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Doxycycline-controlled splicing modulation by regulated antisense U7 snRNA expression cassettes

Gene Therapy volume 16, pages 70–77 (2009)Cite this article

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 β-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.

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Acknowledgements

We thank Karin Schranz for technical help, Didier Trono and Isabelle Barde from EPFL Lausanne for the gift of lentiviral vectors and for performing copy number analyses by real-time PCR, respectively. This study was supported by AFM (Association Française contre les Myopathies), EURASNET (European Network of Excellence on Alternative Splicing) and the Kanton Bern.

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Author notes

  1. J Marquis

    Present address: 3Current address: Laboratory of Virology and Genetics, School of Life Sciences, EPFL Lausanne, Switzerland.,

  2. S S Kämpfer

    Present address: 4Current address: Institute of Immunology, University Hospital Bern, Switzerland.,

  3. L Angehrn

    Present address: 5Current address: Swiss Institute for Experimental Cancer Research (ISREC), Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.,

  4. J Marquis and S S Kämpfer: These authors contributed equally to this work and should be considered joint first authors.

Authors and Affiliations

  1. Institute of Cell Biology, University of Bern, Bern, Switzerland

    J Marquis, S S Kämpfer, L Angehrn & D Schümperli

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  1. J Marquis
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  4. D Schümperli
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Correspondence to D Schümperli.

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Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)

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Marquis, J., Kämpfer, S., Angehrn, L. et al. Doxycycline-controlled splicing modulation by regulated antisense U7 snRNA expression cassettes. Gene Ther 16, 70–77 (2009). https://doi.org/10.1038/gt.2008.138

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