Muscular dystrophy (MD) refers to a group of genetic, hereditary muscle diseases that cause progressive muscle weakness. Muscular dystrophies are characterised by progressive skeletal muscle weakness, defects in muscle proteins, and the death of muscle cells and tissue. This leads to muscle wasting, and probable death in early adulthood – the age of mortality depends on the type of MD.
Nine diseases are always classed as MD: Duchenne, Becker, limb girdle, congenital, facioscapulohumeral, myotonic, oculopharyngeal, distal, and Emery-Dreifuss. However, there are more than 100 diseases with similarities to MD. Most types of MD are multi-system disorders that manifest in various organs such as the heart, gastrointestinal and nervous systems, endocrine glands, skin, and eyes, amongst others.
Some forms of MD, including the most common, are sex-linked and so predominantly affect boys; others are autosomal dominant meaning that only one copy of the gene variant needs to be passed on from a parent, or autosomal recessive where both parents must pass on a mutant copy of the gene.
Two boys are born every week in the UK with the commonest form, Duchenne muscular dystrophy (DMD), which first appears at ages 2–16 and causes death in early adulthood. Although all forms of MD are still incurable, recent research has opened many promising avenues of treatment.
DMD is caused by errors in the largest known gene in the human genome, dystrophin. Without dystrophin, muscle cells weaken with continuous contraction and eventually die. There is no cure yet, but researchers are optimistic that animal work will lead to gene therapy and stem cell therapy which could have a major impact on patients’ quality of life.
Miyoshi myopathy and limb-girdle MD arise when the protein dysferlin, responsible for repairing muscle damage, is mutated.
Some forms of MD are caused by mutations that disrupt an assembly of muscle proteins called the dystrophin-glycoprotein complex.
Various other mouse models are throwing light on how damage is caused in MD.
A strain of mice and some breeds of dogs suffer 'naturally' from muscular dystrophy. Together with GM mice, these animals are being used to understand more about the disease and its treatment .
The mdx mouse is a naturally occurring strain with a mutation similar to that in DMD. There is also a naturally occurring mutation in golden retrievers that exactly matches the human DMD mutation in the dystrophin gene. These models make a major contribution to research into the physiopathology of DMD, ie how the disease disrupts normal body functions; and are invaluable to scientists for testing a variety of potential therapies.
The membranes surrounding muscle cells are frequently damaged, and this causes an influx of calcium to the injured site, where it forms vesicles that plug the damage. Dysferlin is a protein responsible for repairing muscle damage, and mice lacking the gene that makes it developed a progressive form of muscle dystrophy. At a microscopic level, muscle membranes that were damaged were unable to repair themselves .
Some forms of MD are caused by mutations that disrupt an assembly of muscle proteins called the dystrophin-glycoprotein complex. When one of these proteins, dystroglycan, was removed from the muscles of MD mice, the disease was far milder than in controls because so-called satellite cells stepped in and repaired some of the damage . Some forms of MD are caused by mutations in enzymes called glycosyltransferases, which lead to defects in the addition of sugars to dystroglycan. By restoring levels of a sugar-transferring enzyme called LARGE, or by over-expressing the gene that makes it, the disease was prevented in MD mice.
Subtle defects in the processing of a single protein that protects muscle cells from damage can lead to rare but devastating forms of MD, found in humans and mice. In muscle-eye-brain disease and Fukuyama congenital MD, defects in enzymes that process the structural protein dystroglycan affect muscles and cause additional developmental brain abnormalities including mental retardation. Two studies in mice demonstrated that dystroglycan is defective, and this will help doctors to provide accurate diagnosis and genetic counselling to patients and their families .
In several kinds of congenital MD half the patients lack a protein called merosin, or laminin m; a laminin-deficient MD mouse model has been useful for investigating gene therapy .
Mice lacking the sarcoglycan gene mimic limb-girdle muscular dystrophy in humans; in contrast to some other MD animal models, they show ongoing muscle necrosis with age, a hallmark of the human disease .
Gene therapy aims to deliver the normal dystrophin gene to the affected muscle in DMD, producing dystrophin in sufficient quantities to halt the wasting process
A successful approach has been to use the gene for a related protein, utrophin, which is smaller. When it was given to affected mice, their muscles produced utrophin and reduced muscle damage. A smaller dystrophin 'minigene' has also been engineered. It even reversed progression of the disease in mice .
Muscle development is restrained in normal animals by a protein called myostatin, and blocking this protein causes muscles to over-develop. This has been seen for 200 years in Belgian Blue cattle. The myostatin gene in mice was sequenced in 1997. When the gene for myostatin was knocked out in mdx mice using an antibody, their body weight and muscle mass increased and they showed less muscle degeneration. This has the potential to slow Duchenne and Becker muscular dystrophy .
