Abstract
More than one hundred different mutations in the gene encoding rhodopsin are associated with a group of retinal degenerations including retinitis pigmentosa, congenital stationary night blindness and retinitis punctata albescens. Given this large heterogeneity of mutations, it would be ideal to develop mutation-independent therapies for these diseases. We describe use of RNA interference (RNAi) and specifically short hairpin RNAs (shRNAs) expressed from DNA templates to silence both normal and mutant (P23H) human rhodopsin alleles by 94.34±2.17 and 94.9±1.9%, respectively, in human embryonic retinoblasts. Degeneracy of the genetic code was used to engineer a codon-exchanged mRNA (cmRNA) that demonstrated complete resistance to silencing by the shRNA. Simulation of autosomal dominant retinitis pigmentosa in cell culture through triple transfection of DNAs expressing a cmRNA, a P23H mRNA and an shRNA revealed shRNA-mediated silencing, specifically of P23H rhodopsin by 90.64±5.19% and no loss of rhodopsin translation from the cmRNA in those cells. In addition, we present data on two alternative shRNA sequences targeting human rhodopsin. Our results have implications for the treatment of a very large variety of retinal degenerations in a mutation-independent manner.
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Acknowledgements
We thank Beverly Davidson (University of Iowa) for providing the plasmid pmCMVmpAGFPi, Robert Molday (University of British Columbia) for providing antibody 1D4 and Jeremy Nathans (Johns Hopkins) for providing the human rhodopsin cDNA. We would also like to thank Laura McCullough for technical assistance. This study was supported by grants to RK-S from the National Eye Institute (1R01EY013837 and 1R01EY014991), a career development award from Research to Prevent Blindness and generous funding from The Foundation Fighting Blindness (FFB) and the Knight's Templar Eye Foundation.
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Cashman, S., Binkley, E. & Kumar-Singh, R. Towards mutation-independent silencing of genes involved in retinal degeneration by RNA interference. Gene Ther 12, 1223–1228 (2005). https://doi.org/10.1038/sj.gt.3302512
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DOI: https://doi.org/10.1038/sj.gt.3302512
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