title: Allele-specific siRNA silencing for the common keratin 12 founder mutation in Meesmann epithelial corneal dystrophy.
abstract: OBJECTIVE
  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 IC(50) 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.
authors.affiliation: School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland BT52 1SA, UK.|Dermatology and Genetic Medicine, Colleges of Life Sciences and Medicine, Dentistry & Nursing, University of Dundee, Dundee DD1 5EH, Scotland.
authors.forename: Edwin H A
authors.initials: EHA
authors.lastname: Allen
authors.affiliation: School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland BT52 1SA, UK.|Dermatology and Genetic Medicine, Colleges of Life Sciences and Medicine, Dentistry & Nursing, University of Dundee, Dundee DD1 5EH, Scotland.
authors.forename: Sarah D
authors.initials: SD
authors.lastname: Atkinson
authors.affiliation: Dermatology and Genetic Medicine, Colleges of Life Sciences and Medicine, Dentistry & Nursing, University of Dundee, Dundee DD1 5EH, Scotland.
authors.forename: Haihui
authors.initials: H
authors.lastname: Liao
authors.affiliation: School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland BT52 1SA, UK.
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Moore
authors.affiliation: Dermatology and Genetic Medicine, Colleges of Life Sciences and Medicine, Dentistry & Nursing, University of Dundee, Dundee DD1 5EH, Scotland.
authors.forename: Deena M Leslie
authors.initials: DML
authors.lastname: Pedrioli
authors.affiliation: Dermatology and Genetic Medicine, Colleges of Life Sciences and Medicine, Dentistry & Nursing, University of Dundee, Dundee DD1 5EH, Scotland.
authors.forename: Frances J D
authors.initials: FJD
authors.lastname: Smith
authors.affiliation: Dermatology and Genetic Medicine, Colleges of Life Sciences and Medicine, Dentistry & Nursing, University of Dundee, Dundee DD1 5EH, Scotland.
authors.forename: W H Irwin
authors.initials: WHI
authors.lastname: McLean
authors.affiliation: School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland BT52 1SA, UK.|Dermatology and Genetic Medicine, Colleges of Life Sciences and Medicine, Dentistry & Nursing, University of Dundee, Dundee DD1 5EH, Scotland.
authors.forename: C B Tara
authors.initials: CBT
authors.lastname: Moore
mesh_terms: D000483:Alleles; D018929:Cell Culture Techniques; D053559:Corneal Dystrophy, Juvenile Epithelial of Meesmann / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D004247:DNA / Q000235:genetics*; D005091:Exons; D020868:Gene Silencing; D006801:Humans; D053551:Keratin-12 / Q000235:genetics* / Q000378:metabolism; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D034741:RNA, Small Interfering / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D053551:Keratin-12; D050505:Mutant Proteins; D034741:RNA, Small Interfering; D004247:DNA