Morpholino antisense oligonucleotide induced dystrophin exon 23 skipping in mdx mouse muscle

Themdxmouse model of muscular dystrophy arose due to a nonsense mutation in exon 23 of the dystrophin gene. We have previously demonstrated that 20-O-methyl phosphorothioate antisense oligonucleotides (AOs) can induce removal of exon 23 during processing of the primary transcript. This results in an in-frame mRNA transcript and subsequent expression of a slightly shorter dystrophin protein in mdx muscle. Refinement of AO design has allowed efficient exon skipping to be induced in mdx mouse muscle cultures at nanomolar concentrations. In contrast, splicing intervention by morpholino AOs has been applied to the b-globin gene pre-mRNA in cultured cells to correct aberrant splicing when delivered in the micromolar range. The morpholino chemistry produces a neutral molecule that has exceptional biological stability but poor cellular delivery. We present data showing that exon skipping in mdx cells may be induced by morpholino AOs at nanomolar concentrations when annealed to a sense oligonucleotide or ‘leash’, and delivered as a cationic lipoplex. We have investigated a number of leash designs and chemistries, including mixed backbone oligonucleotides, and their ability to influence delivery and efficacy of the morpholino AO. Significantly, we detected dystrophin protein synthesis and correct sarcolemmal localisation after intramuscular injection of morpholino AO: leash lipoplexes in mdx muscle in vivo. We show enhanced delivery of a morpholino AO, enabling the advantageous properties to be exploited for potentially therapeutic outcomes