1. ¹TransDerm Inc., Santa Cruz, California, USA
2. ²Human Genetics Unit, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK
3. ³Molecular Imaging Program at Stanford (MIPS), and Departments of Radiology, Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, USA
4. ⁴Departments of Pediatrics and Pathology, Stanford University School of Medicine, Stanford, California, USA
5. ⁵Thermo Fisher Scientific, Dharmacon Products, Lafayette, Colorado, USA
6. ⁶Department of Dermatology, University of Utah, Salt Lake City, Utah, USA
7. ⁷Department of Dermatology, Yale University, New Haven, Connecticut, USA

Correspondence: Roger L. Kaspar, 2161 Delaware Avenue, Suite D, Santa Cruz, California 95060, USA. E-mail: roger.kaspar@tansderminc.com

Received 29 May 2007; Revised 27 June 2007; Accepted 1 July 2007; Published online 11 October 2007.

Abstract

RNA interference offers a novel approach for developing therapeutics for dominant-negative genetic disorders. The ability to inhibit expression of the mutant allele without affecting wild-type gene expression could be a powerful new treatment option. Targeting the single-nucleotide keratin 6a (K6a) N171K mutation responsible for the rare monogenic skin disorder pachyonychia congenita (PC), we demonstrate that small interfering RNAs (siRNAs) can potently and selectively block expression of mutant K6a. To test whether lead siRNAs could discriminate mutant mRNA in the presence of both wild-type and mutant forms, a dominant-negative PC cell culture model was developed. As predicted for a dominant-negative disease, simultaneous expression of both wild-type and mutant K6a resulted in defective keratin filament formation. Addition of mutant-specific siRNAs allowed normal filament formation, suggesting selective inhibition of mutant K6a. The effectiveness of our siRNA in skin was tested by co-delivering a firefly luciferase/mutant K6a bicistronic reporter construct and mutant-specific siRNAs to mouse footpads. Potent inhibition of the fluorescent reporter was demonstrated using the Xenogen IVIS200 in vivo imaging system. Additionally, wild type-specific siRNAs knocked down the expression of pre-existing endogenous K6a in human keratinocytes. These results suggest that efficient delivery of these "designer siRNAs" may allow effective treatment of numerous genetic disorders including PC.