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Rescue of common exon‐skipping mutations in cystic fibrosis with modified U1 snRNAs
Author(s) -
Donegà Stefano,
Rogalska Malgorzata Ewa,
Pianigiani Giulia,
Igreja Susana,
Amaral Margarida Duarte,
Pagani Franco
Publication year - 2020
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.24116
Subject(s) - minigene , rna splicing , exon , exon skipping , biology , alternative splicing , genetics , mutation , exonic splicing enhancer , mutagenesis , rna , microbiology and biotechnology , gene
In cystic fibrosis (CF), the correction of splicing defects represents an interesting therapeutic approach to restore normal CFTR function. In this study, we focused on 10 common mutations/variants 711+3A>G/C, 711+5G>A, TG13T3, TG13T5, TG12T5, 1863C>T, 1898+3A>G, 2789+5G>A, and 3120G>A that induce skipping of the corresponding CFTR exons 5, 10, 13, 16, and 18. To rescue the splicing defects we tested, in a minigene assay, a panel of modified U1 small nuclear RNAs (snRNAs), named Exon Specific U1s (ExSpeU1s), that was engineered to bind to intronic sequences downstream of each defective exon. Using this approach, we show that all 10 splicing mutations analyzed are efficiently corrected by specific ExSpeU1s. Using complementary DNA‐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.