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Title: Modulation of dystrophin pre-mRNA splicing by antisense oligonucleotides : a potential therapy for Duchenne muscular dystrophy
Author: Thorogood, Francesca Clare
Awarding Body: Royal Holloway, University of London
Current Institution: Royal Holloway, University of London
Date of Award: 2007
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Abstract:
Duchenne muscular dystrophy (DMD) is an X-linked muscle wasting disorder caused by mutations in the gene for dystrophin, a 427kDa cytoskeletal protein important for maintaining the integrity of muscle fibres. A number of DMD mutations result in an absence of functional protein due to disruption of the translational reading frame. It has been shown previously that antisense oligonucleotide (AON) reagents can modulate dystrophin pre-mRNA splicing to specifically exclude an out-of-frame exon from the mRNA. This restores the open reading frame resulting in production of a semi-functional internally-truncated dystrophin protein, mimicking what occurs in the milder allelic Becker muscular dystrophy (BMD). Previous work in this laboratory demonstrated successful exclusion of nonsense mutation carrying exon 23 in the mdx mouse model of DMD. This thesis is concerned with extension of the investigation by examining the bioactivity of alternative AON backbone chemistries 2' -O-methyl phosphorothioate (20Me), peptide nucleic acid (PNA) and phosphorodiamidate morpholino oligomer (PMO) targeting the 5'splice site of exon 23 in vitro and in vivo. In cultured murine muscle cells both the 20Me and PMO-based AON reagents induced detectable exon 23 exclusion. In the mdx mouse model intramuscular delivery of the 20Me-based AON reagent resulted in de novo dystrophin expression correctly localised at the sarcolemma that persisted for up to 4 weeks after a single dose. The PMO-based reagent resulted in de novo dystrophin expression that persisted for up to 10 weeks after a single intramuscular dose and for up to 8 weeks after a single intravenous dose. To broaden the investigation nine additional murine dystrophin exons were selected and AON regents designed targeting the 5'splice site and putative exonic splicing enhancer (ESE) sequences. Overall the results demonstrate that the AONs employed here induce detectable, reproducible exclusion of exon 23. The PMO-based reagent is currently superior for modulation of dystrophin pre-mRNA splicing.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.504809  DOI: Not available
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