title: Doxycycline-controlled splicing modulation by regulated antisense U7 snRNA expression cassettes.
abstract: Many diseases affect pre-mRNA splicing, and alternative splicing is a major source of proteome diversity and an important mechanism for modulating gene expression. The ability to regulate a specific splicing event is therefore desirable; for example, to understand splicing-associated pathologies. We have developed methods based on modified U7 snRNAs, which allow us to induce efficient skipping or inclusion of selected exons. Here, we have adapted these U7 tools to a regulatable system that relies on a doxycycline-sensitive version of the Krüppel-associated box (KRAB)/KAP1 transcriptional silencing. Co-transduction of target cells with two lentiviral vectors, one carrying the KRAB protein and the other the regulatable U7 cassette, allows a tight regulation of the modified U7 snRNA. No leakage is observed in the repressed state, whereas full induction can be obtained with doxycycline in ng ml(-1) concentrations. Only a few days are necessary for a full switch, and the induction/repression can be repeated over several cycles without noticeable loss of control. Importantly, the U7 expression correlates with splicing correction, as shown for the skipping of an aberrant beta-globin exon created by a thalassaemic mutation and the promotion of exon 7 inclusion in the human SMN2 gene, an important therapeutic target for spinal muscular atrophy.
authors.affiliation: Institute of Cell Biology, University of Bern, Bern, Switzerland.
authors.forename: J
authors.initials: J
authors.lastname: Marquis
authors.forename: S S
authors.initials: SS
authors.lastname: Kämpfer
authors.forename: L
authors.initials: L
authors.lastname: Angehrn
authors.forename: D
authors.initials: D
authors.lastname: Schümperli
mesh_terms: D017398:Alternative Splicing; D004318:Doxycycline / Q000494:pharmacology*; D005091:Exons; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects*; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D009134:Muscular Atrophy, Spinal / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000235:genetics*; D055540:Survival of Motor Neuron 2 Protein / Q000235:genetics; D055544:beta-Globins / Q000235:genetics; D017086:beta-Thalassemia / Q000235:genetics
chemical_list: D016376:Oligonucleotides, Antisense; D012342:RNA, Small Nuclear; C526477:SMN2 protein, human; D055540:Survival of Motor Neuron 2 Protein; C405487:U7 small nuclear RNA; D055544:beta-Globins; D004318:Doxycycline