38. Morpholino Antisense Oligonucleotide Induced Exon Skipping Efficiently Restores Dystrophin Expression in the mdx Mouse

Although antisense oligonucleotides (AO) have been used most commonly to inhibit gene expression, they can also be used to modify splicing of the primary transcript by targeting the exon splicing enhancers or the 3'/5' splice sites. Recent studies have shown that the morpholino chemistry is particularly useful as such AOs are not readily degraded. The mdx mouse is dystrophin deficient due to a premature stop codon in exon 23. Using an AO that targets exon 23, we have examined the longevity of exon skipping by RT-PCR and dystrophin transcript can still be detected 14 weeks after a single intramuscular injection of morpholino AO and dystrophin is clearly present at least 4 months post treatment. This is substantially longer than the expression seen in similar studies using a 2-O-methyl phosphorothioate AO. We have used similar techniques to examine the dose/volume relationship in preparation for clinical trials of this approach in Duchenne muscular dystrophy (DMD). As the diaphragm of the mdx mouse is the muscle that most closely resembles the pathology in DMD, exhibiting substantial fibrosis and fibre loss, we have used the diaphragm to test the effect of increased fibrosis in reducing AO uptake by the muscle fibres by treating mice at different ages. Delivery of AO in mice younger than 2 weeks can largely prevent the development of dystrophic pathology and treatment at later ages reduces the membrane fragility as determined by uptake of Evans Blue dye. These pre-clinical studies demonstrate the effectiveness of the morpholino based AO in inducing long-lived expression of dystrophin and this approach is now being taken forward to a UK clinical trial in DMD by the MDEX Consortium.