Restoration of dystrophin expression in mdx muscle cells by chimeraplast-mediated exon skipping.

The most common types of dystrophin gene mutations that cause Duchenne muscular dystrophy (DMD) are large deletions that result in a shift of the translational reading frame. Such mutations generally lead to a complete absence of dystrophin protein in the muscle cells of affected individuals. Any therapeutic modality that could restore the reading frame would have the potential to substantially reduce the severity of the disease by allowing the production of an internally deleted, but partially functional, dystrophin protein as occurs in Becker muscular dystrophy (BMD). One approach to restoring the reading frame would be to alter the splicing of the pre-mRNA to produce an in-frame transcript. We have tested the ability of chimeric RNA/DNA oligonucleotides (chimeraplasts) to alter key bases in specific splice sequences in the dystrophin gene to induce exon skipping. Using cells from the mdx mouse as a model system, we show that chimeraplast-mediated base conversion in the intron 22/exon 23 splice junction induces alternative splicing and the production of in-frame transcripts. Interestingly, multiple alternative transcripts were induced by this targeted splice site mutation. Direct sequencing indicated that several of these were predicted to produce in-frame dystrophin transcripts with internal deletions. Indeed, multiple forms of dystrophin protein were observed by western blot analysis, and the functionality of the products was demonstrated by the restoration of expression and localization of a dystrophin-associated protein, alpha-dystroglycan, in differentiated cells. These data demonstrate that chimeraplasts can induce exon skipping by altering splice site sequences at the genomic level. As such, chimeraplast-mediated exon skipping has the potential to be used to transform a severe DMD phenotype into a much milder BMD phenotype.