¹DMD Genetic Therapy Group, Center for Human and Clinical Genetics, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, The Netherlands

Correspondence: Judith C. T. van Deutekom, Fax: +31 71 5268285. E-mail: deutekom@lumc.nl

Received 21 November 2005; Revised 20 February 2006; Accepted 22 February 2006.

Abstract

Through antisense-induced single-, double-, and multiexon skipping, we have previously demonstrated restoration of dystrophin expression in Duchenne muscular dystrophy (DMD) patient-derived muscle cells in vitro. In this study we further explored the frontiers of this strategy by using specific combinations of 2'-O-methyl phosphorothioate antisense oligonucleotides (AONs) targeting either one or multiple exons. We show that skipping efficiencies may indeed be improved by targeting two putative splicing regulatory sequences within one exon. In particular, such double targeting was effective for the thus far "unskippable" exons 47 and 57. We previously reported the feasibility of multiexon skipping spanning exon 45 to exon 51, using a combination of AONs targeting both outer exons (45 and 51). This would be applicable to 13% of all DMD patients. We here explored the frontiers of multiexon skipping both to increase the number of patients that can be treated with the same set of AONs and to mimic large deletions found in relatively mildly affected BMD patients. We aimed at inducing larger multiexon-skipping stretches, such as exons 17–51, exons 42–55, and exons 45–59. However, this appeared complicated and may be dependent on cotranscriptional splicing and the size of the flanking introns.