Redirecting dystrophin pre-mRNA splicing using antisense oligonucleotides: An overview

We are developing an alternative therapy for Duchenne muscular dystrophy (DMD) using antisense oligonucleotides (AO) to displace factors involved in the normal splicing of the dystrophin exons during pre-mRNA processing. Masking selected motifs involved in splicing prevents normal splicesome assembly so that specific exons are omitted from the mature mRNA along with the flanking introns. In this manner, an exon containing a nonsense mutation could be by-passed as long as the reading frame was not disrupted. The most common type of mutations in the dystrophin gene are genomic deletions of one or more exons which disrupt the reading frame. Specific removal of one or more exons flanking the genomic deletions could restore the reading frame. The dystrophin gene should be a most amenable target for this type of AO approach as it has been well established that the complete dystrophin protein is not required for near normal activity. A milder allelic version of DMD, Becker Muscular Dystrophy, arises from in-frame deletions in the dystrophin gene, which allows synthesis of a shorter but still functional protein. Furthermore, dystrophin-positive revertant fibres, which arise from some naturally occurring exon skipping mechanism, have been detected in dystrophic tissue of many DMD patients and animal models of the disease. The mdx mouse model of muscular dystrophy carries a nonsense mutation in exon 23 of the dystrophin gene. AO-induced removal of this exon will by-pass the premature termination codon without disrupting the reading frame so that a dystrophin protein missing only 71 amino acids can be produced. Refinement of AO design and targeting has resulted in high levels of induced exon 23 skipping in the induced dystrophin mRNA, thereby restoring dystrophin synthesis in transfected cultured cells. In vivo administration through intramuscular injection of AOs into the tibialis anterior of mdx mice has resulted in strong and sustained dystrophin expression with correct sub-sarcolemmal localization. AO treated muscle shows significant improvement in muscle strength over untreated dystrophic mdx muscle after as few as a two injections of 1 ug AO:liposome complex. Other AO chemistries (morpholino and peptide nucleic acids) can also be used to displace splicing factors. These have uncharged backbones and, although very resistant to nuclease degradation, have been limited by poor cellular/nuclear uptake. We have annealed sense-strand oligonucleotide leashes to the uncharged AO to allow complex formation with cationic liposomes. Although the PNA:leash:liposome complex was unable to induce any exon skipping, the morpholino AO:leash:liposome complex induced exon skipping at concentrations more than three orders of magnitude lower than other groups have demonstrated antisense effects with uncomplexed morpholino AOs in cultured cells