Designing Efficient Double RNA trans-Splicing Molecules for Targeted RNA Repair
AbstractRNA 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
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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.
Hüttner C, Murauer EM, Hainzl S, Kocher T, Neumayer A, Reichelt J, Bauer JW, Koller U. Designing Efficient Double RNA trans-Splicing Molecules for Targeted RNA Repair. International Journal of Molecular Sciences. 2016; 17(10):1609.Chicago/Turabian Style
Hüttner, Clemens; Murauer, Eva M.; Hainzl, Stefan; Kocher, Thomas; Neumayer, Anna; Reichelt, Julia; Bauer, Johann W.; Koller, Ulrich. 2016. "Designing Efficient Double RNA trans-Splicing Molecules for Targeted RNA Repair." Int. J. Mol. Sci. 17, no. 10: 1609.
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