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A. Georgiadis, M. Tschernutter, J. W. Bainbridge, K. S. Balaggan, F. Mowat, M. S. Balda, K. Matter, R. R. Ali; Lentivirus-Mediated RNA Interference Disrupts Junctional Signalling in the RPE. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5361.
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RNA interference is a potentially powerful tool for in vivo modulation of gene function. In this study we aimed to assess it utility in the retina by examining the efficiency and kinetics of the knock-down of RPE-specific genes using lentiviral short hairpin (shRNA) expression cassettes. Epithelial cell cycle modulators, ZO-1 and ZONAB, are part of an epithelial tight junction pathway cascade that regulates gene expression and RPE differentiation. They were chosen as candidate genes to be targeted separately in a wild-type murine retina as their down-regulation can be assessed through the disruption of the RPE monolayer.
Short hairpins targeting ZO-1, ZONAB and eGFP were cloned into a lentiviral backbone driven by a U6 promoter. Lentiviral preparations were subretinally injected into young adult wild-type mice at two different concentrations (1 x 10e7 and 1 x 10e8 particles/ml) and the treated eyes were analysed at 5, 10, 50 and 100 days post injection. We analysed the eyes by light and electron microscopy as well as by immunohistochemistry for ZO-1, ZONAB, RPE65 and BrdU incorporation.
Lentiviral-mediated delivery of shRNA cassettes leads to a rapid knock-down of the target gene expression. Robust phenotypic changes are apparent 5 days after injection at a low titre. Downregulation of ZO-1 leads to retinal disorganisation and RPE proliferation and migration. Downregulation of ZONAB leads to retinal disorganisation and possible RPE dedifferentiation marked by downregulation of the RPE-specific gene RPE65. Delivery of reduced titre lentivirus results in patchy RPE transduction and enables phenotypic analysis of individual, transduced RPE cells in a wild-type monolayer.
Lentiviral RPE transduction leads to inhibition of gene expression through a U6-based RNAi cassette. Our data suggest that as early as 5 days after subretinal delivery the transcribed shRNA reaches physiologically significant concentrations in the transduced cells and enables us to demonstrate through the modulation of the ZO-1/ZONAB pathway the importance of junctional signalling in RPE proliferation and differentiation.
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