Exon skipping that restores the levels of dystrophin protein is currently in clinical trials for Duchenne muscular dystrophy. Professor George Dickson and his colleagues at Royal Holloway set out to develop a new exon skipping strategy that blocks the action of a protein called myostatin, in addition to restoring dystrophin levels. They found that this combined approach was more effective at improving muscle function and strength than a single exon skipping strategy that only restores dystrophin. This finding provides an important proof-of-concept that will support the development of clinical reagents and improved therapeutic strategies for Duchenne muscular dystrophy.
What were the researchers aiming to do?
Duchenne muscular dystrophy is caused by mutations in the dystrophin gene. This gene contains the instructions for making dystrophin protein which acts as a shock absorber to prevent damage when the muscle contracts. The loss of dystrophin in Duchenne muscular dystrophy leads to wasting of the muscle, with the muscle fibres gradually being replaced by fat and scar tissue.
This project focused on the use of a technology called exon skipping, which has been tested in several clinical trials for Duchenne muscular dystrophy. This technology uses a small piece of DNA called a ‘molecular patch’ or antisense oligonucleotide, to specifically mask the area of a gene where there is a mutation. When this molecular patch is applied to the dystrophin gene, the production of dystrophin protein is restored. However, depending on the target gene and the molecular patch used, molecular patches can also be used to stop genes producing a functional protein.
Professor Dickson and his team have tested a range of molecular patches that targeted part of the coding sequence of the myostatin gene. This prevents the cell from reading the gene properly so that no myostatin protein can be produced (see figure below). Myostatin is an inhibitor of muscle growth and, together with other proteins that promote muscle growth, it works to keep the size and strength of muscle within the normal range. Researchers have shown in animals that blocking myostatin activity causes muscles to increase in strength and size, as well as reducing fat and scar tissue. Targeting myostatin with exon skipping might therefore be a useful way to bulk up muscles in people with muscle-wasting conditions, helping to increase their muscle strength.
After identifying the most effective molecular patches for exon skipping of myostatin, Professor Dickson planned to test them the mdx mouse model; first alone and then in combination with a different molecular patch that restores dystrophin production. It was thought that this combination of exon skipping strategies might prove more effective at rescuing muscle mass and strength than a single exon skipping approach.
What did their research show?
Professor Dickson and his team found that the molecular patches could induce exon skipping of myostatin by at least 80% in cells grown in the lab and up to 50% in muscles from the mdx mouse. Healthy mice that were injected with some of the patches over a four month period showed increased muscle strength and size, which confirmed that blocking myostatin production was beneficial. These experiments also showed which molecular patches were the most effective, and at what dose they should be used.
In the final part of the project, the researchers injected young and old mdx mice with molecular patches that targeted either just the dystrophin gene, or both the dystrophin and myostatin genes. They found that the combined molecular patch treatment improved muscle strength more than the single molecular patch targeting dystrophin. The therapeutic effects of the combined treatment lasted for at least four months of the study and were seen in muscles across the body; this included the heart and diaphragm (breathing muscle), which are often affected in Duchenne muscular dystrophy. The combined treatment was more efficient in the young mice than in the older mice, which suggests that it might be more effective at early stages of the condition.
What are the next steps?
Future work for Professor Dickson and his team will include investigating the mechanism underlying the therapeutic effects of blocking myostatin production. They will also investigate how often and at what stage the molecular patches should be delivered in order to provide the most therapeutic benefits and least side effects.
How will the outcomes of the research benefit people with muscle-wasting conditions?
Exon skipping that targets dystrophin is currently in clinical trial for Duchenne muscular dystrophy. This research suggests that the clinical benefits could be enhanced if myostatin was targeted in addition to dystrophin. Exon skipping that targets myostatin is yet to be tested in humans but this study shows that it could benefit people with a range of muscle-wasting conditions.
Project leader: Professor George Dickson
Location: Royal Holloway
Duration of project: 3 years (from 2012)
Total project cost: £124,696
Official project title: Combination Antisense Treatment for Duchenne Muscular Dystrophy: Open Reading Frame Rescue of Dystrophin in conjunction with Destructive Exon Skipping of Myostatin mRNA
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