K14 mRNA reprogramming for dominant epidermolysis bullosa simplex.

[epidermolysis bullosa simplex]

The major challenge to a successful gene therapy of autosomal dominant genetic diseases is a highly efficient and specific knock-down or repair of the disease-causing allele . In epidermolysis bullosa simplex -type Dowling-Meara (EBS -DM ), a single amino acid exchange in exon 1 of the keratin 14 gene (K 14 ) triggers a severe skin phenotype , characterized by blistering of the skin and mucous membranes after minor trauma . We chose spliceosome-mediated RNA trans-splicing to specifically replace exons 1 -7 of the K 14 gene . In this approach , the mutated coding region is replaced by an RNA-trans-splicing molecule (RTM ) that incorporates a binding domain (BD ) and the wild-type sequence of K 14 . Since the BD is crucial for the trans-splicing functionality , we developed a fluorescence-based RTM screen consisting of an RTM library containing random BDs . Co -transfection of the library with a target molecule enabled us to identify highly functional RTMs . The best RTMs were adapted for endogenous trans-splicing in an EBS -DM patient cell line . In this cell line , we were able to detect functional , efficient and correct trans-splicing on RNA and protein levels . Scratch assays confirmed phenotypic reversion in vitro . Owing to concomitant knock-down and repair of the mutated allele , we assume that trans-splicing is a promising tool for the treatment of autosomal dominant genetic disease .