Induced non-productive splicing to study muscle gene expression

RNA silencing has been applied to suppress gene expression, with varying degrees of specificity and efficiency reported. Endogenous alternative splicing can regulate gene expression through a process called Regulated Unproductive Splicing and Translation (RUST), by either incorporating an exon carrying a nonsense mutation, or excising an exon to disrupt the reading frame. As a result, the mature gene transcripts cannot be translated into functional products. We show that it is possible to efficiently disrupt the normal dystrophin mRNA reading frame and ablate dystrophin expression. Total suppression of dystrophin gene expression can be induced and maintained for several weeks in vivo, and a severe dystrophic pathology observed within 4 weeks of commencing treatment in neonatal normal mice. This approach to gene down-regulation is very efficient and specific. Disruption of gene expression by selected exon exclusion could be applied to many different genes, and offers the opportunity to induce transient mouse models to study the consequences of gene suppression in vivo. In addition, selected exon removal to yield in-frame transcripts can allow mapping of functional protein domains, based upon exon boundaries, and provide a possible alternative to transgenic mouse models for the study of muscle gene expression. We are applying this approach to defining dystrophin functional domains, to optimise exon-skipping strategies for the treatment of Duchenne Muscular Dystrophy