A bridge over troubled waters

At 2.5 megabases, the dystrophin gene is the largest gene in the human genome, according to Professor Steve Wilton, head of the Experimental Molecular Medicine Group at the Centre for Neuromuscular and Neurological Disorders at the University of Western Australia. Almost 100 times longer than the average-sized human gene (27,000bp), it is a barn-door target for mutation's slings and arrows.

Wilton says one in three Duchenne muscular dystrophy (DMD) cases results from de novo frameshift mutations or deletions.

The first symptoms of muscle degeneration appear between ages three and five. The adult male body is at least 30 per cent muscle and as affected boys grow, progressive muscle weakness confines most to a wheelchair by age 12. Until the implementation of better management, including corticosteroid treatment and night-assisted ventilation, 90 per cent of DMD boys die from respiratory or cardiac problems before 20.

Wilton describes the dystrophin protein as a 'molecular shock absorber' that links the muscle-cell membrane to the flexible cytoskeleton, so they move in unison with the stresses imposed by contracting muscles. In DMD-affected muscle, the loss of dystrophin means the full force of the contracting muscle fibre is applied to the unstabilised membrane, causing it to tear.

The dystrophin gene has 79 exons, which, when spliced together, form a messenger RNA ~14,000 bases in length. In a mild form of the disorder, Becker dystrophy, internal deletions in exons leave the reading frame intact, resulting in an internally truncated but still functional protein.

There is a very high rate of wastage of messenger RNAs transcribed from the dystrophin gene; while some are alternative transcripts, more than 99.5 per cent of pre-mature mRNA is discarded as intronic sequences during processing of the full-length isoforms.

In rare cases, researchers are able to detect dystrophin-positive muscle fibres in the muscles of DMD patients with protein truncating mutations. In these 'revertant' fibres, a phenomenon called 'exon skipping' removes the segment of the mRNA containing the disease associated exon, leaving an in-frame transcript that can be translated into a semi-functional protein.

Wilton and his colleagues are experimenting with a targeted exon-skipping therapy to remove damaged exons from dystrophin mRNAs in DMD patients.

The therapy uses antisense oligonucleotides (AONs) that will bind to motifs in the damaged exons, masking them off from the spliceosome complex as it all removes the introns from the pre-mRNA, and splices the remaining exons together.

In effect, the spliceosome will 'see' the damaged exon as part of a single, extended intron formed by the two introns that originally flanked it. The extended intron will be spliced out and degraded, along with all the other intronic RNA in the pre-mRNA. The spliceosome will then join up the exposed ends of the neighbouring exons. The resulting mature mRNA will code for an internally truncated but functional dystrophin protein.

Wilton says a large number of AONs will be required to effect such repairs across the dystrophin gene transcript. There are hotspots for deletion mutations involving exons 3-7 and 44-55 and AONs have been designed to target these regions. The deletion of exons 3-7 disrupts the reading frame and leads to DMD. If exons 8 and 9 could be excised from this particular mutation during pre-mRNA processing, it should be possible to induce a protein identified in a particularly mild BMD patient.

"One Becker dystrophy patient, who was diagnosed at age 65 had a deletion of exons 3-9, which involved a loss of more than 200 kilobases," Wilton says. "Despite this, he was still playing badminton in his 60s and was described as a very active bushwalker."

He said the efficacy of exon skipping is influenced both by the design and the chemistry of the AONs. A panel of AONs have been developed that will induce specific removal of the majority of dystrophin exons.

He said exon skipping has proved very promising in animal studies using the Mdx mouse model of muscular dystrophy, but targeted dystrophin exon skipping has yet to be demonstrated in human muscle.

As a member of an international consortium, Wilton's team is involved in phase I clinical trials planned for later this year to test the safety of AON therapy, and confirm proof of principle. The first trial will involve nine boys with DMD aged 14 to 18, from London and Newcastle on Tyne, and will use a construct developed in Wilton's laboratory.

If the trials prove safe and confirm targeted exon skipping in a localised area after injection of the AON, he says, further trials will test the efficacy of systemic administration, as only whole body delivery can address the devastating disease.