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Int. J. Mol. Sci. 2016, 17(10), 1609; doi:10.3390/ijms17101609

Designing Efficient Double RNA trans-Splicing Molecules for Targeted RNA Repair

1
EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, 5020 Salzburg, Austria
2
Department of Dermatology, University Hospital of the Paracelsus Medical University Salzburg, 5020 Salzburg, Austria
*
Authors to whom correspondence should be addressed.
Academic Editor: Constantinos Stathopoulos
Received: 14 June 2016 / Revised: 24 August 2016 / Accepted: 14 September 2016 / Published: 22 September 2016
(This article belongs to the Special Issue Pre-mRNA Splicing 2016)
View Full-Text   |   Download PDF [3831 KB, uploaded 22 September 2016]   |  

Abstract

RNA trans-splicing is a promising tool for mRNA modification in a diversity of genetic disorders. In particular, the substitution of internal exons of a gene by combining 3′ and 5′ RNA trans-splicing seems to be an elegant way to modify especially large pre-mRNAs. Here we discuss a robust method for designing double RNA trans-splicing molecules (dRTM). We demonstrate how the technique can be implemented in an endogenous setting, using COL7A1, the gene encoding type VII collagen, as a target. An RTM screening system was developed with the aim of testing the replacement of two internal COL7A1 exons, harbouring a homozygous mutation, with the wild-type version. The most efficient RTMs from a pool of randomly generated variants were selected via our fluorescence-based screening system and adapted for use in an in vitro disease model system. Transduction of type VII collagen-deficient keratinocytes with the selected dRTM led to accurate replacement of two internal COL7A1 exons resulting in a restored wild-type RNA sequence. This is the first study demonstrating specific exon replacement by double RNA trans-splicing within an endogenous transcript in cultured cells, corroborating the utility of this technology for mRNA repair in a variety of genetic disorders. View Full-Text
Keywords: double RNA trans-splicing; genetic diseases; RNA therapy; type VII collagen; epidermolysis bullosa double RNA trans-splicing; genetic diseases; RNA therapy; type VII collagen; epidermolysis bullosa
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Hüttner, C.; Murauer, E.M.; Hainzl, S.; Kocher, T.; Neumayer, A.; Reichelt, J.; Bauer, J.W.; Koller, U. Designing Efficient Double RNA trans-Splicing Molecules for Targeted RNA Repair. Int. J. Mol. Sci. 2016, 17, 1609.

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