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Rescue of common exon skipping mutations in Cystic Fibrosis with modified U1 snRNAs
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  • Stefano Donega',
  • Malgorzata Rogalska,
  • Giulia Pianigiani,
  • Susana Igreja,
  • Margarida Amaral,
  • Franco Pagani
Stefano Donega'
International Centre for Genetic Engineering and Biotechnology
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Malgorzata Rogalska
International Centre for Genetic Engineering and Biotechnology
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Giulia Pianigiani
International Centre for Genetic Engineering and Biotechnology
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Susana Igreja
University of Lisbon Faculty of Sciences
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Margarida Amaral
University of Lisbon Faculty of Sciences
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Franco Pagani
International Centre for Genetic Engineering and Biotechnology
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Abstract

In Cystic Fibrosis (CF), correction of splicing defects represents an interesting therapeutic approach to restore normal CFTR function. In this study, we focused on ten common mutations/variants, 711+3A>G/C, 711+5G>A, 1863C>T, 1898+3A>G, 2789+5G>A, TG13T3, TG13T5, TG12T5 and 3120G>A that induce skipping of the corresponding CFTR exons 5, 9, 13, 16 and 18. To rescue the splicing defects we tested, in a minigene assay, a panel of modified U1 snRNAs, named Exon Specific U1s (ExSpeU1) that were engineered to bind to intronic sequences downstream of each defective exon. Using this approach, we show that all ten splicing mutations analysed are efficiently corrected by specific ExSpeU1s. Using cDNA-splicing competent minigenes, we also show that the ExspeU1-mediated splicing correction at the RNA level recovered the full-length CFTR protein for 1863C>T, 1898+3A>G, 2789+5G>A variants. In addition, detailed mutagenesis experiments performed on exon 13 led us to identify a novel intronic regulatory element involved in the ExSpeU1-mediated splicing rescue. These results provide a common strategy based on modified U1 snRNAs to correct exon skipping in a group of disease-causing CFTR mutations.

Peer review status:ACCEPTED

10 Jul 2020Submitted to Human Mutation
11 Jul 2020Submission Checks Completed
11 Jul 2020Assigned to Editor
14 Jul 2020Reviewer(s) Assigned
29 Jul 2020Review(s) Completed, Editorial Evaluation Pending
08 Aug 2020Editorial Decision: Revise Minor
28 Aug 20201st Revision Received
01 Sep 2020Submission Checks Completed
01 Sep 2020Assigned to Editor
01 Sep 2020Reviewer(s) Assigned
01 Sep 2020Review(s) Completed, Editorial Evaluation Pending
07 Sep 2020Editorial Decision: Accept