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Morpholino antisense oligonucleotides for inducing exon skipping

Gebski, B.L., Mann, C.J., Fletcher, S. and Wilton, S.D. (2003) Morpholino antisense oligonucleotides for inducing exon skipping. In: 3rd Meeting of Australasian Gene Therapy Society, 30 April - 2 May 2003, Queensland Institute of Medical Research, Brisbane.


The mdx mouse model of muscular dystrophy arose due to a nonsense mutation in exon 23 of the dystrophin gene. We have previously demonstrated 2’ O-methyl phosphorothioate antisense oligonucleotides (AOs) induced the removal of exon 23 from the mRNA during processing of the primary gene transcript, producing an in-frame transcript which results in expression of dystrophin in mdx muscle. Refinement of AO design has allowed efficient exon skipping to be induced in mdx mouse muscle cultures at nanomolar concentrations.

Another chemistry that is fast gaining recognition as a potential therapeutic oligonucleotide is the morpholino AO which have been reported to overcome some of the limitations faced when using phosphodiester (PO) and the phosphorothioate (PS) AOs. The backbone modifications of the morpholino structural type produce an AO that is neutral and has greater biological stability. Splicing intervention by morpholino AOs has been successfully applied in vitro to the β-globin gene pre-mRNA to correct aberrant splicing when delivered in the micromolar concentration.

We present data showing that dystrophin exon skipping in mdx muscle cells may be induced by morpholino AOs at nanomolar concentrations when annealed to a ‘leash’ or sense oligonucleotide and then complexed with a cationic liposome. We have investigated a number of leash designs and chemistries, including mixed backbone oligonucleotides, and their ability to influence the ability of a 25-mer morpholino AO to induce specific exon skipping. Typically, each leash has a central phosphodiester core, which contains between 15-25 complimentary bases that anneal to the morpholino AO. Flanking the core region are 5 to 10 nucleotides on a PS backbone which generate overhangs at the 5’ or 3’ terminus of the morpholino AO. Varying the length and design of the leashes has enabled the morpholino AO to induce targeted pre-mRNA skipping and protein synthesis at concentrations several magnitudes below those required by uncomplexed morpholino AO, presumably due to improved delivery. The superior stability of the morpholino chemistry is expected to provide efficient and prolonged exon skipping and is currently under investigation in vivo.

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