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Optimisation of antisense oligonucleotide cocktails using in silico and in vitro techniques for targeted exon skipping in the dystrophin central rod domain

Barrett, L., Pinniger, G., Fall, A.M., Fletcher, S. and Wilton, S.D. (2009) Optimisation of antisense oligonucleotide cocktails using in silico and in vitro techniques for targeted exon skipping in the dystrophin central rod domain. In: 6th Australasian Gene Therapy Society Meeting, 29 April - 1 May 2009, Kerry Packer Education Centre. Royal Prince Alfred Hospital, Sydney, NSW.

Abstract

Becker muscular dystrophy (BMD), an allelic disorder to the severe condition Duchenne muscular dystrophy (DMD) presents with variable severity and age of onset. BMD is caused by mutations in the dystrophin gene that maintain the reading frame, resulting in a shorter, but still partially functional protein. BMD mutations are commonly found in the central rod domain, which consists of 25 triple-helical repeats similar to spectrin, with recent reports suggesting these repeats are not equivalent. One spectrin repeat is encoded by approximately 2 exons, hence excluding a single exon will leave an imperfect spectrin repeat. Antisense oligomer (AO) cocktails are currently being optimized for targeted removal of exons 23+24 in the mdx mouse, an animal model of DMD with a nonsense mutation in exon 23, to compare the effect on phenotype of taking out a half or a whole spectrin repeat. In silico analysis of oligomer cocktails is consistent with our hypothesis that the annealing of one compound to the pre-mRNA facilitates binding of the second oligomer. Individually, components of some cocktails induce no or only low levels of exon skipping. Hence it appears binding of one oligomer to a region not involved in spliceosome formation may permit another oligomer to anneal and mask crucial splice-control motifs not otherwise accessible. Optimized AO cocktails are being used in mice to induce multiple dystrophin isoforms for detailed molecular histological and physiological studies. Functional muscle testing in 12-week-old mice indicates that mdx muscles are weaker and more susceptible to stretch-induced damage when compared to normal mice, especially the diaphragm, which is most reflective of the human DMD phenotype.

Publication Type: Conference Item
Notes: Poster presentation
URI: http://researchrepository.murdoch.edu.au/id/eprint/23665
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