Allele-specific siRNA silencing for the common keratin 12 founder mutation in Meesmann epithelial corneal dystrophy

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

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    Abstract

    PURPOSE. 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.

    METHODS. 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.

    RESULTS. 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.

    CONCLUSIONS. 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. (Invest Ophthalmol Vis Sci. 2013;54:494-502) DOI:10.1167/iovs.12-10528

    Original languageEnglish
    Pages (from-to)494-502
    Number of pages9
    JournalInvestigative Ophthalmology & Visual Science
    Volume54
    Issue number1
    DOIs
    Publication statusPublished - 2013

    Keywords

    • DISEASES
    • EPIDERMOLYSIS-BULLOSA SIMPLEX
    • CELL-LINE
    • RNAI THERAPEUTICS
    • GENE-THERAPY
    • KNOCKING
    • INCLUDING PACHYONYCHIA-CONGENITA
    • DISORDERS
    • INTERFERENCE
    • DELIVERY

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