Another way of sidestepping the size of the dystrophin gene has been to use a small ‘antisense’ RNA molecule injected into the muscle to 'patch up' the mutation. This molecule binds to specific sequences in the dystrophin RNA and interfers with a process called splicing, resulting in near-normal dystrophin production and relieving many of the symptoms. A single injection gave near-normal dystrophin production for over three months – a long time to a mouse . Obviously, it is vitally important not only to test for dystrophin protein production, but also to find out if it has a long-term effect on the symptoms. Human clinical trials are now underway (see Clinical trials of gene therapies).
Targeted gene repair has been successful in golden retrievers that suffer naturally from DMD. This approach used a manufactured molecule containing both DNA and RNA, which enabled the cell to use its own repair mechanism to correct the gene defect .
Gene therapy has also been successful in restoring normal muscle function in mice with laminin-deficient MD. Scientists replaced the missing gene with a 'minigene' containing elements of the agrin gene, as agrin has a similar effect to laminin on nerves and muscles. Offspring with the agrin gene showed weight gain, growth patterns and movement ability similar to that of normal mice. Another route to congenital MD treatment may lie in drugs that raise agrin concentrations .
Muscle stem cells taken from newborn mice and grown in tissue culture, then injected into mdx mice (a DMD model) appeared to develop into muscle, nerve and blood vessel cells. An added marker gene proved that the new cells came from the injected stem cells, and the new cells made dystrophin
Using MD mice lacking the sarcoglycan gene (equivalent to limb-girdle muscular dystrophy in humans), scientists have used mesangioblasts, stem cells taken from blood vessels, and found they transdifferentiated into muscle fibres . The scientists went on to try this with the golden retriever DMD model, and also had promising results: the dogs with the crippling genetic disorder were able to walk and even jump again . If mesangioblasts can be collected from human MD patients, they may offer a new avenue for treating the disease. The approach would involve collecting mesangioblasts from a patient's blood, genetically ‘correcting’ the cells in the laboratory, allowing them to multiply, and injecting them back into the patient's bloodstream. The cells would then migrate to the patient's muscles, and begin producing healthy muscle cells. Because the cells would be from the patient's own body, their immune system wouldn't reject them.
Muscle has also been regenerated in MD mice by injecting bone marrow ('hematopoietic') stem cells, which normally gives rise to blood cells .
Another potential therapeutic approach is the use of muscle growth factors. Researchers using MD mice have discovered that stem cells travel long distances to reach an injury and that they are summoned by a substance called mIGF-1 .
Antisense RNA 'patches' are now being tested in human muscle cells, reducing reliance on animal research. Muscle cells isolated from biopsies taken when the condition is diagnosed are grown in culture to assess the effectiveness of newly developed patches.
However, although cell culture experiments can help to assess the potential of novel approaches to treatment, only by understanding and testing such therapies in the complex environment of the whole body can we develop clinically useful treatments. To develop clinical trials into effective treatments, the DMD community will continue to rely heavily on the mdx mouse.
Clinical trials of gene therapies
Targeting all skeletal muscles is complicated, but experimental clinical studies are in progress in three areas
Two early stage gene-therapy clinical trials using adeno-associated viral (AAV) vectors have recently started; one in limb-girdle muscular dystrophy and one in DMD. They will assess the safety of the intramuscular administration (ie injected into the muscle) of the AAV vectors, and how well it works.
Two more early stage studies are also underway in DMD, testing antisense RNA patches (See Gene therapy, above). These are administered by injection into a small muscle on the top of the foot. Human studies have shown that this small foot muscle is relatively well preserved compared to other muscles in boys up to the age of around 15.
A fifth study is evaluating a drug that allows the dystrophin gene sequence of DMD patients to be 'read', even though they have nonsense mutations that would normally prevent this.
These studies should provide 'proof-of-principle' of these experimental approaches in humans, but it is likely that further work will be needed to refine the procedures before they can be used to treat MD.
Talking about one retriever, Dr Giulio Cossu of the San Raffaele Scientific Institute, said: “One of the most emotional moments I had was when I saw the severely impaired dog running again. I couldn’t have anticipated it going so well. I hope that this result can be transferred to humans.”
Professor Dominic Wells of Imperial College, London, points out: “Although cell culture experiments can help us to assess the potential of novel approaches to treatment, only by understanding and testing such therapies in the complex environment – the whole organism – can we develop clinically useful protocols. As clinical trials develop into effective treatments, the DMD community will have a lot to thank the mdx mouse for.”
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