Generic and personalized RNAi-based therapeutics for a dominant-negative epidermal fragility disorder |
| |
Authors: | Leslie Pedrioli Deena M Fu Dun Jack Gonzalez-Gonzalez Emilio Contag Christopher H Kaspar Roger L Smith Frances J D McLean W H Irwin |
| |
Institution: | 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 |
| |
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. |
| |
Keywords: | |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|