Feature: Developing a genetic therapy for Duchenne muscular dystrophy

By Susan Williamson

This feature appeared in the November/December 2010 issue of Australian Life Scientist. To subscribe to the magazine, go here.

Duchenne muscular dystrophy (DMD) is a progressive X-linked muscle wasting disease that occurs in about 1 in 3500 live male births. The disease has no known treatment and, if it takes its natural course, boys are confined to a wheel chair by age 12, and 90 per cent die in their late teens from cardiovascular disease or respiratory problems.

Current treatments, such as steroids and assisted ventilation, can delay disease progression and patients with DMD now live to their mid 20s or early 30s, some even to their 40s.

Finding a treatment for this debilitating disorder is the focus of Professor Steve Wilton, Head of the Molecular Genetic Therapy Group at the Australian Neuromuscular Research Institute based at the University of Western Australia.

Wilton and his team are working with antisense oligomers to modify gene expression, in particular to promote exon skipping, in the hope that these molecules can be used to reduce the severity of DMD.

An initial trial on one of these molecules generated such promising results during the proof of concept stage that a clinical trial using systemic treatment was begun before the initial results were published. This trial has now demonstrated safety and confirmed that this technique may provide a viable treatment for patients with DMD.

DMD is caused by mutations in the dystrophin gene that result the premature truncation of dystrophin mRNA and a dysfunctional protein. The dystrophin protein resides at the inner surface of muscle cells and acts as a shock absorber, stabilising the cells during contraction. Muscle fibres that lack functional dystrophin are fragile and more vulnerable to breaking than normal muscle fibres.

The dystrophin gene is large – it has 79 exons that span 2.4 megabases (Mb), whereas the average gene has eight or nine exons. The mature full-length dystrophin mRNA is 14 kilobases long, which means that a lot (99.6 per cent) of the pre-mRNA contains intronic sequences that are discarded during splicing.

One in three cases of DMD are de novo; that is, there is no family history of the disease. This de novo occurrence makes screening for DMD problematic, especially considering the size of the gene.

“Finding a point mutation in a 2.4 Mb long gene is a challenge,” says Wilton. “Having a family history can make it easier to find because the gene has been well characterised and new molecular screening systems allow rapid detection of exonic deletions, the most common type of gene defect.”

A milder form of DMD, called Becker muscular dystrophy (BMD), involves in-frame deletions – that is, the ends of the protein are present but they are closer together, resulting in a shorter protein that retains some function. Patients with BMD are defined as restricted to a wheel chair after 16 years of age; however, a wide spectrum of disease severity can occur depending upon the nature and position of the mutation and hence the functionality of the dystrophin isoform.

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Exon skipping therapy

The aim of Wilton’s work is to target specific mutations in the dystrophin gene transcript and modify the splicing process with antisense oligonucelotides as the pre-mRNA is processed. These antisense oligonucleotides bind to a mutated exon, or one or more exons flanking a genomic deletion, effectively masking the mutations from the splicing machinery. Thus, when the introns are removed from the primary mRNA, so are the targeted exons, generating a final mRNA that is translated into a BMD-like dystrophin isoform. In this way, this so-called exon-skipping enables a functional dystrophin to be produced from a DMD gene transcript that would otherwise have undergone premature termination of translation.

Frame-shift mutations (whole exon deletions or duplications) are the most common type of re-arrangement in the dystrophin gene, and two in three in patients with DMD have deletion of one or more exons.

“The major deletion hot spot exists between dystrophin exons 45–55,” said Wilton. “If this entire section of the gene is deleted, the reading frame is not disrupted and a functional protein results. Individuals with a deletion of exons 45–55 have only mild BMD. In contrast, a deletion of only exon 50 disrupts the dystrophin reading frame and the protein produced is dysfunctional and quickly degraded.”

Currently, trying to treat patients with DMD with a deletion of exon 50 by targeted skipping of exon 45–55 is too challenging. However, restoring the reading frame by skipping exon 51 is a simpler and more efficient option and has been the basis of the ongoing clinical trials in which Wilton is involved.

Wilton’s team has a number of antisense oligonucleotides ready for preclinical testing. They have transfected these compounds into normal human myogenic cells and assessed them with RT–PCR to show the exon has been removed. An international consortium, MDEX, led by Professor Francesco Muntoni, Imperial College London, has published proof of concept studies and shown that a compound developed in Wilton’s laboratory restores dystrophin expression and correct localisation after direct injection into muscles of boys with DMD. This work was published in Lancet Neurology last year.

The proof of concept study involved injecting low or high doses of a phosphorodiamidate morpholino oligomer called AVI-4658, which was designed to excise exon 51, into the extensor digitalis brevis muscle on the top of one foot in boys with DMD. The other foot was injected with saline.

After one month, the muscles, which are relatively redundant as far as function goes, were removed from both feet and tested for the presence of dystrophin with immunohistochemistry. Dystrophin was present in each injection site, but not outside the site and not in the control saline-injected muscles.

A systemic study was then begun that involved an intravenous injection. This open-label dose-escalation study involved giving six different doses of the compound to 20 patients with DMD in 12 consecutive weekly doses. The dosing was staggered and started at 0.5 mg/kg/week for cohort one and increased to 20 mg/kg/week for cohort six. The drug was well tolerated with no adverse events reported.

“All the boys who received doses of 10 mg/kg and 20 mg/kg made dystrophin,” said Wilton. “The responses varied but some were spectacular responders. For example, in one boy over half the fibres in the muscle biopsy taken were dystrophin-positive.”

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A flexible approach

Ethics approval is required for clinical studies and the rational and conventional approach is to conduct a placebo-controlled trial to prove there is an effect. Wilton hopes regulatory bodies will be able step outside the box and consider personalised genetic treatment in a different way so that all amenable patients can be treated in trials.

Because of the many different antisense oligonucleotides that will be needed to treat the variety of mutations involved in DMD, one patient will need a treatment tailored to his specific genetic mutation which, under the current regulations, would require extensive safety testing. The compound in current clinical trials will only be relevant to about one in 10 boys with DMD.

Wilton met with the Therapeutic Goods Administration earlier this year and was very encouraged that Australia might lead the way in revising guidelines for DMD and hopes other regulatory agencies may recognise this as well.

He and his colleagues hope the regulatory bodies will agree to treating the antisense oligonucleotides as a class-specific type of drug. Then, if they show that one specific antisense oligonucleotide is safe to use for one mutation this should accelerate approval to use other oligonucleotides against other mutations, rather than undergoing safety testing for each one.

Wilton has been looking at the cost of clinical trials and is determined to work out a way of making it sustainable. But in reality, the prohibitive cost of clinical trials, including producing these drugs, makes commercial involvement a necessity.

“Half the DMD boys alive when we published our first exon skipping paper 10 years ago have now succumbed to their disease, and most of those still alive are now in wheelchairs. All researchers and clinicians in this area know the urgency: if exon skipping does not prove viable, we must fail it as quickly as possible to direct attention and resources elsewhere. However, if exon skipping is shown to be a clinically effective treatment for some mutations, we must push it as fast as morally and ethically possible and make this splice switching therapy available to all amenable DMD mutations, not just the ‘commercially attractive ones’.”

This feature appeared in the November/December 2010 issue of Australian Life Scientist. To subscribe to the magazine, go here.