Rescue of common exon skipping mutations in Cystic Fibrosis with
modified U1 snRNAs
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.