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P. F. Kenna, A. Palfi, N. Chadderton, M. O'Reilly, S. Millington-Ward, M. Humphries, P. Humphries, J. Farrar; RNAi-Based Suppression of Rhodopsin in vivo. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1978.
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Retinitis Pigmentosa is the most prevalent cause of registered blindness in those of working age in the developed world, affecting approximately 1 in 3,000 people. The autosomal dominant forms of the disease are genetically and mutationally extremely heterogeneous. We previously described strategies requiring mutation-independent suppression of the mutant allele and replacement with a gene construct altered such that it escapes suppression in order to overcome mutational heterogeneity (Millington Ward et al, Hum Mol Genet. 1997 Sep;6(9):1415-26). The present study was designed to see if efficient RNAi-based suppression of rhodopsin could be achieved in vivo.
Adult mice carrying a normal human rhodopsin (NHR) transgene on a murine rhodopsin negative (rho-/-) background were sub-retinally injected with either an eGFP-tagged AAV2/5 construct incorporating a H1 promoter driven short hairpin RNA (shRNA) targeting NHR (N = 14 eyes) or an eGFP-tagged non-targeting construct (N = 12 eyes). Retinas were harvested two weeks post injection, dissociated, and FACS sorted to select for cells expressing eGFP. NHR mRNA levels in these cells were analysed using real-time PCR (rt-PCR).
rt-PCR analysis of NHR mRNA levels indicated that suppression of the order of 90% was achieved in eGPF positive retinal cells from eyes which received the targeting vector compared with eGFP positive retinal cells from eyes which received the non-targeting vector.
We have previously shown that shRNAs can efficiently reduce murine rhodopsin and rds mRNA expression levels in COS 7 cells and in electroporated retinal explants by up to 80% and can discriminate, in a mutation independent manner, between wild-type and mutant genes (Kiang et al, Mol Ther. 2005 Sep;12(3):555-61; Palfi et al, Hum Mutat. 2006 Mar;27(3):260-8;). In this study we demonstrate, in vivo, similar suppression levels following sub-retinal injection of AAV-shRNA targeting normal human rhodopsin.
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