siRNA-mediated allele-specific inhibition of mutant KRT5 and KRT14 transcripts carrying frequent dominant mutations in epidermolysis bullosa simplex (EBS)
Lay summary
Epidermolysis bullosa simplex (EBS) is characterized by intra-epidermal blistering upon mild trauma. Most EBS cases are caused by dominant missense mutations in KRT5 or KRT14 encoding keratin 5 and keratin 14. Keratin 5 and keratin 14 constitute the intermediate filament network of the basal keratinocytes of the skin and form obligate heteropolymers. Like a multi-stranded rope, when one strand is weak the resistance of the entire rope is compromised, dominant mutations within either of these keratin genes impair the formation and function of the keratin filament network.
This project aims at developing a new therapeutic approach for EBS using RNA interference to knock down mutant alleles of KRT5 and KRT14 carrying recurrent mutations underlying severe forms of EBS. RNA interference allows to selectively destroy the mutant messenger RNA of the KRT5 or KRT14 genes, thus preventing the synthesis of the dominant keratin protein (the weak strands), while maintaining the synthesis of the normal keratin protein (the normal strands). That way, only strong strands will be used by the cell to form the keratin filament network.
Scientific Summary
This project aims to target frequent KRT5 and KRT14 dominant mutations in EBS by silencing RNA (siRNA) technology. Several efficient siRNAs have been selected using an in vitro screening employing an allele-specific luciferase reporter system. The most efficient molecules will be used on EBS patient cells in culture. Efficiency of allele-specific knockdown will be quantified by digital droplet PCR while functional correction will be demonstrated by the reduction in keratin aggregates in treated cells. Effectiveness of subcutaneous or systemic injections or topical applications will be compared on EBS skin equivalents transplanted in immune deficient mice.