title: Generic and personalized RNAi-based therapeutics for a dominant-negative epidermal fragility disorder.
abstract: Epidermolytic palmoplantar keratoderma (EPPK) is one of >30 autosomal-dominant human keratinizing disorders that could benefit from RNA interference (RNAi)-based therapy. EPPK is caused by mutations in the keratin 9 (KRT9) gene, which is exclusively expressed in thick palm and sole skin where there is considerable keratin redundancy. This, along with the fact that EPPK is predominantly caused by a few hotspot mutations, makes it an ideal proof-of-principle model skin disease to develop gene-specific, as well as mutation-specific, short interfering RNA (siRNA) therapies. We have developed a broad preclinical RNAi-based therapeutic package for EPPK containing generic KRT9 siRNAs and allele-specific siRNAs for four prevalent mutations. Inhibitors were systematically identified in vitro using a luciferase reporter gene assay and validated using an innovative dual-Flag/Strep-TagII quantitative immunoblot assay. siKRT9-1 and siKRT9-3 were the most potent generic K9 inhibitors, eliciting >85% simultaneous knockdown of wild-type and mutant K9 protein synthesis at picomolar concentrations. The allele-specific inhibitors displayed similar potencies and, importantly, exhibited strong specificities for their target dominant-negative alleles with little or no effect on wild-type K9. The most promising allele-specific siRNA, siR163Q-13, was tested in a mouse model and was confirmed to preferentially inhibit mutant allele expression in vivo.
authors.affiliation: Dermatology and Genetic Medicine, Division of Molecular Medicine, Colleges of Life Sciences and Medicine, Dentistry & Nursing, University of Dundee, Dundee, UK. d.lesliepedrioli@dundee.ac.uk
authors.forename: Deena M
authors.initials: DM
authors.lastname: Leslie Pedrioli
authors.forename: Dun Jack
authors.initials: DJ
authors.lastname: Fu
authors.forename: Emilio
authors.initials: E
authors.lastname: Gonzalez-Gonzalez
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Contag
authors.forename: Roger L
authors.initials: RL
authors.lastname: Kaspar
authors.forename: Frances J D
authors.initials: FJ
authors.lastname: Smith
authors.forename: W H Irwin
authors.initials: WH
authors.lastname: McLean
mesh_terms: D000818:Animals; D002460:Cell Line; D004195:Disease Models, Animal; D004817:Epidermis / Q000502:physiology; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D053534:Keratin-9 / Q000235:genetics*; D015603:Keratinocytes / Q000166:cytology; D053546:Keratoderma, Palmoplantar, Epidermolytic / Q000235:genetics* / Q000628:therapy*; D007668:Kidney / Q000166:cytology; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008815:Mice, Inbred Strains; D057285:Precision Medicine / Q000379:methods*; D034741:RNA, Small Interfering / Q000494:pharmacology*
chemical_list: C507440:KRT9 protein, human; C507441:KRT9 protein, mouse; D053534:Keratin-9; D034741:RNA, Small Interfering; D008156:Luciferases