Correction of Hair Shaft Defects through Allele-Specific Silencing of Mutant Krt75

Dominant mutations in keratingenes can cause a number of inheritable skin disorders characterized by intraepidermal blistering, epidermal hyperkeratosis, or abnormalities in skin appendages, such as nail platedystrophy and structural defects in hair. Allele-specific silencing of mutant keratinsthrough RNA interferenceis a promising therapeutic approach for suppressing the expression of mutant keratinsand related phenotypesin the epidermis. However, its effectiveness on skin appendagesremains to be confirmed in vivo. In this study, we developed allele-specific small interfering RNAscapable of selectively suppressing the expression of a mutantKrt75 , which causes hair shaft structural defects characterized by the development of blebs along the hair shaft in mice. Hair regenerated from epidermal keratinocyte progenitor cells isolated from mutantKrt75 mouse models reproduced the blebbing phenotypewhen grafted in vivo. In contrast, mutantcells manipulated with a lentiviral vector expressing mutantKrt75 -specific short hairpin RNA (shRNA) persistently suppressed this phenotype. The phenotypiccorrection was associated with a significant reduction of mutantKrt75 mRNA in the skin grafts. Thus, data obtained from this study demonstrated the feasibility of utilizing RNA interferenceto achieve durable correction of hair structural phenotypesthrough allele-specific silencing of mutant keratingenes.