An ex vivo RNA trans-splicing strategy to correct human generalized severe epidermolysis bullosa simplex

Generalized severe epidermolysis bullosa simplex (EBS-gen sev) is a genetic blistering skin disease in which autosomal dominant mutations in either the keratin KRT5 or KRT14 genes lead to impaired function of the intermediate filament cytoskeleton in the basal epidermis. Here we present an ex vivo RNA trans-splicing-based therapeutic approach to correct the phenotype.

To correct a mutation within exon 1 of the KRT14 gene, using a 5'-trans-splicing approach, where any mutation within the first seven exons could be replaced by a single therapeutic molecule.

A therapeutic RNA trans-splicing molecule containing wild-type exons 1-7 was stably transduced into an EBS patient-derived keratinocyte line. Trans-splicing was confirmed via reverse-transcriptase polymerase chain reaction, Western blotting and immunofluorescence microscopy. Skin equivalents generated from corrected keratinocytes were grafted onto nude mice and analysed about 8 weeks post-transplantation for regular epidermal stratification, trans-splicing-induced green fluorescent protein expression and blistering.

Transplanted skin equivalents generated from trans-splicing-corrected patient keratinocytes showed a stable and blister-free epidermis. KRT14 correction disrupted EBS-gen sev-associated proinflammatory signalling, as shown at the mRNA and protein levels. Disruption of the pathogenic feedback loop in addition to overall downregulation of KRT14 expression highlighted the effect of KRT14 correction on the EBS pathomechanism.

Our data demonstrate that trans-splicing-mediated mRNA therapy is an effective method for the correction of dominantly inherited KRT14 mutations at the transcriptional level. This results in the rescue of the EBS-gen sev phenotype and stabilization of the epidermis in a xenograft mouse model.