title: Correction of a Cystic Fibrosis Splicing Mutation by Antisense Oligonucleotides.
abstract: Cystic fibrosis (CF), the most common life-threatening genetic disease in Caucasians, is caused by ∼2,000 different mutations in the CF transmembrane conductance regulator (CFTR) gene. A significant fraction of these (∼13%) affect pre-mRNA splicing for which novel therapies have been somewhat neglected. We have previously described the effect of the CFTR splicing mutation c.2657+5G>A in IVS16, showing that it originates transcripts lacking exon 16 as well as wild-type transcripts. Here, we tested an RNA-based antisense oligonucleotide (AON) strategy to correct the aberrant splicing caused by this mutation. Two AONs (AON1/2) complementary to the pre-mRNA IVS16 mutant region were designed and their effect on splicing was assessed at the RNA and protein levels, on intracellular protein localization and function. To this end, we used the 2657+5G>A mutant CFTR minigene stably expressed in HEK293 Flp-In cells that express a single copy of the transgene. RNA data from AON1-treated mutant cells show that exon 16 inclusion was almost completely restored (to 95%), also resulting in increased levels of correctly localized CFTR protein at the plasma membrane (PM) and with increased function. A novel two-color CFTR splicing reporter minigene developed here allowed the quantitative monitoring of splicing by automated microscopy localization of CFTR at the PM. The AON strategy is thus a promising therapeutic approach for the specific correction of alternative splicing.
authors.affiliation: University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Lisboa, Portugal.
authors.forename: Susana
authors.initials: S
authors.lastname: Igreja
authors.affiliation: University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Lisboa, Portugal.
authors.forename: Luka A
authors.initials: LA
authors.lastname: Clarke
authors.affiliation: University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Lisboa, Portugal.
authors.forename: Hugo M
authors.initials: HM
authors.lastname: Botelho
authors.affiliation: University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Lisboa, Portugal.
authors.forename: Luís
authors.initials: L
authors.lastname: Marques
authors.affiliation: University of Lisboa, Faculty of Sciences, BioISI - Biosystems & Integrative Sciences Institute, Lisboa, Portugal.
authors.forename: Margarida D
authors.initials: MD
authors.lastname: Amaral
mesh_terms: D001483:Base Sequence; D003550:Cystic Fibrosis / Q000175:diagnosis* / Q000235:genetics* / Q000473:pathology / Q000628:therapy*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D005091:Exons; D015316:Genetic Therapy; D057809:HEK293 Cells; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D009154:Mutation; D016376:Oligonucleotides, Antisense / Q000235:genetics* / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing; D017422:Sequence Analysis, DNA
chemical_list: C505032:CFTR protein, human; D016376:Oligonucleotides, Antisense; D012322:RNA Precursors; D019005:Cystic Fibrosis Transmembrane Conductance Regulator
keywords: CFTR; antisense oligonucleotide; cystic fibrosis; exon skipping; splicing mutation