Abstract for presentation at 11th International Congress of Human Genetics

Morpholino antisense oligonucleotide induced dystrophin expression in the mdx mouse

  • Sue Fletcher, University of Western Australia, Australia
  • Ms Kaite Honeyman, University of Western Australia, Australia
  • Ms Abbie Fall, University of Western Australia, Australia
  • Ms Penny Harding, University of Western Australia, Australia
  • Mr Joshua Steinhaus, University of Western Australia, Australia
  • Mr Russell Johnsen, University of Western Australia, Australia
  • Dr Patrick Iversen, AVI Biopharma, United States
  • A/Prof Steve Wilton, University of Western Australia, Australia

    • Purpose: We are evaluating antisense oligonucleotides (AOs) to by-pass protein truncating mutations in the dystrophin gene. Defects preventing the production of dystrophin cause Duchenne muscular dystrophy (DMD), a fatal and common childhood muscular dystrophy. In-frame dystrophin mutations typically result in the milder allelic disorder, Becker muscular dystrophy (BMD), clearly demonstrating that some regions of the dystrophin protein are variably dispensable. The large size of the dystrophin gene, complex transcript processing, tissue-specific isoforms and expression of the predominant isoforms in non-dividing cells has hindered gene replacement strategies. These features render the dystrophin transcript an ideal candidate for splicing intervention. Splicing manipulation allows dystrophin expression to remain under the natural control of tissue-specific promoters.
    Methods: AO analogues have been used to exclude exon 23 of the dystrophin pre-mRNA, which carries a nonsense mutation, in the mdx mouse model of muscular dystrophy. We report in vivo studies evaluating phosphorodiamidate morpholino oligonucleotides (PMO), 2’-O-methyl phosphorothioate AOs and peptide nucleic acids (PNAs) directed at the same dystrophin splice site in this model of muscular dystrophy.
    Results: The PMO, with or without a conjugated peptide to enhance delivery, induced exon 23 removal, with subsequent dystrophin expression in mdx mouse muscle. Intramuscular injection of the PMO into mdx mice reduced central nucleation and induced substantial dystrophin expression in the treated muscle. Systemic administration of the peptide-conjugated PMO to neonatal mdx mice resulted in near-normal dystrophin expression and muscle architecture in diaphragm and limb muscles, but not in the myocardium. The treatment reduced serum creatine kinase levels and did not cause any overt adverse effects in the mdx mice.
    Conclusion: The morpholino chemistry appears safe and, if administered to neonatal mdx mice, prevents most manifestations of the dystrophinopathy. The efficient uptake of PMOs as a simple formulation in vivo may be considered a major advantage when applying these compounds in a clinical setting.

    Conference Organiser - ICMS Pty Ltd