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S. Cashman, C. Kubo, R. Kumar-Singh; Progress on Mutation Independent Silencing of Rhodopsin Using RNA Interference (RNAi) and Trans Complementation by an RNAi -Resistant Rhodopsin Transgene. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4612.
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More than one hundred different mutations in the gene encoding rhodopsin are associated with autosomal dominant retinitis pigmentosa. Given this large heterogeneity of mutations, it would be ideal to develop mutation-independent therapies for these diseases.
We developed RNA interference (RNAi), or more specifically, short hairpin RNAs expressed from DNA templates that target and silence both normal and mutant (P23H) human rhodopsin mRNA. Using the degeneracy of the genetic code, we also developed a rhodopsin transgene that was completely resistant to silencing by these shRNAs. These constructs were transfected transiently into human embryonic retinoblasts, simulating adRP disease and rescue. Subsequently, we developed stable cell lines expressing normal and P23H rhodopsin mRNAs and adeno associated virus (AAV) and adenovirus (Ad) vectors expressing shRNAs targeting rhodopsin and an RNAi-resistant rhodopsin transgene.
Co transfection of DNAs expressing a RNAi-resistant rhodopsin mRNA, a P23H rhodopsin mRNA and a shRNA revealed shRNA mediated silencing specifically of P23H rhodopsin by 90% and no loss of rhodopsin translation from the RNAi-resistant mRNA in those cells. Using AAV and Ad vectors in stable cell lines expressing rhodopsin validated the above results from transient transfections, albeit we found that silencing of rhodopsin was more robust in transient transfections than in stable cell lines. Finally, we introduced these viral vectors expressing shRNA and RNAi-resistant rhodopsin transgenes into animal models of adRP and preliminary data from these in vivo experiments will be presented.
Mutation independent approaches using RNA interference may have application in the treatment of retinal degeneration in humans.
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