K14 mRNA reprogramming for dominant 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 (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.
Wally
Verena
V
Division of Molecular Dermatology and EB-house Austria, Department of Dermatology, Paracelsus Medical University, Salzburg, Austria.
Brunner
Marietta
M
Lettner
Thomas
T
Wagner
Martin
M
Koller
Ulrich
U
Trost
Andrea
A
Murauer
Eva M
EM
Hainzl
Stefan
S
Hintner
Helmut
H
Bauer
Johann W
JW
D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D016110:Epidermolysis Bullosa Simplex / Q000235:genetics* / Q000628:therapy*; D015870:Gene Expression; D015723:Gene Library; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D053547:Keratin-14 / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017381:Spliceosomes / Q000235:genetics; D020040:Trans-Splicing
D053547:Keratin-14; D012333:RNA, Messenger