Research Article

Cellular and Molecular Life Sciences CMLS

, Volume 60, Issue 3, pp 557-566

First online:

U7 snRNAs induce correction of mutated dystrophin pre-mRNA by exon skipping

  • C. BrunAffiliated withDivision of Neuropathology, Institute of Pathology, University of Bern, Murtenstrasse 31, 3010 Bern (Switzerland), Fax +41 31 632 9872, e-mail: joachim.weis@pathology.unibe.ch
  • , D. SuterAffiliated withDivision of Neuropathology, Institute of Pathology, University of Bern, Murtenstrasse 31, 3010 Bern (Switzerland), Fax +41 31 632 9872, e-mail: joachim.weis@pathology.unibe.ch
  • , C. PauliAffiliated withDivision of Neuropathology, Institute of Pathology, University of Bern, Murtenstrasse 31, 3010 Bern (Switzerland), Fax +41 31 632 9872, e-mail: joachim.weis@pathology.unibe.ch
  • , P. DunantAffiliated withGene Center and Friedrich-Baur-Institute, Ludwig-Maximilians-University, Munich (Germany)
  • , H. LochmüllerAffiliated withGene Center and Friedrich-Baur-Institute, Ludwig-Maximilians-University, Munich (Germany)
  • , J.-M. BurgunderAffiliated withDepartment of Neurology, Inselspital University Hospital, Bern (Switzerland)
  • , D. SchümperliAffiliated withInstitute of Cell Biology, University of Bern, Bern (Switzerland)
  • , J. WeisAffiliated withDivision of Neuropathology, Institute of Pathology, University of Bern, Murtenstrasse 31, 3010 Bern (Switzerland), Fax +41 31 632 9872, e-mail: joachim.weis@pathology.unibe.ch

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Abstract.

Most cases of Duchenne muscular dystrophy are caused by dystrophin gene mutations that disrupt the mRNA reading frame. Artificial exclusion (skipping) of a single exon would often restore the reading frame, giving rise to a shorter, but still functional dystrophin protein. Here, we analyzed the ability of antisense U7 small nuclear (sn)RNA derivatives to alter dystrophin pre-mRNA splicing. As a proof of principle, we first targeted the splice sites flanking exon 23 of dystrophin pre-mRNA in the wild-type muscle cell line C2C12 and showed precise exon 23 skipping. The same strategy was then successfully adapted to dystrophic immortalized mdx muscle cells where exon-23-skipped dystrophin mRNA rescued dystrophin protein synthesis. Moreover, we observed a stimulation of antisense U7 snRNA expression by the murine muscle creatine kinase enhancer. These results demonstrate that alteration of dystrophin pre-mRNA splicing could correct dystrophin gene mutations by expression of specific U7 snRNA constructs.

Key words. Duchenne muscular dystrophy; dystrophin; exon skipping; gene therapy; pre-mRNA; U7 snRNA.