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K14 mRNA reprogramming for dominant epidermolysis bullosa simplex

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 DowlingMeara (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 17 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Human Molecular Genetics Oxford University Press

K14 mRNA reprogramming for dominant epidermolysis bullosa simplex

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References (40)

Publisher
Oxford University Press
Copyright
The Author 2010. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissionsoxfordjournals.org
Subject
ARTICLES
ISSN
0964-6906
eISSN
1460-2083
DOI
10.1093/hmg/ddq405
pmid
20861136
Publisher site
See Article on Publisher Site

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 DowlingMeara (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 17 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

Human Molecular Genetics – Oxford University Press

Published: Dec 1, 2010

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