title: Development of therapeutic siRNAs for pachyonychia congenita.
abstract: Pachyonychia congenita (PC) is an autosomal-dominant keratin disorder where the most painful, debilitating aspect is plantar keratoderma. PC is caused by mutations in one of four keratin genes; however, most patients carry K6a mutations. Knockout mouse studies suggest that ablation of one of the several K6 genes can be tolerated owing to compensatory expression of the others. Here, we have developed potent RNA interference against K6a as a paradigm for treating a localized dominant skin disorder. Four small interfering RNAs (siRNAs) were designed against unique sequences in the K6a 3'-untranslated region. We demonstrated near-complete ablation of endogenous K6a protein expression in two keratinocyte cell lines, HaCaT and NEB-1, by transient transfection of each of the four K6a siRNAs. The siRNAs were effective at very low, picomolar concentrations. One potent lead K6a inhibitor, which was highly specific for K6a, was tested in a mouse model where reporter gene constructs were injected intradermally into mouse paw and luciferase activity was used as an in vivo readout. Imaging in live mice using the Xenogen IVIS system demonstrated that the K6a-specific siRNA strongly inhibited bicistronic K6a-luciferase gene expression in vivo. These data suggest that siRNAs can specifically and very potently target mutated genes in the skin and support development of these inhibitors as potential therapeutics.
authors.affiliation: Epithelial Genetics Group, Human Genetics Unit, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK. f.j.d.smith@dundee.ac.uk
authors.forename: Frances J D
authors.initials: FJ
authors.lastname: Smith
authors.forename: Robyn P
authors.initials: RP
authors.lastname: Hickerson
authors.forename: Jane M
authors.initials: JM
authors.lastname: Sayers
authors.forename: Robert E
authors.initials: RE
authors.lastname: Reeves
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Contag
authors.forename: Devin
authors.initials: D
authors.lastname: Leake
authors.forename: Roger L
authors.initials: RL
authors.lastname: Kaspar
authors.forename: W H Irwin
authors.initials: WH
authors.lastname: McLean
mesh_terms: D020413:3' Untranslated Regions / Q000737:chemistry / Q000235:genetics; D000818:Animals; D002460:Cell Line; D005260:Female; D006801:Humans; D053553:Keratin-6 / Q000037:antagonists & inhibitors* / Q000235:genetics; D015603:Keratinocytes / Q000378:metabolism; D051379:Mice; D053549:Pachyonychia Congenita / Q000628:therapy*; D034741:RNA, Small Interfering / Q000627:therapeutic use*
chemical_list: D020413:3' Untranslated Regions; D053553:Keratin-6; C507567:Krt6a protein, mouse; D034741:RNA, Small Interfering