title: Development of allele-specific therapeutic siRNA for keratin 5 mutations in epidermolysis bullosa simplex.
abstract: Epidermolysis bullosa simplex (EBS) is an incurable, inherited skin-blistering disorder predominantly caused by dominant-negative mutations in the genes encoding keratins K5 or K14. RNA interference, particularly in the form of small interfering RNA (siRNA), offers a potential therapy route for EBS and related keratin disorders by selectively silencing the mutant allele. Here, using a systemic screening system based on a luciferase reporter gene assay, we have developed mutant-specific siRNAs for two independent EBS-causing missense mutations in the K5 gene (p.Ser181Pro and p.Asn193Lys). The specificity of the allele-specific inhibitors identified in the screen was subsequently confirmed at the protein level, where the lead inhibitors were shown to strongly knock down the expression of mutant proteins with negligible effect on wild-type K5 expression. In a cell-based model system, the lead inhibitors were able to significantly reverse the cytoskeletal aggregation phenotype. Overall, this approach shows promise for the treatment of EBS and paves the way for future clinical trials.
authors.affiliation: Epithelial Genetics Group, Division of Molecular Medicine, Medical Sciences Institute, Colleges of Life Sciences and Medicine, Dentistry & Nursing, University of Dundee, Dundee, UK.
authors.forename: Sarah D
authors.initials: SD
authors.lastname: Atkinson
authors.forename: Victoria E
authors.initials: VE
authors.lastname: McGilligan
authors.forename: Haihui
authors.initials: H
authors.lastname: Liao
authors.forename: Ildiko
authors.initials: I
authors.lastname: Szeverenyi
authors.forename: Frances J D
authors.initials: FJ
authors.lastname: Smith
authors.forename: C B Tara
authors.initials: CB
authors.lastname: Moore
authors.forename: W H Irwin
authors.initials: WH
authors.lastname: McLean
mesh_terms: D000483:Alleles; D000818:Animals; D004173:Dipodomys; D016110:Epidermolysis Bullosa Simplex / Q000235:genetics* / Q000628:therapy*; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D053547:Keratin-14 / Q000235:genetics; D053555:Keratin-5 / Q000235:genetics*; D009154:Mutation; D010641:Phenotype; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism*; D014162:Transfection
chemical_list: D053547:Keratin-14; D053555:Keratin-5; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins