Development of allele-specific therapeutic siRNA for keratin 5 mutations in epidermolysis bullosa simplex.

[epidermolysis bullosa simplex]

Epidermolysis bullosa simplex (EBS ) is an incurable , inherited skin -blistering disorder predominantly caused by dominant-negative mutations in the genes encoding keratins K 5 or K 14 . 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 K 5 gene (p .Ser 181 Pro and p .Asn 193 Lys ). 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 K 5 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 .