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Edwin H. A. Allen, Sarah D. Atkinson, Haihui Liao, Jonathan E. Moore, Deena M. Leslie Pedrioli, Frances J. D. Smith, William H. Irwin McLean, C. B. Tara Moore; Allele-Specific siRNA Silencing for the Common Keratin 12 Founder Mutation in Meesmann Epithelial Corneal Dystrophy. Invest. Ophthalmol. Vis. Sci. 2013;54(1):494-502. doi: 10.1167/iovs.12-10528.
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To identify an allele-specific short interfering RNA (siRNA), against the common KRT12 mutation Arg135Thr in Meesmann epithelial corneal dystrophy (MECD) as a personalized approach to treatment.
siRNAs against the K12 Arg135Thr mutation were evaluated using a dual luciferase reporter gene assay and the most potent and specific siRNAs were further screened by Western blot. Off-target effects on related keratins were assessed and immunological stimulation of TLR3 was evaluated by RT-PCR. A modified 5′ rapid amplification of cDNA ends method was used to confirm siRNA-mediated mutant knockdown. Allele discrimination was confirmed by quantitative infrared immunoblotting.
The lead siRNA, with an IC50 of thirty picomolar, showed no keratin off-target effects or activation of TLR3 in the concentration ranges tested. We confirmed siRNA-mediated knockdown by the presence of K12 mRNA fragments cleaved at the predicted site. A dual tag infrared immunoblot showed knockdown to be allele-specific, with 70% to 80% silencing of the mutant protein.
A potent allele-specific siRNA against the K12 Arg135Thr mutation was identified. In combination with efficient eyedrop formulation delivery, this would represent a personalized medicine approach, aimed at preventing the pathology associated with MECD and other ocular surface pathologies with dominant-negative or gain-of-function pathomechanisms.
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