Last Monday the Muscular Dystrophy Campaign announced that the antisense oligonucleotide drug AVI-4658 had performed well in its second clinical trial in 19 boys with Duchenne Muscular Dystrophy (DMD). DMD is one of the most common hereditary disorders affecting the skeletomuscular system, and which results in progressive muscle degeneration, difficulty in walking and breathing, and ultimately death. Because DMD is a recessive condition linked to range of mutations in the dystrophin gene found on the X-chromosome, almost all its victims are boys. AVI-4658 is one of the first antisense oligomers – also known as morpholinos – to be evaluated in clinical trials, and is the result of more than a decade of research using animal models of DMD.
While the mutations responsible for causing DMD are scattered through many regions of the DMD gene – a cluster of mutations in exons 45-52 of the dystrophin gene has been found to be responsible for about 13% of cases. Exons are the portion of a gene which code for a protein, and a single gene can consist of many exons separated by stretches of DNA known as introns; the dystrophin gene has a total of 79 exons. The first step in the process of gene expression – getting from a gene to a protein – is transcription of the DNA sequence – both exons and introns – into a pre-messenger RNA sequence. The pre-messenger RNA sequence is then spliced to remove the introns, and the resulting spliced messenger RNA is translated into an amino acid sequence, which then normally undergoes post-translational processing to yield the mature protein. The mutations in exons 45-52 of the dystrophin gene result in either the translation of the dystrophin mRNA being terminated prematurely, or in the production of a defective dystrophin protein that is quickly broken down, rather than undergoing processing to yield a mature functional dystrophin protein. It is this absence of a functioning dystrophin protein that results in the symptoms of DMD. The antisense oligomer AVI-4658 works by binding to and blocking a specific sequence within exon 51 of the pre-messanger RNA, causing the splicing machinery to skip the affected exons and yield a shortened dystrophin protein, missing a segment within it, which nevertheless includes the key functional portions at either end of the protein and thus restores most of the dystrophin functionality.
The trial reported last week – funded by the UK Medical Research Council and AVI BioPharma – found that when AVI-4658 was given intravenously to DMD patients resulted in the expression of dystrophin protein , and that the dystrophin was interacting with other proteins as it should. At the highest doses tested there was an apparent reduction in muscle damage in some patients. The primary aim of the trial was to assess the safety and tolerability of the treatment – it performed well on both counts – with a secondary aim of assessing the ability of intravenous AVI-4658 to induce dystrophin expression. The trial was of an insufficient duration to evaluate if the treatment improved muscle strength, this key outcome will need to be evaluated in future trials.
In the above video Prof. Francesco Muntoni of University College London, who led the project, stresses the interdisciplinary nature of the research that resulted in the development of AVI-4658, and the crucial contribution made by animal research. In its press release on Monday the Muscular Dystrophy Campaign agrees, stating:
So far scientists have shown this technique to be effective in a dog and a mouse model of Duchenne muscular dystrophy. These animal models have been essential for the development of the therapy prior to testing in humans.”
This is very true, in mdx mice a mutation in exon 23 of the dystrophin gene halts production of the protein, and the first in-vitro demonstration that exon-skipping could restore dystrophin expression was performed in mdx mice (Dunckley MG, 1998). Over the next few years a variety of antisense oligonucleotides were used to restore dystrophin expression and function in the mdx mouse model (Aartsma-Rus A., 2007) and further support for this approach was obtained when antisense oligonucleotides were used to successfully restore dystrophin expression in a canine model that more closely mimics human DMD than the mdx mouse (McClorey G, 2006).
A Lancet Neurology paper published in 2009 by Prof. Muntoni’s group on the 1st clinical trial of AVI-4658 highlights the key role played by studies in the mdx mouse in determining the design of the morpholino.
Other chemically modified oligonucleotides have been used in preclinical models and clinical trials. Phosphorodiamidate morpholino oligomers (PMOs; figure 1) are non-toxic, and in the mdx mouse model of DMD they were the most effective oligomer chemistry for inducing exon skipping and restoring long-lasting (weeks) dystrophin expression after intravenous or intramuscular injection.21–24 PMOs, unlike other antisense oligonucleotides, are uncharged, not metabolised, and in preclinical or clinical studies were not associated with activation of the immune system, anaphylaxis, hypotension, or anti-arrhythmias.25 On the basis of these data, we have studied the safety and biochemical efficacy of AVI-4658, a PMO designed to target exon 51 that is delivered by intramuscular injection. Here, we report the results of a single-blind, placebo-controlled, dose-escalation safety and efficacy study of PMOs in patients with DMD.”
But the role of animal models of muscular dystrophy in developing treatment with AVI-4658 has not ended with the start of clinical trials, the Muscular Dystrophy Campaign point to recent research using the mdx mouse to optimize the AVI-4658 dosing regime. And the work on developing exon skipping technologies for the treatment of DMD goes on; in 2009 we discussed more advanced morpholino technologies , in particular morpholino cocktails that could target multiple mutations in the dystrophin gene, and novel peptide-linked morpholino designs that could restore dystrophin expression in the heart – something AVI-4658 cannot do. The development of peptide-linked morpholinos that can target dystrophin expression in the heart is also among the more advanced strategies under active investigation by the muscular dystrophy campaign, and as you would expect animal research is key to this work.
All in all this is a very exciting time for DMD research, with at least two promising exon-skipping therapies in advanced clinical trials and improved therapies following hot on their heels, it is increasingly likely that those diagnosed with DMD today face a far brighter future than previous generations.