Journal Article

K14 mRNA reprogramming for dominant epidermolysis bullosa simplex

Verena Wally, Marietta Brunner, Thomas Lettner, Martin Wagner, Ulrich Koller, Andrea Trost, Eva M. Murauer, Stefan Hainzl, Helmut Hintner and Johann W. Bauer

in Human Molecular Genetics

Volume 19, issue 23, pages 4715-4725
Published in print December 2010 | ISSN: 0964-6906
Published online September 2010 | e-ISSN: 1460-2083 | DOI: http://dx.doi.org/10.1093/hmg/ddq405
K14 mRNA reprogramming for dominant epidermolysis bullosa simplex

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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 (K14) 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 K14 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 K14. 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.

Journal Article.  6100 words.  Illustrated.

Subjects: Genetics and Genomics

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