Abstract
SummaryBackgroundGeneralized 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.ObjectivesTo 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.MethodsA 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.ResultsTransplanted 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.ConclusionsOur 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.
Highlights
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 keratin 14 gene (KRT14) genes lead to impaired function of the intermediate filament cytoskeleton in the basal epidermis
The keratinocyte lines used in this study are the previously published EBS-gen sev cell line EBDM-1 (KRT14, c.373C>T, p.R125H) and the human wild-type keratinocyte lines hKc and NEB-1.24,28 Cells were cultured in a humidified incubator at 37 °C and 5% CO2 using EpiLife medium (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.) supplemented with Human Keratinocyte Growth Supplement (Cascade Biologics, Bromborough, U.K.) and 10% penicillin–streptomycin (Biochrom AG, Berlin, Germany)
EBDM-1 keratinocytes were used in order to confirm that this construct is functional in another EBS-gen sev cell line upon retroviral transduction (RV_RTM163_GFP)
Summary
To correct a mutation within exon 1 of the KRT14 gene, using a 50-transsplicing approach, where any mutation within the first seven exons could be replaced by a single therapeutic molecule. Methods A therapeutic RNA trans-splicing molecule containing wild-type exons 1–7 was stably transduced into an EBS patient-derived keratinocyte line. Transsplicing 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-splicinginduced green fluorescent protein expression and blistering
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