An ex vivo RNA trans‐splicing strategy to correct human generalized severe epidermolysis bullosa simplex. (7th October 2018)

• Record Type: Journal Article
• Authors: Peking, P.; Breitenbach, J.S.; Ablinger, M.; Muss, W.H.; Poetschke, F.J.; Kocher, T.; Koller, U.; Hainzl, S.; Kitzmueller, S.; Bauer, J.W.; Reichelt, J.; Lettner, T.; Wally, V.
• Is Part Of: British journal of dermatology. Volume 180:Number 1(2019)
• Page Start: 141
• Page End: 148
• Publication Date: 2018-10-07
• Subjects: Dermatology -- Periodicals; Skin -- Diseases -- Periodicals; 616.5
• DOI: 10.1111/bjd.17075
• Languages: English
• ISSNs: 0007-0963

Abstract

Summary: Background: 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. Objectives: 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. Methods: 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. Results: 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. Conclusions: 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. Abstract : What's already known about this topic? RTM163, described in this study, was previously used in a transient in vitro transfection system, where the ability to correct KRT14 at the mRNA level was demonstrated. What does this study add? In this study, we stably transduced RTM163 in a second patient‐derived keratinocyte line. Successful trans ‐splicing was confirmed in this cell line. The expression of disease‐related marker genes, which are characteristically deregulated in epidermolysis bullosa simplex, were analysed. For the first time this study showed that RNA trans ‐splicing molecule‐transduced patient keratinocytes can differentiate into a phenotypically normal and blister‐free epidermis in a xenograft mouse model. What is the translational message? This study shows the feasibility of using spliceosome‐mediated RNA trans ‐splicing to generate a stable and blister‐free epidermis in vivo . Combined with pre‐existing ex vivo gene therapeutic methods, this might be a valid option for future treatments of dominantly inherited genodermatoses. Linked Comment: Bremer and van den Akker. Br J Dermatol 2019;180 :17–19 . Respond to this article