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Successful use of Out-of-Frame Exon 2 Skipping induces IRES-Driven expression of the N-Truncated dystrophin isoform: promising approach for treating other 5 ' Dystrophin Mutations

Wein, N., Vulin, A., Falzanaro, M.S., Szigyarto, C.A.K., Maiti, B., Findlay, A., Heller, K.N., Uhlen, M., Bakthavachalu, B., Messina, S., Vita, G.L., Gualandi, F., Wilton, S.D., Yang, L., Dunn, D.M., Schoenberg, D., Weiss, R.B., Howard, M.T., Ferlini, A. and Flanigan, K.M. (2014) Successful use of Out-of-Frame Exon 2 Skipping induces IRES-Driven expression of the N-Truncated dystrophin isoform: promising approach for treating other 5 ' Dystrophin Mutations. Molecular Therapy, 22 (Supp. 1). S294-S295.

Link to Published Version: http://dx.doi.org/10.1038/mt.2014.64
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Abstract

Most mutations that truncate the reading frame of the DMD gene result in loss of dystrophin expression and lead to the most common childhood muscle disease, the severe and progressive Duchenne muscular dystrophy. However, frame-truncating mutations within the first five exons of the DMD gene typically do not result in Duchenne muscular dystrophy, but instead result in milder dystrophinopathy syndromes as originally observed in patients with very mild clinical features despite nonsense mutations in exon 1. We have previously shown that amelioration of disease severity result from the expression of a highly functional N-truncated dystrophin beginning in exon 6 of the DMD. Here we demonstrate that this protein represents a novel dystrophin isoform resulting from usage of an internal ribosome entry site (IRES) within exon 5 that is glucocorticoid-inducible. In vitro studies with bicistronic reporter assays demonstrate translation at levels approximately 60% of the well-known viral IRES (eMCV), suggesting a relatively strong activity. Activity in humans was confirmed in patient muscle tissues using ribosome profiling and mass-spectrometric peptide sequencing. The resultant N-truncated dystrophin protein produced from this IRES, lacks the first calponin homology domain of the canonical actin binding domain 1. Nevertheless, it is highly functional, raising the possibility of the therapeutic use of this isoform. We use a novel out-of-frame exon-skipping approach to generate a truncated reading frame upstream of the IRES in both patient-derived cell lines and in a new DMD mouse model, leading to synthesis of a functional N-truncated isoform. In the mouse, this expression protects muscle from contraction-induced injury and corrects muscle force to the same level as control mice. Together these results support a novel therapeutic approach for patients with mutations within the 5' exons of DMD.

Publication Type: Journal Article
Murdoch Affiliation: Centre for Comparative Genomics
School of Engineering and Information Technology
Publisher: Nature Publishing Group
Notes: Abstract Only: ASGCT 17th Annual Meeting, Washington DC, May 21 - 24 2014
URI: http://researchrepository.murdoch.edu.au/id/eprint/23111
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