title: An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects.
abstract: A significant proportion of disease-causing mutations affect precursor-mRNA splicing, inducing skipping of the exon from the mature transcript. Using F9 exon 5, CFTR exon 12 and SMN2 exon 7 models, we characterized natural mutations associated to exon skipping in Haemophilia B, cystic fibrosis and spinal muscular atrophy (SMA), respectively, and the therapeutic splicing rescue by using U1 small nuclear RNA (snRNA). In minigene expression systems, loading of U1 snRNA by complementarity to the normal or mutated donor splice sites (5'ss) corrected the exon skipping caused by mutations at the polypyrimidine tract of the acceptor splice site, at the consensus 5'ss or at exonic regulatory elements. To improve specificity and reduce potential off-target effects, we developed U1 snRNA variants targeting non-conserved intronic sequences downstream of the 5'ss. For each gene system, we identified an exon-specific U1 snRNA (ExSpeU1) able to rescue splicing impaired by the different types of mutations. Through splicing-competent cDNA constructs, we demonstrated that the ExSpeU1-mediated splicing correction of several F9 mutations results in complete restoration of secreted functional factor IX levels. Furthermore, two ExSpeU1s for SMA improved SMN exon 7 splicing in the chromosomal context of normal cells. We propose ExSpeU1s as a novel therapeutic strategy to correct, in several human disorders, different types of splicing mutations associated with defective exon definition.
authors.affiliation: Human Molecular Genetics, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
authors.forename: Eugenio
authors.initials: E
authors.lastname: Fernandez Alanis
authors.forename: Mirko
authors.initials: M
authors.lastname: Pinotti
authors.forename: Andrea
authors.initials: A
authors.lastname: Dal Mas
authors.forename: Dario
authors.initials: D
authors.lastname: Balestra
authors.forename: Nicola
authors.initials: N
authors.lastname: Cavallari
authors.forename: Malgorzata E
authors.initials: ME
authors.lastname: Rogalska
authors.forename: Francesco
authors.initials: F
authors.lastname: Bernardi
authors.forename: Franco
authors.initials: F
authors.lastname: Pagani
mesh_terms: D017398:Alternative Splicing; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism; D005091:Exons; D002836:Hemophilia B / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007438:Introns; D009134:Muscular Atrophy, Spinal / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D022821:RNA Splice Sites; D012326:RNA Splicing / Q000502:physiology; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D012322:RNA Precursors; D022821:RNA Splice Sites; D012342:RNA, Small Nuclear; C405483:U1 small nuclear RNA; D019005:Cystic Fibrosis Transmembrane Conductance Regulator