Date of Award

1999

Degree Type

Thesis

Degree Name

Bachelor of Science Honours

Faculty

Faculty of Communication, Health and Science

First Advisor

Dr Steve Wilton

Second Advisor

Dr Sue Fletcher

Abstract

The development of an effective therapy for Duchenne Muscular Dystrophy (DMD) is one of the primary goals of all DMD/Becker Muscular Dystrophy (BMD) research. Golden Retriever Muscular Dystrophy (GRMD), an animal model of DMD is a fatal degenerative myopathy. Unlike the mdx model, the GRMD dog more accurately reflects the phenotype shown by human DMG patients, making the model better suited for the investigation and assessment of potential therapeutic approaches. The GRMD mutation, a base change from A to G in the 3' splice acceptor site of intron 6, results in exon 7 skipping which disrupts the translational reading frame. As a result, affected dogs have drastically reduced levels of dystrophin and its mRNA transcript. Recently, genetic therapy with antisense oligonucleotides (AOs) has attracted special interest as a novel therapeutic approach for DMD. AOs may be introduced into myotubes to redirect the splicing of GRMD dystrophin pre-mRNA to restore the reading frame. Ideally, this would increase the quantity of functional dystrophin in affected DMD tissues to levels of therapeutic value. The objective of this project was to identify intronic sequences from the canine dystrophin gene in the region of the mutation to allow the design AOs to be trialled as a therapy for GRMD. lntronic sequences that needed to be identified for the design of AOs were at the boundaries of intron 5/ exon 6, exon 6/ intron 7, exon 8/ intron 8 and intron 8/ exon 9. It was hoped that the application of the appropriate AOs would induce the processing/splicing of the dystrophin gene to exclude exons 6 and 8 (exon 7 is omitted due to the GRMD mutation) to restore the reading frame. To detennine these intronic sequences, several intronic sequencing strategies were attempted. These included long range PCR amplification, inverse PCR, PCR screening of a plagemid GR library, construction of a GR cosmid library for hybridization screening and PCR screening of a phage canine genomic library. This project determined the intronic boundaries of exon6/intron 6, intron 7/exon 8, exon 8(1ntron 8 and intron 8/exon9. Successful methods that identified intronic sequences included long-range PCR amplification and ihe PCR screening of both the phagemid and phage genomic DNA libraries. As it eventuated, this thesis also reported the first attempt at AO-induced GR dystrophin ex on skipping in a GR primary myoblast tissue culture.

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