removed at 0, 1, 2, 3, 4 , 7, 30 and 180 days after vector application and fixed in 2% formaldehyde then cryosectioned. Slides were analyzed for direct fluorescence using confocal microscopy. Images were analyzed using Optimas Imaging software and were analyzed for statistical significance by student t-test using GraphPad prism.
The GFP fluorescence was first detected 24 hours after application and the peak fluorescence detected occurred at 7 days. Day 7 fluorescence was 22 times greater than day 0. Levels of the fluorescence from day 180 were statistically the same as day 0 levels of fluorescence. The transgene was expressed in all cell types of the cornea; epithelium, keratocytes and endothelium.
These data indicate that topically applied scAAV vectors rapidly express transgenes in all cell types in the cornea. Transduction of corneal cells with scAAV vector expressing ribozymes or shRNA targeting profibrotic genes may provide an optimal therapy to reduce scar formation in the cornea.
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