K14 mRNA reprogramming for dominant epidermolysis bullosa simplex

Hum Mol Genet. 2010 Dec 1;19(23):4715-25. doi: 10.1093/hmg/ddq405. Epub 2010 Sep 22.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Blotting, Western
  • Cell Line
  • Epidermolysis Bullosa Simplex / genetics*
  • Epidermolysis Bullosa Simplex / therapy*
  • Gene Expression
  • Gene Library
  • Genetic Therapy / methods
  • Humans
  • Keratin-14 / genetics*
  • Mutation / genetics
  • Phenotype
  • Polymerase Chain Reaction
  • RNA, Messenger / genetics*
  • RNA, Messenger / metabolism
  • Sequence Analysis, DNA
  • Spliceosomes / genetics
  • Trans-Splicing*

Substances

  • Keratin-14
  • RNA, Messenger